D-glucose optical probe as well as preparation method and application thereof
By modifying the amino acid content of D-glucose-binding protein at specific sites and binding it with fluorescent protein to form an optical probe, the problems of high cost, poor stability and real-time monitoring in existing D-glucose detection technologies have been solved, achieving high-throughput and quantitative D-glucose detection.
Patent Information
- Application Number
- CN202410557236.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-07
- Publication Date
- 2025-11-07
AI Technical Summary
Existing D-glucose detection methods suffer from high cost, low repeatability, poor stability, and difficulty in achieving in situ real-time monitoring within living cells.
Develop a D-glucose optical probe by modifying or mutating amino acids at specific sites of the D-glucose binding protein to form a D-glucose-sensitive polypeptide and an optically active polypeptide by binding to a fluorescent protein, and construct a B1-A-B2 probe structure to achieve high-throughput and quantitative detection.
This technology enables real-time localization and quantitative detection of D-glucose inside and outside cells, improving the sensitivity and stability of the detection and meeting the need for in-situ monitoring of dynamic changes in D-glucose within living cells.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of optical probe, in particular to a D-glucose optical probe and a preparation method and application thereof. BACKGROUND
[0002] D-glucose (D-glc) is the most important and basic energy source known to all life forms on earth, from bacteria to humans, and is the main energy source for the human brain and nervous system. D-glucose plays an important role in the field of biology and is an essential nutrient for living organisms. It is not only a rich potential energy source, but also a precursor of a wide range of metabolic intermediates in biosynthetic pathways. Plants can also produce glucose through photosynthesis. D-glucose is the main energy source for most tissues, especially the nervous system, red blood cells, renal medulla, and skeletal muscle. Failure to regulate this nutrient can lead to hypoglycemia and hyperglycemia, and can even lead to death.
[0003] The most commonly used methods for detecting D-glucose at present include enzymatic probes, electrochemical methods, nanomaterial probes, various wearable glucose detection devices developed based on the above principles, and genetically encoded glucose probes. Among them, although the enzymatic probe has high selectivity for glucose and is feasible for large-scale production, it has high manufacturing cost, low repeatability, and poor stability; the electrochemical method is difficult to balance the cost and the selectivity for glucose; the genetically encoded fluorescent probe developed at present has limited specificity and cannot complete in vivo real-time monitoring of the affinity of the substrate, so it is urgent to develop a genetically encoded fluorescent probe that can monitor the dynamic changes of D-glucose in living cells in situ. SUMMARY
[0004] The purpose of the present application is to provide a probe and a method for real-time positioning, high-throughput, and quantitative detection of D-glucose in cells.
[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:
[0006] The first aspect of the present application provides a D-glucose binding protein variant, which:
[0007] (a) has the sequence shown in SEQ ID NO: 1 and has a mutation at 1, 2, 3 or more positions selected from W8, W9, A42, H66, K312, V347, H348, the mutation including modification, substitution or deletion of amino acids,
[0008] (b) is a sequence having at least 70% sequence identity with the sequence of (a) and having the mutation of (a) and retaining the binding ability to D-glucose.
[0009] In one or more embodiments, the mutation comprises a mutation at a site selected from any one of the following groups:
[0010] (1) V347, H348, (2) V347, H348 and K312, (3) V347, H348 and H66, (4) V347, H348 and W9, (5) V347, H348 and E13, (6) V347, H348 and W8, (7) V347, H348 and A42.
[0011] In one or more embodiments, W8 is mutated to R, H, E, A, V, L, F, I, M, C, N, G, K, D or T; in one or more embodiments, W9 is mutated to R, H, E, A, V, L, F, I, M, C, P, D, N, G, K, Y, S or T; in one or more embodiments, A42 is mutated to R, E, W, F, M, C, P, N, G, K, Y or T; in one or more embodiments, H66 is mutated to R, E, A, F, M, C, P, Q or T; in one or more embodiments, K312 is mutated to R, A, Q, G, H or S; in one or more embodiments, V347 is mutated to W; in one or more embodiments, H348 is mutated to T, G or S.
[0012] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W, H348T and H66M, (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W, H348T and W9H, (15) V347W, H348T and W9E, (16) V347W, H348T and W9A, (17) V347W, H348T and W9V, (18) V347W, H348T and W9L, (19) V347W, H348T and W9F, (20) V347W, H348T and W9I, (21) V347W, H348T and W9M, (22) V347W, H348T and W9C, (23) V347W, H348T and W9P, (24) V347W, H348T and W9N, (25) V347W, H348T and W9G, (26) V347W, H348T and W9K, (27) V347W, H348T and W9Y, (28) V347W, H348T and W9S, (29) V347W, H348T and W9T, (30) V347W, H348T and W9D, (31) V347W, H348T and W8R, (32) V347W, H348T and W8H, (33) V347W, H348T and W8E, (34) V347W, H348T and W8A, (35) V347W, H348T and W8V, (36) V347W, H348T and W8L, (37) V347W, H348T and W8F, (38) V347W, H348T and W8I, (39) V347W, H348T and W8M, (40) V347W, H348T and W8C, (41) V347W, H348T and W8N, (42) V347W, H348T and W8G, (43) V347W, H348T and W8K, (44) V347W, H348T and W8T, (45) V347W, H348T and W8D, (46) V347W, H348T and A42R, (47) V347W, H348T and A42E, (48) V347W, H348T and A42W, (49) V347W, H348T and A42F,(50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H348T and A42Y, (57) V347W, H348T and A42T, (58) V347W, H348T and K312R, (59) V347W, H348T and K312A, (60) V347W, H348T and K312Q, (61) V347W, H348T and K312G, (62) V347W, H348T and K312H, (63) V347W, H348T and K312S.
[0013] In another aspect, the present application provides a D-glucose optical probe comprising a D-glucose sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the D-glucose sensitive polypeptide. The D-glucose sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.
[0014] In one or more embodiments, the D-glucose optical probe comprises a D-glucose sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the D-glucose sensitive polypeptide B, dividing the D-glucose sensitive polypeptide B into a first part B1 and a second part B2, forming a probe structure of the formula B1-A-B2.
[0015] In one or more embodiments, the optically active polypeptide is located between residues 117-123, 266-288, and / or 340-353 of the D-glucose sensitive polypeptide, numbering corresponding to the full length of the D-glucose sensitive polypeptide. Preferably, the optically active polypeptide is located at any one or more of the following positions selected from the group consisting of: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288,271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353, 347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 34 6, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 35 1 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.
[0016] In one or more embodiments, the D-glucose-sensitive polypeptide is a D-glucose-binding protein or a functional variant thereof.
[0017] In one or more embodiments, the D-glucose-sensitive polypeptide has:
[0018] (1) The sequence shown in SEQ ID NO:1, or a sequence that has at least 70% sequence identity with them and retains D-glucose binding activity,
[0019] (2) The sequence of the D-glucose-binding protein variant described in any embodiment of the first aspect of this document, or
[0020] (3) has at least 70% sequence identity with the sequence described in (2) and has the mutation described in (2) and retains the sequence sensitive to D-glucose.
[0021] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof, wherein the functional variant of the fluorescent protein has a mutation within 3 amino acids from the attachment site of the optically active polypeptide.
[0022] In one embodiment, the fluorescent protein is selected from the group consisting of yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein. In one embodiment, the fluorescent protein has a sequence as set forth in any one of SEQ ID NOs: 2-9. Preferably, the fluorescent protein has a sequence as set forth in any one of SEQ ID NOs: 2, 6, 7, 9.
[0023] In one or more embodiments, the functional variant of the fluorescent protein has a mutation at amino acid position 1-3, preferably position 1. Preferably, the functional variant of the fluorescent protein comprises a mutation of the amino acid at position 1 to A or G.
[0024] In one or more embodiments, the functional variant of the fluorescent protein has a sequence as set forth in SEQ ID NO: 2 and a mutation at position Y1. Preferably, the mutation is Y1A or Y1G.
[0025] In one or more embodiments, the fluorescent protein has a sequence as set forth in SEQ ID NO: 2 or is a variant Y1A or Y1G having any one of the following mutations at position 1, and the optically active polypeptide is located at position 348 / 352 of the D-glucose sensitive polypeptide.
[0026] In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the present application can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide is flanked by a linker of no more than 5 amino acids, such as a linker of 0, 1, 2, 3, 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises the amino acid Y. In one embodiment, the linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as set forth below: first portion of the D-glucose sensitive polypeptide B1, Y, optically active polypeptide A, second portion of the D-glucose sensitive polypeptide B2. In one embodiment, the optical probe of the present application does not comprise a linker.
[0027] In one embodiment, the optical probe of the present application further comprises a localization sequence for localizing the probe to a specific organelle, such as a cell.
[0028] In one or more embodiments, the sequence of the D-glucose sensitive polypeptide is set forth in SEQ ID NO: 1, the optically active polypeptide is set forth in any one of SEQ ID NOs: 2, 6, 7, 9, and the optically active polypeptide is located at any one or more of the positions selected from the group consisting of: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267,271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.
[0029] In one or more embodiments, the D-glucose-sensitive polypeptide is as set forth in SEQ ID NO: 1, the optically active polypeptide is as set forth in any one of SEQ ID NOs: 2, 6, 7, 9 or a variant thereof having a mutation at the amino acid corresponding to amino acid position 1 of SEQ ID NO: 2 selected from any one or more of the following: Y1A or Y1G, the optically active polypeptide is located between residues 117-123, 266-288 and / or 340-353 of the D-glucose-sensitive polypeptide, the numbering corresponding to the full length of the D-glucose-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located between any one or more of the following positions selected from 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271, 271 / 272, 271 / 273, 271 / 274, 271 / 275, 271 / 276, 271 / 277, 271 / 278, 271 / 279, 271 / 280, 271 / 281, 271 / 282, 271 / 283, 271 / 284, 271 / 285, 271 / 286, 271 / 287, 271 / 288, 272 / 267, 272 / 268, 272 / 269, 272 / 270, 272 / 271, 272 / 272, 272 / 273, 272 / 274, 272 / 275, 272 / 276, 272 / 277, 272 / 278, 272 / 279, 272 / 280, 272 / 281, 272 / 282, 272 / 283, 272 / 284, 272 / 285, 272 / 286, 272 / 287, 272 / 288, 273 / 267, 273 / 268, 273 / 269, 273 / 270, 273 / 271, 273 / 272, 273 / 273, 273 / 274, 273 / 275, 273 / 276, 273 / 277, 273 / 278, 273 / 279, 273 / 280, 273 / 281, 273 / 282, 273 / 283, 273 / 284, 273 / 285, 273 / 286, 273 / 287, 273 / 288, 274 / 267, 274 / 268, 274 / 269, 274 / 270, 274 / 271, 274 / 272, 274 / 273, 274 / 274, 274 / 275, 274 / 276, 274 / 277, 274 / 278, 274 / 279, 274 / 280, 274 / 281, 274 / 282, 274 / 283, 274 / 284, 274 / 285, 274 / 286, 274 / 287, 274 / 288, 275 / 267, 275 / 268, 275 / 269, 275 / 270, 275 / 271, 275 / 272, 275 / 273, 275 / 274, 275 / 275, 275 / 276, 275 / 277, 275 / 278, 275 / 279, 275 / 280, 275 / 281, 275 / 282, 275 / 283, 275 / 284, 275 / 285, 275 / 286, 275 / 287, 275 / 288, 276 / 267, 276 / 268, 276 / 269, 276 / 270, 276 / 271, 276 / 272, 276 / 273, 276 / 274, 276 / 275, 276 / 276, 276 / 277, 276 / 278, 276 / 279, 276 / 280, 276 / 281, 276 / 282, 276 / 283, 276 / 284, 276 / 285, 276 / 286, 276 / 287, 276 / 288, 277 / 267, 277 / 268, 277 / 269, 277 / 270, 277 / 271, 277 / 272, 277 / 273, 277 / 274, 277 / 275, 277 / 276, 277 / 277, 277 / 278, 277 / 279, 277 / 280, 277 / 281, 277 / 282, 277 / 283, 277 / 284, 277 / 285, 277 / 286, 277 / 287, 277 / 288, 278 / 267, 278 / 268, 278 / 269, 278 / 270, 278 / 271, 278 / 272, 278 / 273, 278 / 274, 278 / 275, 278 / 276, 278 / 277, 278 / 278, 278 / 279, 278 / 280, 278 / 281, 278 / 282, 278 / 283, 278 / 284, 278 / 285, 278 / 286, 278 / 287, 278 / 288, 279 / 267, 279 / 268, 279 / 269, 279 / 270, 279 / 271, 279 / 272, 279 / 273, 279 / 274, 279 / 275, 279 / 276, 279 / 277, 279 / 278, 279 / 279, 279 / 280, 279 / 281, 279 / 282, 279 / 283, 279 / 284, 279 / 285, 279 / 286, 279 / 287, 279 / 288, 280 / 267, 280 / 268, 280 / 269, 280 / 270, 280 / 271, 280 / 272, 280 / 273, 280 / 274, 280 / 275, 280 / 276, 280 / 277, 280 / 278, 280 / 279, 280 / 280, 280 / 281, 280 / 282, 280 / 283, 280 / 284, 280 / 285, 280 / 286, 280 / 287, 280 / 288, 281 / 267, 281 / 268, 281 / 269, 281 / 270, 281 / 271, 281 / 272, 281 / 273, 281 / 274, 281 / 275, 281 / 276, 281 / 277, 281 / 278, 281 / 279, 281 / 280, 281 / 281, 281 / 282, 281 / 283, 281 / 284, 281 / 285, 281 / 286, 281 / 287, 281 / 288, 282 / 267, 282 / 268, 282 / 269, 282 / 270, 282 / 271, 282 / 272, 282 / 273, 282 / 274, 282 / 275, 282 / 276, 282 / 277, 282 / 278, 282 / 279, 282 / 280, 282 / 281, 282 / 282, 282 / 283, 282 / 284, 282 / 285, 282 / 286, 282 / 287, 282 / 288270 / 275、270 / 276、270 / 277、270 / 278、270 / 279、270 / 280、270 / 281、270 / 282、270 / 283、270 / 284、270 / 285、270 / 286、270 / 287、270 / 288、271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352, 345 / 353, 346 / 341, 346 / 342, 346 / 343, 346 / 344, 346 / 345, 346 / 346, 346 / 347, 346 / 348, 346 / 349, 346 / 350, 346 / 351, 346 / 352, 346 / 353, 347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.
[0030] In one or more embodiments, the optical probe, the D-glucose sensitive polypeptide is as set forth in SEQ ID NO: 1 and has one or more mutations of: W8, W9, A42, H66, K312, V347, H348. The optically active polypeptide is as set forth in SEQ ID NO: 2, 6, 7, 9 or is a variant thereof having at the amino acid corresponding to amino acid position 1 of SEQ ID NO: 2 one or more mutations selected from any one or more of: Y1A or Y1G, the optically active polypeptide is located at 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271, 271 / 272, 271 / 273, 271 / 274, 271 / 275, 271 / 276, 271 / 277, 271 / 278, 271 / 279, 271 / 280, 271 / 281, 271 / 282, 271 / 283, 271 / 284, 271 / 285, 271 / 286, 271 / 287, 271 / 288, 272 / 267, 272 / 268, 272 / 269, 272 / 270, 272 / 271, 272 / 272, 272 / 273, 272 / 274, 272 / 275, 272 / 276, 272 / 277, 272 / 278, 272 / 279, 272 / 280, 272 / 281, 272 / 282, 272 / 283, 272 / 284, 272 / 285, 272 / 286, 272 / 287, 272 / 288, 273 / 267, 273 / 268, 273 / 269, 273 / 270, 273 / 271, 273 / 272, 273 / 273, 273 / 274, 273 / 275, 273 / 276, 273 / 277, 273 / 278, 273 / 279, 273 / 280, 273 / 281, 273 / 282, 273 / 283, 273 / 284, 273 / 285, 273 / 286, 273 / 287, 273 / 288, 274 / 267, 274 / 268, 274 / 269, 274 / 270, 274 / 271, 274 / 272, 274 / 273, 274 / 274, 274 / 275, 274 / 276, 274 / 277, 274 / 278, 274 / 279, 274 / 280, 274 / 281, 274 / 282, 274 / 283, 274 / 284, 274 / 285, 274 / 286, 274 / 287, 274 / 288, 275 / 267, 275 / 268, 275 / 269, 275 / 270, 275 / 271, 275 / 272, 275 / 273, 275 / 274, 275 / 275, 275 / 276, 275 / 277, 275 / 278, 275 / 279, 275 / 280, 275 / 281, 275 / 282, 275 / 283, 275 / 284, 275 / 285, 275 / 286, 275 / 287, 275 / 288, 276 / 267, 276 / 268, 276 / 269, 276 / 270, 276 / 271, 276 / 272, 276 / 273, 276 / 274, 276 / 275, 276 / 276, 276 / 277, 276 / 278, 276 / 279, 276 / 280, 276 / 281, 276 / 282, 276 / 283, 276 / 284, 276 / 285, 276 / 286, 276 / 287, 276 / 288, 277 / 267, 277 / 268, 277 / 269, 277 / 270, 277 / 271, 277 / 272, 277 / 273, 277 / 274, 277 / 275, 277 / 276, 277 / 277, 277 / 278, 277 / 279, 277 / 280, 277 / 281, 277 / 282, 277 / 283, 277 / 284, 277 / 285, 277 / 286, 277 / 287, 277 / 288, 278 / 267, 278 / 268, 278 / 269, 278 / 270, 278 / 271, 278 / 272, 278 / 273, 278 / 274, 278 / 275, 278 / 276, 278 / 277, 278 / 278, 278 / 279, 278 / 280, 278 / 281, 278 / 282, 278 / 283, 278 / 284, 278 / 285, 278 / 286, 278 / 287, 278 / 288, 279 / 267, 279 / 268, 279 / 269, 279 / 270, 279 / 271, 279 / 272, 279 / 273, 279 / 274, 279 / 275, 279 / 276, 279 / 277, 279 / 278, 279 / 279, 279 / 280, 279 / 281, 279 / 282, 279 / 283, 279 / 284, 279 / 285, 279 / 286, 279 / 287, 279 / 288, 280 / 267, 280 / 268, 280 / 269, 280 / 270, 280 / 271, 280 / 272, 280 / 273, 280 / 274, 280 / 275, 280 / 276, 280 / 277, 280 / 278, 280 / 279, 280 / 280, 280 / 281, 280 / 282, 280 / 283, 280 / 284, 280 / 285, 280 / 286, 280 / 287, 280 / 288, 281 / 267, 281 / 268, 281 / 269, 281 / 270, 281 / 271, 281 / 272, 281 / 273, 281 / 274, 281 / 275, 281 / 276, 281 / 277, 281 / 278, 281 / 279, 281 / 280, 281 / 281, 281 / 282, 281 / 283, 281 / 284, 281 / 285, 281 / 286, 281 / 287, 281 / 288, 282 / 267, 282 / 268, 282 / 269, 282 / 270, 282 / 271, 282 / 272, 282 / 273, 282 / 274, 282 / 275, 282 / 276, 282 / 277, 282 / 278, 282 / 279, 282 / 280, 282 / 281, 282 / 282, 282 / 283, 282 / 284, 282 / 285, 282 / 286, 282 / 287, 282 / 288, 283 / 267, 283 / 268, 283 / 269, 283 / 270, 283 / 271, 283 / 272, 283 / 273, 283 / 274270 / 280、270 / 281、270 / 282、270 / 283、270 / 284、270 / 285、270 / 286、270 / 287、270 / 288、271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344, 346 / 345, 346 / 346, 346 / 347, 346 / 348, 346 / 349, 346 / 350, 346 / 351, 346 / 352, 346 / 353, 347 / 341, 347 / 342, 347 / 343, 347 / 344, 347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353. Preferably, the mutation of the D-glucose sensitive polypeptide comprises a mutation selected from any one of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W, H348T and H66M, (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W, H348T and W9K, (15) V347W, H348T and W9Q, (16) V347W, H348T and W9E, (17) V347W, H348T and W9A, (18) V347W, H348T and W9F, (19) V347W, H348T and W9M, (20) V347W, H348T and W9C, (21) V347W, H348T and W9P, (22) V347W, H348T and W9Q, (23) V347W, H348T and W9T, (24) V347W, H348T and W9S, (25) V347W, H348T and W9H, (26) V347W, H348T and W9Y, (27) V347W, H348T and W9V, (28) V347W, H348T and W9L, (29) V347W, H348T and W9I, (30) V347W, H348T and W9N, (31) V347W, H348T and W9D, (32) V347W, H348T and W9E, (33) V347W, H348T and W9K, (34) V347W, H348T and W9R, (35) V347W, H348T and W9Q, (36) V347W, H348T and W9A, (37) V347W, H348T and W9M, (38) V347W, H348T and W9C, (39) V347W, H348T and W9P, (40) V347W, H348T and W9Q, (41) V347W, H348T and W9T, (42) V347W, H348T and W9S, (43) V347W, H348T and W9H, (44) V347W, H348T and W9Y, (45) V347W, H348T and W9V, (46) V347W, H348T and W9L, (47) V347W, H348T and W9I, (48) V347W, H348T and W9N, (49) V347W, H348T and W9D, (50) V347W, H348T and W9E, (51) V347W, H348T and W9K, (52) V347W, H348T and W9R, (53) V347W, H348T and W9Q, (54) V347W, H348T and W9A, (55) V347W, H348T and W9M, (56) V347W, H348T and W9C, (57) V347W, H348T and W9P, (58) V347W, H348T and W9Q, (59) V347W, H348T and W9T, (60) V347W, H348T and W9S, (61) V347W, H348T and W9H, (62) V347W, H348T and W9Y, (63) V347W, H348T and W9V, (64) V347W, H348T and W9L, (65) V347W, H348T and W9I, (66) V347W, H348T and W9N, (67) V347W, H348T and W9D, (68) V347W, H348T and W9E, (69) V347W, H348T and W9K, (70) V347W, H348T and W9R, (71) V347W, H348T and W9Q, (72) V347W, H348T and W9A, (73) V347W, H348T and W9M, (74) V347W, H348T and W9C, (75) V347W, H348T and W9P, (76) V347W, H348T and W9Q, (77) V347W, H348T and W9T, (78) V347W, H348T and W9S, (79) V347W, H348T and W9H, (80) V347W, H348T and W9Y, (81) V347W, H348T and W9V, (82) V347W, H348T and W9L, (83) V347W, H348T and W9I, (84) V347W, H348T and W9N, (85) V347W, H348T and W9D, (86) V347W, H348T and W9E, (87) V347W, H348T and W9K, (88) V347W, H348T and W9R, (89) V347W, H348T and W9Q, (90) V347W, H348T and W9A, (91) V347W, H348T and W9M, (92) V347W, H348T and W9C, (93) V347W, H348T and W9P, (94) V347W, H348T and W9Q, (95) V347W, H348T and W9T, (96) V347W, H348T and W9S, (97) V347W, H348T and W9H, (98) V347W, H348T and W9Y, (99) V347W, H348T and W9V, (100) V347W, H348T and W9L, (101) V347W, H348T and W9I, (102) V347W, H348T and W9N, (103) V347W, H348T and W9D, (104) V347W, H348T and W9E, (105) V347W, H348T and W9K, (106) V347W, H348T and W9R, (107) VH348T and W9H, (15) V347W, H348T and W9E, (16) V347W, H348T and W9A, (17) V347W, H348T and W9V, (18) V347W, H348T and W9L, (19) V347W, H348T and W9F, (20) V347W, H348T and W9I, (21) V347W, H348T and W9M, (22) V347W, H348T and W9C, (23) V347W, H348T and W9P, (24) V347W, H348T and W9N, (25) V347W, H348T and W9G, (26) V347W, H348T and W9K, (27) V347W, H348T and W9Y, (28) V347W, H348T and W9S, (29) V347W, H348T and W9T, (30) V347W, H348T and W9D, (31) V347W, H348T and W8R, (32) V347W, H348T and W8H, (33) V347W, H348T and W8E, (34) V347W, H348T and W8A, (35) V347W, H348T and W8V, (36) V347W, H348T and W8L, (37) V347W, H348T and W8F, (38) V347W, H348T and W8I, (39) V347W, H348T and W8M, (40) V347W, H348T and W8C, (41) V347W, H348T and W8N, (42) V347W, H348T and W8G, (43) V347W, H348T and W8K, (44) V347W, H348T and W8T, (45) V347W, H348T and W8D, (46) V347W, H348T and A42R, (47) V347W, H348T and A42E, (48) V347W, H348T and A42W, (49) V347W, H348T and A42F, (50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H348T and A42Y, (57) V347W, H348T and A42T, (58) V347W, H348T and K312R, (59) V347W, H348T and K312A, (60) V347W, H348T and K312Q, (61) V347W, H348T and K312G, (62) V347W,H348T and K312H, (63)V347W, H348T and K312S.
[0031] In one or more embodiments, the optical probe, wherein the D-glucose sensitive polypeptide is as set forth in SEQ ID NO: 1, the optically active polypeptide is as set forth in SEQ ID NO: 2, 6, 7, 9, the optically active polypeptide is located at the 348 / 352 site of the D-glucose sensitive polypeptide, and the optical probe has the following mutations: (1) V347W, H348T of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (2) V347W, H348G of the D-glucose sensitive polypeptide, and 1G of the optically active polypeptide, (3) V347W, H348S of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (4) V347W, H348S of the D-glucose sensitive polypeptide, and 1G of the optically active polypeptide, (5) V347W, H348T of the D-glucose sensitive polypeptide, and 1G of the optically active polypeptide, (6) V347W, H348T, H66R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (7) V347W, H348T, H66E of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (8) V347W, H348T, H66A of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (9) V347W, H348T, H66F of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (10) V347W, H348T, H66M of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (11) V347W, H348T, H66C of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (12) V347W, H348T, H66P of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (13) V347W, H348T, H66Q of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (14) V347W, H348T, H66T of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (15) V347W, H348T, W9R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (16) V347W, H348T, W9H of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (17) V347W, H348T, W9E of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (18) V347W, H348T, W9A of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (19) V347W, H348T, W9V of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (20) V347W, H348T, W9L of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (21) V347W, H348T, W9F of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (22) V347W, H348T, W9I of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide.(23) V347W, H348T, W9M of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (24) V347W, H348T, W9C of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (25) V347W, H348T, W9P of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (26) V347W, H348T, W9N of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (27) V347W, H348T, W9G of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (28) V347W, H348T, W9K of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (29) V347W, H348T, W9Y of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (30) V347W, H348T, W9S of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (31) V347W, H348T, W9T of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (32) V347W, H348T, W9D of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (33) V347W, H348T, W8R of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (34) V347W, H348T, W8H of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (35) V347W, H348T, W8E of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (36) V347W, H348T, W8A of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (37) V347W, H348T, W8V of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (38) V347W, H348T, W8L of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (39) V347W, H348T, W8F of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (40) V347W, H348T, W8I of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (41) V347W, H348T, W8M of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (42) V347W, H348T, W8C of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (43) V347W, H348T, W8N of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (44) V347W, H348T, W8G of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (45) V347W, H348T, W8K of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (46) V347W, H348T, W8T of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide,(47) V347W, H348T, W8D of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (48) V347W, H348T, A42R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (49) V347W, H348T, A42E of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (50) V347W, H348T, A42W of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (51) V347W, H348T, A42F of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (52) V347W, H348T, A42M of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (53) V347W, H348T, A42C of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (54) V347W, H348T, A42P of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (55) V347W, H348T, A42N of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (56) V347W, H348T, A42G of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (57) V347W, H348T, A42K of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (58) V347W, H348T, A42Y of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (59) V347W, H348T, A42T of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (60) V347W, H348T, K312R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (61) V347W, H348T, K312A of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (62) V347W, H348T, K312Q of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (63) V347W, H348T, K312G of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (64) V347W, H348T, K312H of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (65) V347W, H348T, K312S of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide.
[0032] In one or more embodiments, the optical probe is set forth in SEQ ID NO: 11.
[0033] In another aspect, the present application provides fusion polypeptides comprising the optical probes described herein and other polypeptides. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the optical probes. In some embodiments, the other polypeptides include localization sequences (e.g., polypeptides that localize the optical probes to different organelles or subcellular compartments), tags that facilitate purification, or tags for use in immunological reactions (e.g., immunoblotting). Linkers can be present between the optical probes and the other polypeptides in the fusion polypeptides described herein.
[0034] In another aspect, the present application provides nucleic acid molecules comprising: (a) a coding sequence of the polypeptides or probes described in any of the embodiments herein, or (b) a complement of (a), or (c) a fragment of (a) or (b). The fragments are primers.
[0035] The present application also relates to variants of the nucleic acid molecules described above, including nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments, and variants of the optical probes or fusion proteins of the present application, or complements thereof.
[0036] In another aspect, the present application provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequences encode the optical probes or fusion polypeptides described herein.
[0037] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.
[0038] In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.
[0039] In some embodiments, the expression vector is selected from the group consisting of a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.
[0040] In another aspect, the present application provides a host cell that: (1) expresses the optical probes or fusion polypeptides described in any of the embodiments herein; (2) comprises the nucleic acid molecules described in any of the embodiments herein; or (3) comprises the nucleic acid constructs described in any of the embodiments herein. The host cell is preferably E. coli.
[0041] In another aspect, the present application provides a D-glucose detection kit comprising the optical probes or fusion polypeptides or polynucleotides described herein or the optical probes prepared according to the methods described herein.
[0042] In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of buffers, media, D-glucose standards.
[0043] In another aspect, the present application provides a method of making an optical probe described herein, comprising: providing a host cell expressing an optical probe or fusion polypeptide described herein, culturing the host cell under conditions in which the cell expresses the optical probe or fusion polypeptide, and isolating the optical probe or fusion polypeptide.
[0044] In one or more embodiments, the method comprises the steps of: 1) incorporating a nucleic acid molecule encoding a D-glucose optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the D-glucose optical probe.
[0045] In another aspect, the present application provides a method of detecting D-glucose in a sample, comprising: contacting an optical probe or fusion polypeptide or host cell described herein with the sample, and detecting a change in the optically active polypeptide. The detection can be in vivo, in vitro, subcellular, or in situ. The sample is, for example, blood.
[0046] In another aspect, the present application provides a method of quantifying D-glucose in a sample, comprising: contacting an optical probe or fusion polypeptide or host cell described herein with the sample, detecting an optical change in the optically active polypeptide, and quantifying the D-glucose in the sample based on the optical change in the optically active polypeptide.
[0047] In another aspect, the present application provides a method of screening for a compound (e.g., a drug), comprising: contacting an optical probe or fusion polypeptide or host cell described herein with a candidate compound in a D-glucose-containing system, detecting an optical change in the optically active polypeptide, and screening for the compound based on the optical change in the optically active polypeptide. The method can screen for the compound in high throughput.
[0048] In one or more embodiments, a host cell described herein is contacted with a candidate compound in a D-glucose-containing system, and an optical change in the optically active polypeptide indicates whether the candidate compound is capable of modulating cellular uptake of D-glucose.
[0049] In another aspect, the present application provides a method of intracellular and / or extracellular localization of the D-glucose, comprising: contacting a D-glucose-containing system with the optical probe or the host cell, and detecting an optical change in the optically active polypeptide.
[0050] In one or more embodiments, the system is a solution system, a cellular system, a subcellular system.
[0051] In another aspect, the present application provides use of a D-glucose optical probe or fusion polypeptide or host cell described herein in detecting D-glucose in a sample, in screening for a compound, or in intracellular and / or extracellular localization of D-glucose. In one or more embodiments, the localization is real-time localization.
[0052] In another aspect, the present application provides use of the D-glucose optical probe, or the fusion polypeptide, or the polynucleotide, or the nucleic acid construct, or the host cell as described herein in the preparation of a kit for detecting D-glucose in a sample, screening compounds, or intracellular and / or extracellular localization of D-glucose.
[0053] The D-glucose optical probe provided by the present application is easy to mature, has large fluorescence dynamic change, good specificity, and can be expressed in cells by genetic manipulation, and can be used for real-time localization, high-throughput, and quantitative detection of D-glucose in and out of cells, thereby saving time-consuming sample processing steps. Experimental results show that the D-glucose optical probe provided by the present application has a response to D-glucose that is more than 10 times that of a control, and can be used for localization, qualitative and quantitative detection of cells in cytoplasm, nucleus, cell membrane, mitochondria and other subcellular structures, and can be used for high-throughput compound screening and quantitative detection of D-glucose in blood. BRIEF DESCRIPTION OF DRAWINGS
[0054] Figure 1 SDS-PAGE of an exemplary D-glucose optical probe;
[0055] Figure 2 Fluorescence spectrum property of an exemplary D-glucose optical probe;
[0056] Figure 3 Titration curve of an exemplary D-glucose optical probe for different concentrations of D-glucose;
[0057] Figure 4 Specificity bar chart of an exemplary D-glucose optical probe for another substrate and sugar metabolism intermediates and analogs;
[0058] Figure 5 Subcellular organ localization photo of an exemplary D-glucose optical probe in mammalian cells;
[0059] Figure 6 Schematic diagram of dynamic monitoring of D-glucose concentration in cytoplasm in mammalian cells by an exemplary D-glucose optical probe;
[0060] Figure 7 Dot plot of high-throughput compound screening at the level of living cells by an exemplary D-glucose optical probe;
[0061] Figure 8 Bar chart of quantitative detection of D-glucose in mouse and human blood by an exemplary D-glucose optical probe. DETAILED DESCRIPTION
[0062] The term "about," as used herein when referring to a numerical value or range, means that the numerical value or range is within 20%, within 10%, and within 5% of the given numerical value or range.
[0063] The terms "comprising," "including," and "having," as used herein, are inclusive, i.e., "comprising," "including," and "having" are to be construed with a meaning that includes more than the stated specification, e.g., a composition "comprising" X can consist exclusively of X or can include something additional, e.g., X + Y.
[0064] The term "D-glucose-sensitive polypeptide," as used herein, refers to a polypeptide that responds to D-glucose, including any response of a chemical, biological, electrical, or physiological parameter of the polypeptide associated with the interaction of the sensitive polypeptide. The response includes small changes, e.g., changes in the orientation of an amino acid or peptide fragment of the polypeptide, as well as changes in, e.g., the primary, secondary, or tertiary structure of the polypeptide, including changes in, e.g., protonation, electrochemical potential, and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary, and tertiary structure of a molecule that includes side groups; a change in conformation occurs when the three-dimensional structure of the molecule changes. Examples of changes in conformation include a change from an alpha-helix to a beta-sheet or a change from a beta-sheet to an alpha-helix. It is understood that the detectable change need not be a change in conformation, so long as the fluorescence of the fluorescent protein moiety is altered. The D-glucose-sensitive polypeptides described herein can also include functional variants thereof. Functional variants of the D-glucose-sensitive polypeptides include, but are not limited to, variants that can interact with D-glucose to undergo the same or similar changes as the parent D-glucose-sensitive polypeptide.
[0065] The D-glucose-sensitive polypeptides described herein include, but are not limited to, the glucose-galactose binding protein TtGBP from T. thermophilus or variants having more than 90% homology thereto. The D-glucose binding protein can sense changes in D-glucose concentration, and the spatial conformation of the D-glucose binding protein can change during dynamic changes in D-glucose concentration.
[0066] The term "optical probe," as used herein, refers to a D-glucose-sensitive polypeptide fused to an optically active polypeptide. The inventors have discovered that the conformational change that occurs upon specific binding of D-glucose to a D-glucose-sensitive polypeptide, such as a D-glucose binding protein, causes a conformational change in the optically active polypeptide (e.g., a fluorescent protein), which in turn causes a change in the optical properties of the optically active polypeptide. By plotting a standard curve of the fluorescence of the fluorescent protein measured at different concentrations of D-glucose, the presence and / or level of D-glucose can be detected and analyzed. When describing the optical probes of the application (e.g., when describing the insertion site or mutation site), reference to the amino acid residue number is with reference to SEQ ID NO: 1.
[0067] In the optical probes of the present application, an optically active polypeptide (e.g., a fluorescent protein) is operably inserted into the D-glucose-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of optically active polypeptides that can be used as a means to detect responsiveness of the optical probes of the present application. As used herein, the term "fluorescent property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation or emission spectrum, the excitation and emission wavelength maxima, the amplitude of excitation at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy. A measurable difference in any of these properties between the active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present application in an activity assay. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, e.g., the amount of fluorescence at a particular wavelength or the integral of the fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are easily distinguishable between the unactivated and activated conformational states. The optically active polypeptides described herein can also include functional variants thereof. Functional variants of optically active polypeptides include, but are not limited to, variants that can undergo the same or similar changes in fluorescent properties as the parent optically active polypeptide.
[0068] The term "fluorescent protein" as used herein refers to a protein that emits fluorescence under irradiation of excitation light. Fluorescent proteins are used as a fundamental detection means in the field of biological science, such as the green fluorescent protein GFP commonly used in the field of biotechnology and the circularly permuted blue fluorescent protein (cpBFP), the circularly permuted green fluorescent protein (cpGFP), the circularly permuted yellow fluorescent protein (cpYFP), and the like derived from mutation of the protein; and the red fluorescent protein RFP commonly used in the technical field, and the circularly permuted proteins such as cpmApple, cpmOrange, cpmKate, and the like derived from the protein. Illustratively, cpYFP is shown in SEQ ID NO: 2, cpGFP is shown in SEQ ID NO: 6, cpBFP is shown in SEQ ID NO: 7, and cpmApple is shown in SEQ ID NO: 9.
[0069] The fluorescent protein in the optical probe also includes functional variants having mutations, including but not limited to fluorescent proteins having mutations at the 1st amino acid corresponding to SEQ ID NO: 2. The mutation at the 1st position is preferably a mutation to A or G. In some embodiments, the functional variant of the fluorescent protein has the sequence shown in any one of SEQ ID NO: 2, 6, 7, 9 and has a mutation at the 1st amino acid corresponding to SEQ ID NO: 2 selected from any one of the following groups: Y1A or Y1G. 1S indicates that the 1st amino acid is mutated to S, and so on.
[0070] In the optical probe of the present invention, the optically active polypeptides are located in the NC direction along the residues 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, and 267 / 270 of the D-glucose-sensitive polypeptide. 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271, 271 / 272, 271 / 273, 271 / 274, 271 / 275,271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345、347 / 346、347 / 347、347 / 348、347 / 349、347 / 350、347 / 351、347 / 352、347 / 353、348 / 341、348 / 342、348 / 343、348 / 344、348 / 345、348 / 346、348 / 347、348 / 348、348 / 349、348 / 350、348 / 351、348 / 352、348 / 353、349 / 341、349 / 342、349 / 343、349 / 344、349 / 345、349 / 346、349 / 347、349 / 348、349 / 349、349 / 350、349 / 351、349 / 352、349 / 353、350 / 341、350 / 342、350 / 343、350 / 344、350 / 345、350 / 346、350 / 347、350 / 348、350 / 349、350 / 350、350 / 351、350 / 352、350 / 353、351 / 341、351 / 342、351 / 343、351 / 344、351 / 345、351 / 346、351 / 347、351 / 348、351 / 349、351 / 350、351 / 351、351 / 352、351 / 353、352 / 341、352 / 342、352 / 343、352 / 344、352 / 345、352 / 346、352 / 347、352 / 348、352 / 349、352 / 350、352 / 351、352 / 352 and 352 / 353 or residues substituted therein, the numbering corresponds to the full length of the D-glucose-sensitive polypeptide. Herein, in the site represented in the form of "X / Y", the two ends of the optically active polypeptide have a part of the D-glucose-sensitive polypeptide, respectively, wherein the N-terminal of the optically active polypeptide is the N-terminal starting amino acid (e.g., any amino acid from 1st to 266th) of the D-glucose-sensitive polypeptide sequence to the 1st amino acid of X, and the C-terminal of the optically active polypeptide is the 1st amino acid of Y of the D-glucose-sensitive polypeptide sequence to the C-terminal end amino acid (e.g., any amino acid from Y to 394th) thereof. Among them, if the two numbers in the site represented in the form of "X / Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids described by the numbers, for example, the insertion site 266 / 267 means that the optically active polypeptide is located between the amino acids 266 and 267 of the D-glucose-sensitive polypeptide; if the two numbers in the site represented in the form of "X / Y" are not consecutive integers and X is less than Y, it means that the optically active polypeptide replaces the amino acids between the amino acids indicated by the numbers, for example, the insertion site 266 / 269 means that the optically active polypeptide replaces the amino acids 267-268 of the D-glucose-sensitive polypeptide; if the two numbers in the site represented in the form of "X / Y" are X greater than Y, it means that the part of the D-glucose-sensitive polypeptide located at the N-terminal of the optically active polypeptide ends at the 1st amino acid of X of the D-glucose-sensitive polypeptide sequence, and the part of the D-glucose-sensitive polypeptide located at the C-terminal of the optically active polypeptide starts from the 1st amino acid of Y of the D-glucose-sensitive polypeptide sequence; for example, the insertion site 346 / 344 means that the N-terminal of the optically active polypeptide is fused with the N-terminal starting amino acid (e.g., any amino acid from 1st to 394th) of the D-glucose-sensitive polypeptide sequence to the 1st amino acid of 346, and the C-terminal of the optically active polypeptide is fused with the 1st amino acid of 344 of the D-glucose-sensitive polypeptide sequence to the C-terminal end amino acid (e.g., 394th amino acid), and the exemplary structure is: (1st to 346th amino acids of the D-glucose-sensitive polypeptide sequence)-(optically active polypeptide)-(344th to 394th amino acids of the D-glucose-sensitive polypeptide sequence); if the two numbers in the site represented in the form of "X / Y" are not consecutive integers and X is equal to Y, it means that the optically active polypeptide is inserted between the amino acids indicated by the numbers, for example, the insertion site 267 / 267 means that the optically active polypeptide is inserted between the amino acids 267 and 268 of the myo-inositol-sensitive polypeptide. In an exemplary embodiment, the optically active polypeptide shown in SEQ ID NO: 2, 6, 7 or 9 is located at any one or more of the sites selected from the following sites of the D-glucose-sensitive polypeptide shown in SEQ ID NO: 1: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278,266 / 279、266 / 280、266 / 281、266 / 282、266 / 283、266 / 284、266 / 285、266 / 286、266 / 287、266 / 288、267 / 267、267 / 268、267 / 269、267 / 270、267 / 271、267 / 272、267 / 273、267 / 274、267 / 275、267 / 276、267 / 277、267 / 278、267 / 279、267 / 280、267 / 281、267 / 282、267 / 283、267 / 284、267 / 285、267 / 286、267 / 287、267 / 288、268 / 267、268 / 268、268 / 269、268 / 270、268 / 271、268 / 272、268 / 273、268 / 274、268 / 275、268 / 276、268 / 277、268 / 278、268 / 279、268 / 280、268 / 281、268 / 282、268 / 283、268 / 284、268 / 285、268 / 286、268 / 287、268 / 288、269 / 267、269 / 268、269 / 269、269 / 270、269 / 271、269 / 272、269 / 273、269 / 274、269 / 275、269 / 276、269 / 277、269 / 278、269 / 279、269 / 280、269 / 281、269 / 282、269 / 283、269 / 284、269 / 285、269 / 286、269 / 287、269 / 288、270 / 267、270 / 268、270 / 269、270 / 270、270 / 271、270 / 272、270 / 273、270 / 274、270 / 275、270 / 276、270 / 277、270 / 278、270 / 279、270 / 280、270 / 281、270 / 282、270 / 283、270 / 284、270 / 285、270 / 286、270 / 287、270 / 288、271 / 267、271 / 268、271 / 269、271 / 270、271 / 271、271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345、347 / 346、347 / 347、347 / 348、347 / 349、347 / 350、347 / 351、347 / 352、347 / 353、348 / 341、348 / 342、348 / 343、348 / 344、348 / 345、348 / 346、348 / 347、348 / 348、348 / 349、348 / 350、348 / 351、348 / 352、348 / 353、349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353.
[0071] In one or more embodiments, the optical probe comprises, in order from N-terminus to C-terminus, residues 1-X of SEQ ID NO: 1, an optically active polypeptide or variant thereof as set forth in any one of SEQ ID NOs: 2, 6, 7, 9, and residues Y-394 of SEQ ID NO: 1, wherein X and Y are selected from any one of the following groups:
[0072] (1) X is 266, Y is 267, (2) X is 266, Y is 268, (3) X is 266, Y is 269, (4) X is 266, Y is 270, (5) X is 266, Y is 271, (6) X is 266, Y is 272, (7) X is 266, Y is 273, (8) X is 266, Y is 274, (9) X is 266, Y is 275, (10) X is 266, Y is 276, (11) X is 266, Y is 277, (12) X is 266, Y is 278, (13) X is 266, Y is 279, (14) X is 266, Y is 280, (15) X is 266, Y is 281, (16) X is 266, Y is 282, (17) X is 266, Y is 283, (18) X is 266, Y is 284, (19) X is 266, Y is 285, (20) X is 266, Y is 286, (21) X is 266, Y is 287, (22) X is 266, Y is 288, (23) X is 267, Y is 267, (24) X is 267, Y is 268, (25) X is 267, Y is 269, (26) X is 267, Y is 270, (27) X is 267, Y is 271, (28) X is 267, Y is 272, (29) X is 267, Y is 273, (30) X is 267, Y is 274, (31) X is 267, Y is 275, (32) X is 267, Y is 276, (33) X is 267, Y is 277, (34) X is 267, Y is 278, (35) X is 267, Y is 279, (36) X is 267, Y is 280, (37) X is 267, Y is 281, (38) X is 267, Y is 282, (39) X is 267, Y is 283, (40) X is 267, Y is 284, (41) X is 267, Y is 285, (42) X is 267, Y is 286, (43) X is 267, Y is 287, (44) X is 267, Y is 288, (45) X is 268, Y is 267, (46) X is 268, Y is 268, (47) X is 268, Y is 269, (48) X is 268, Y is 270, (49) X is 268, Y is 271, (50) X is 268, Y is 272, (51) X is 268, Y is 273, (52) X is 268, Y is 274, (53) X is 268, Y is 275, (54) X is 268, Y is 276, (55) X is 268, Y is 277, (56) X is 268, Y is 278, (57) X is 268, Y is 279, (58) X is 268, Y is 280, (59) X is 268, Y is 281, (60) X is 268, Y is 282, (61) X is 268, Y is 283, (62) X is 268, Y is 284, (63) X is 268, Y is 285,(64) X is 268, Y is 286, (65) X is 268, Y is 287, (66) X is 268, Y is 288, (67) X is 269, Y is 267, (68) X is 269, Y is 268, (69) X is 269, Y is 269, (70) X is 269, Y is 270, (71) X is 269, Y is 271, (72) X is 269, Y is 272, (73) X is 269, Y is 273, (74) X is 269, Y is 274, (75) X is 269, Y is 275, (76) X is 269, Y is 276, (77) X is 269, Y is 277, (78) X is 269, Y is 278, (79) X is 269, Y is 279, (80) X is 269, Y is 280, (81) X is 269, Y is 281, (82) X is 269, Y is 282, (83) X is 269, Y is 283, (84) X is 269, Y is 284, (85) X is 269, Y is 285, (86) X is 269, Y is 286, (87) X is 269, Y is 287, (88) X is 269, Y is 288, (89) X is 270, Y is 267, (90) X is 270, Y is 268, (91) X is 270, Y is 269, (92) X is 270, Y is 270, (93) X is 270, Y is 271, (94) X is 270, Y is 272, (95) X is 270, Y is 273, (96) X is 270, Y is 274, (97) X is 270, Y is 275, (98) X is 270, Y is 276, (99) X is 270, Y is 277, (100) X is 270, Y is 278, (101) X is 270, Y is 279, (102) X is 270, Y is 280, (103) X is 270, Y is 281, (104) X is 270, Y is 282, (105) X is 270, Y is 283, (106) X is 270, Y is 284, (107) X is 270, Y is 285, (108) X is 270, Y is 286, (109) X is 270, Y is 287, (110) X is 270, Y is 288, (111) X is 271, Y is 267, (112) X is 271, Y is 268, (113) X is 271, Y is 269, (114) X is 271, Y is 270, (115) X is 271, Y is 271, (116) X is 271, Y is 272, (117) X is 271, Y is 273, (118) X is 271, Y is 274, (119) X is 271, Y is 275, (120) X is 271, Y is 276, (121) X is 271, Y is 277, (122) X is 271, Y is 278, (123) X is 271, Y is 279, (124) X is 271, Y is 280,(125) X is 271, Y is 281, (126) X is 271, Y is 282, (127) X is 271, Y is 283, (128) X is 271, Y is 284, (129) X is 271, Y is 285, (130) X is 271, Y is 286, (131) X is 271, Y is 287, (132) X is 271, Y is 288, (133) X is 272, Y is 267, (134) X is 272, Y is 268, (135) X is 272, Y is 269, (136) X is 272, Y is 270, (137) X is 272, Y is 271, (138) X is 272, Y is 272, (139) X is 272, Y is 273, (140) X is 272, Y is 274, (141) X is 272, Y is 275, (142) X is 272, Y is 276, (143) X is 272, Y is 277, (144) X is 272, Y is 278, (145) X is 272, Y is 279, (146) X is 272, Y is 280, (147) X is 272, Y is 281, (148) X is 272, Y is 282, (149) X is 272, Y is 283, (150) X is 272, Y is 284, (151) X is 272, Y is 285, (152) X is 272, Y is 286, (153) X is 272, Y is 287, (154) X is 272, Y is 288, (155) X is 273, Y is 267, (156) X is 273, Y is 268, (157) X is 273, Y is 269, (158) X is 273, Y is 270, (159) X is 273, Y is 271, (160) X is 273, Y is 272, (161) X is 273, Y is 273, (162) X is 273, Y is 274, (163) X is 273, Y is 275, (164) X is 273, Y is 276, (165) X is 273, Y is 277, (166) X is 273, Y is 278, (167) X is 273, Y is 279, (168) X is 273, Y is 280, (169) X is 273, Y is 281, (170) X is 273, Y is 282, (171) X is 273, Y is 283, (172) X is 273, Y is 284, (173) X is 273, Y is 285, (174) X is 273, Y is 286, (175) X is 273, Y is 287, (176) X is 273, Y is 288, (177) X is 274, Y is 267, (178) X is 274, Y is 268, (179) X is 274, Y is 269, (180) X is 274, Y is 270, (181) X is 274, Y is 271, (182) X is 274, Y is 272, (183) X is 274,Y is 273, (184) X is 274, Y is 274, (185) X is 274, Y is 275, (186) X is 274, Y is 276, (187) X is 274, Y is 277, (188) X is 274, Y is 278, (189) X is 274, Y is 279, (190) X is 274, Y is 280, (191) X is 274, Y is 281, (192) X is 274, Y is 282, (193) X is 274, Y is 283, (194) X is 274, Y is 284, (195) X is 274, Y is 285, (196) X is 274, Y is 286, (197) X is 274, Y is 287, (198) X is 274, Y is 288, (199) X is 275, Y is 267, (200) X is 275, Y is 268, (201) X is 275, Y is 269, (202) X is 275, Y is 270, (203) X is 275, Y is 271, (204) X is 275, Y is 272, (205) X is 275, Y is 273, (206) X is 275, Y is 274, (207) X is 275, Y is 275, (208) X is 275, Y is 276, (209) X is 275, Y is 277, (210) X is 275, Y is 278, (211) X is 275, Y is 279, (212) X is 275, Y is 280, (213) X is 275, Y is 281, (214) X is 275, Y is 282, (215) X is 275, Y is 283, (216) X is 275, Y is 284, (217) X is 275, Y is 285, (218) X is 275, Y is 286, (219) X is 275, Y is 287, (220) X is 275, Y is 288, (221) X is 276, Y is 267, (222) X is 276, Y is 268, (223) X is 276, Y is 269, (224) X is 276, Y is 270, (225) X is 276, Y is 271, (226) X is 276, Y is 272, (227) X is 276, Y is 273, (228) X is 276, Y is 274, (229) X is 276, Y is 275, (230) X is 276, Y is 276, (231) X is 276, Y is 277, (232) X is 276, Y is 278, (233) X is 276, Y is 279, (234) X is 276, Y is 280, (235) X is 276, Y is 281, (236) X is 276, Y is 282, (237) X is 276, Y is 283, (238) X is 276, Y is 284, (239) X is 276, Y is 285, (240) X is 276, Y is 286, (241) X is 276, Y is 287,(242) X is 276, Y is 288, (243) X is 277, Y is 267, (244) X is 277, Y is 268, (245) X is 277, Y is 269, (246) X is 277, Y is 270, (247) X is 277, Y is 271, (248) X is 277, Y is 272, (249) X is 277, Y is 273, (250) X is 277, Y is 274, (251) X is 277, Y is 275, (252) X is 277, Y is 276, (253) X is 277, Y is 277, (254) X is 277, Y is 278, (255) X is 277, Y is 279, (256) X is 277, Y is 280, (257) X is 277, Y is 281, (258) X is 277, Y is 282, (259) X is 277, Y is 283, (260) X is 277, Y is 284, (261) X is 277, Y is 285, (262) X is 277, Y is 286, (263) X is 277, Y is 287, (264) X is 277, Y is 288, (265) X is 278, Y is 267, (266) X is 278, Y is 268, (267) X is 278, Y is 269, (268) X is 278, Y is 270, (269) X is 278, Y is 271, (270) X is 278, Y is 272, (271) X is 278, Y is 273, (272) X is 278, Y is 274, (273) X is 278, Y is 275, (274) X is 278, Y is 276, (275) X is 278, Y is 277, (276) X is 278, Y is 278, (277) X is 278, Y is 279, (278) X is 278, Y is 280, (279) X is 278, Y is 281, (280) X is 278, Y is 282, (281) X is 278, Y is 283, (282) X is 278, Y is 284, (283) X is 278, Y is 285, (284) X is 278, Y is 286, (285) X is 278, Y is 287, (286) X is 278, Y is 288, (287) X is 279, Y is 267, (288) X is 279, Y is 268, (289) X is 279, Y is 269, (290) X is 279, Y is 270, (291) X is 279, Y is 271, (292) X is 279, Y is 272, (293) X is 279, Y is 273, (294) X is 279, Y is 274, (295) X is 279, Y is 275, (296) X is 279, Y is 276, (297) X is 279, Y is 277, (298) X is 279, Y is 278, (299) X is 279, Y is 279, (300) X is 279,Y is 280, (301) X is 279, Y is 281, (302) X is 279, Y is 282, (303) X is 279, Y is 283, (304) X is 279, Y is 284, (305) X is 279, Y is 285, (306) X is 279, Y is 286, (307) X is 279, Y is 287, (308) X is 279, Y is 288, (309) X is 280, Y is 267, (310) X is 280, Y is 268, (311) X is 280, Y is 269, (312) X is 280, Y is 270, (313) X is 280, Y is 271, (314) X is 280, Y is 272, (315) X is 280, Y is 273, (316) X is 280, Y is 274, (317) X is 280, Y is 275, (318) X is 280, Y is 276, (319) X is 280, Y is 277, (320) X is 280, Y is 278, (321) X is 280, Y is 279, (322) X is 280, Y is 280, (323) X is 280, Y is 281, (324) X is 280, Y is 282, (325) X is 280, Y is 283, (326) X is 280, Y is 284, (327) X is 280, Y is 285, (328) X is 280, Y is 286, (329) X is 280, Y is 287, (330) X is 280, Y is 288, (331) X is 281, Y is 267, (332) X is 281, Y is 268, (333) X is 281, Y is 269, (334) X is 281, Y is 270, (335) X is 281, Y is 271, (336) X is 281, Y is 272, (337) X is 281, Y is 273, (338) X is 281, Y is 274, (339) X is 281, Y is 275, (340) X is 281, Y is 276, (341) X is 281, Y is 277, (342) X is 281, Y is 278, (343) X is 281, Y is 279, (344) X is 281, Y is 280, (345) X is 281, Y is 281, (346) X is 281, Y is 282, (347) X is 281, Y is 283, (348) X is 281, Y is 284, (349) X is 281, Y is 285, (350) X is 281, Y is 286, (351) X is 281, Y is 287, (352) X is 281, Y is 288, (353) X is 282, Y is 267, (354) X is 282, Y is 268, (355) X is 282, Y is 269, (356) X is 282, Y is 270, (357) X is 282, Y is 271, (358) X is 282, Y is 272,(359) X is 282, Y is 273, (360) X is 282, Y is 274, (361) X is 282, Y is 275, (362) X is 282, Y is 276, (363) X is 282, Y is 277, (364) X is 282, Y is 278, (365) X is 282, Y is 279, (366) X is 282, Y is 280, (367) X is 282, Y is 281, (368) X is 282, Y is 282, (369) X is 282, Y is 283, (370) X is 282, Y is 284, (371) X is 282, Y is 285, (372) X is 282, Y is 286, (373) X is 282, Y is 287, (374) X is 282, Y is 288, (375) X is 283, Y is 267, (376) X is 283, Y is 268, (377) X is 283, Y is 269, (378) X is 283, Y is 270, (379) X is 283, Y is 271, (380) X is 283, Y is 272, (381) X is 283, Y is 273, (382) X is 283, Y is 274, (383) X is 283, Y is 275, (384) X is 283, Y is 276, (385) X is 283, Y is 277, (386) X is 283, Y is 278, (387) X is 283, Y is 279, (388) X is 283, Y is 280, (389) X is 283, Y is 281, (390) X is 283, Y is 282, (391) X is 283, Y is 283, (392) X is 283, Y is 284, (393) X is 283, Y is 285, (394) X is 283, Y is 286, (395) X is 283, Y is 287, (396) X is 283, Y is 288, (397) X is 284, Y is 267, (398) X is 284, Y is 268, (399) X is 284, Y is 269, (400) X is 284, Y is 270, (401) X is 284, Y is 271, (402) X is 284, Y is 272, (403) X is 284, Y is 273, (404) X is 284, Y is 274, (405) X is 284, Y is 275, (406) X is 284, Y is 276, (407) X is 284, Y is 277, (408) X is 284, Y is 278, (409) X is 284, Y is 279, (410) X is 284, Y is 280, (411) X is 284, Y is 281, (412) X is 284, Y is 282, (413) X is 284, Y is 283, (414) X is 284, Y is 284, (415) X is 284, Y is 285, (416) X is 284, Y is 286, (417) X is 284,Y is 287, (418) X is 284, Y is 288, (419) X is 285, Y is 267, (420) X is 285, Y is 268, (421) X is 285, Y is 269, (422) X is 285, Y is 270, (423) X is 285, Y is 271, (424) X is 285, Y is 272, (425) X is 285, Y is 273, (426) X is 285, Y is 274, (427) X is 285, Y is 275, (428) X is 285, Y is 276, (429) X is 285, Y is 277, (430) X is 285, Y is 278, (431) X is 285, Y is 279, (432) X is 285, Y is 280, (433) X is 285, Y is 281, (434) X is 285, Y is 282, (435) X is 285, Y is 283, (436) X is 285, Y is 284, (437) X is 285, Y is 285, (438) X is 285, Y is 286, (439) X is 285, Y is 287, (440) X is 285, Y is 288, (441) X is 286, Y is 267, (442) X is 286, Y is 268, (443) X is 286, Y is 269, (444) X is 286, Y is 270, (445) X is 286, Y is 271, (446) X is 286, Y is 272, (447) X is 286, Y is 273, (448) X is 286, Y is 274, (449) X is 286, Y is 275, (450) X is 286, Y is 276, (451) X is 286, Y is 277, (452) X is 286, Y is 278, (453) X is 286, Y is 279, (454) X is 286, Y is 280, (455) X is 286, Y is 281, (456) X is 286, Y is 282, (457) X is 286, Y is 283, (458) X is 286, Y is 284, (459) X is 286, Y is 285, (460) X is 286, Y is 286, (461) X is 286, Y is 287, (462) X is 286, Y is 288, (463) X is 287, Y is 267, (464) X is 287, Y is 268, (465) X is 287, Y is 269, (466) X is 287, Y is 270, (467) X is 287, Y is 271, (468) X is 287, Y is 272, (469) X is 287, Y is 273, (470) X is 287, Y is 274, (471) X is 287, Y is 275, (472) X is 287, Y is 276, (473) X is 287, Y is 277, (474) X is 287, Y is 278, (475) X is 287, Y is 279,(476) X is 287, Y is 280, (477) X is 287, Y is 281, (478) X is 287, Y is 282, (479) X is 287, Y is 283, (480) X is 287, Y is 284, (481) X is 287, Y is 285, (482) X is 287, Y is 286, (483) X is 287, Y is 287, (484) X is 287, Y is 288, (485) X is 117, Y is 118, (486) X is 117, Y is 119, (487) X is 117, Y is 120, (488) X is 117, Y is 121, (489) X is 117, Y is 122, (490) X is 117, Y is 123, (491) X is 118, Y is 118, (492) X is 118, Y is 119, (493) X is 118, Y is 120, (494) X is 118, Y is 121, (495) X is 118, Y is 122, (496) X is 118, Y is 123, (497) X is 119, Y is 118, (498) X is 119, Y is 119, (499) X is 119, Y is 120, (500) X is 119, Y is 121, (501) X is 119, Y is 122, (502) X is 119, Y is 123, (503) X is 120, Y is 118, (504) X is 120, Y is 119, (505) X is 120, Y is 120, (506) X is 120, Y is 121, (507) X is 120, Y is 122, (508) X is 120, Y is 123, (509) X is 121, Y is 118, (510) X is 121, Y is 119, (511) X is 121, Y is 120, (512) X is 121, Y is 121, (513) X is 121, Y is 122, (514) X is 121, Y is 123, (515) X is 122, Y is 118, (516) X is 122, Y is 119, (517) X is 122, Y is 120, (518) X is 122, Y is 121, (519) X is 122, Y is 122, (520) X is 122, Y is 123, (521) X is 340, Y is 341, (522) X is 340, Y is 342, (523) X is 340, Y is 343, (524) X is 340, Y is 344, (525) X is 340, Y is 345, (526) X is 340, Y is 346, (527) X is 340, Y is 347, (528) X is 340, Y is 348, (529) X is 340, Y is 349, (530) X is 340, Y is 350, (531) X is 340, Y is 351, (532) X is 340, Y is 352, (533) X is 340, Y is 353, (534) X is 341,Y is 341, (535) X is 341, Y is 342, (536) X is 341, Y is 343, (537) X is 341, Y is 344, (538) X is 341, Y is 345, (539) X is 341, Y is 346, (540) X is 341, Y is 347, (541) X is 341, Y is 348, (542) X is 341, Y is 349, (543) X is 341, Y is 350, (544) X is 341, Y is 351, (545) X is 341, Y is 352, (546) X is 341, Y is 353, (547) X is 342, Y is 341, (548) X is 342, Y is 342, (549) X is 342, Y is 343, (550) X is 342, Y is 344, (551) X is 342, Y is 345, (552) X is 342, Y is 346, (553) X is 342, Y is 347, (554) X is 342, Y is 348, (555) X is 342, Y is 349, (556) X is 342, Y is 350, (557) X is 342, Y is 351, (558) X is 342, Y is 352, (559) X is 342, Y is 353, (560) X is 343, Y is 341, (561) X is 343, Y is 342, (562) X is 343, Y is 343, (563) X is 343, Y is 344, (564) X is 343, Y is 345, (565) X is 343, Y is 346, (566) X is 343, Y is 347, (567) X is 343, Y is 348, (568) X is 343, Y is 349, (569) X is 343, Y is 350, (570) X is 343, Y is 351, (571) X is 343, Y is 352, (572) X is 343, Y is 353, (573) X is 344, Y is 341, (574) X is 344, Y is 342, (575) X is 344, Y is 343, (576) X is 344, Y is 344, (577) X is 344, Y is 345, (578) X is 344, Y is 346, (579) X is 344, Y is 347, (580) X is 344, Y is 348, (581) X is 344, Y is 349, (582) X is 344, Y is 350, (583) X is 344, Y is 351, (584) X is 344, Y is 352, (585) X is 344, Y is 353, (586) X is 345, Y is 341, (587) X is 345, Y is 342, (588) X is 345, Y is 343, (589) X is 345, Y is 344, (590) X is 345, Y is 345, (591) X is 345, Y is 346, (592) X is 345, Y is 347,(593) X is 345, Y is 348, (594) X is 345, Y is 349, (595) X is 345, Y is 350, (596) X is 345, Y is 351, (597) X is 345, Y is 352, (598) X is 345, Y is 353, (599) X is 346, Y is 341, (600) X is 346, Y is 342, (601) X is 346, Y is 343, (602) X is 346, Y is 344, (603) X is 346, Y is 345, (604) X is 346, Y is 346, (605) X is 346, Y is 347, (606) X is 346, Y is 348, (607) X is 346, Y is 349, (608) X is 346, Y is 350, (609) X is 346, Y is 351, (610) X is 346, Y is 352, (611) X is 346, Y is 353, (612) X is 347, Y is 341, (613) X is 347, Y is 342, (614) X is 347, Y is 343, (615) X is 347, Y is 344, (616) X is 347, Y is 345, (617) X is 347, Y is 346, (618) X is 347, Y is 347, (619) X is 347, Y is 348, (620) X is 347, Y is 349, (621) X is 347, Y is 350, (622) X is 347, Y is 351, (623) X is 347, Y is 352, (624) X is 347, Y is 353, (625) X is 348, Y is 341, (626) X is 348, Y is 342, (627) X is 348, Y is 343, (628) X is 348, Y is 344, (629) X is 348, Y is 345, (630) X is 348, Y is 346, (631) X is 348, Y is 347, (632) X is 348, Y is 348, (633) X is 348, Y is 349, (634) X is 348, Y is 350, (635) X is 348, Y is 351, (636) X is 348, Y is 352, (637) X is 348, Y is 353, (638) X is 349, Y is 341, (639) X is 349, Y is 342, (640) X is 349, Y is 343, (641) X is 349, Y is 344, (642) X is 349, Y is 345, (643) X is 349, Y is 346, (644) X is 349, Y is 347, (645) X is 349, Y is 348, (646) X is 349, Y is 349, (647) X is 349, Y is 350, (648) X is 349, Y is 351, (649) X is 349, Y is 352, (650) X is 349, Y is 353, (651) X is 350,(660) X is 350, Y is 350, (661) X is 350, Y is 351, (662) X is 350, Y is 352, (663) X is 350, Y is 353, (664) X is 351, Y is 341, (665) X is 351, Y is 342, (666) X is 351, Y is 343, (667) X is 351, Y is 344, (668) X is 351, Y is 345, (669) X is 351, Y is 346, (670) X is 351, Y is 347, (671) X is 351, Y is 348, (672) X is 351, Y is 349, (673) X is 351, Y is 350, (674) X is 351, Y is 351, (675) X is 351, Y is 352, (676) X is 351, Y is 353, (677) X is 352, Y is 341, (678) X is 352, Y is 342, (679) X is 352, Y is 343, (680) X is 352, Y is 344, (681) X is 352, Y is 345, (682) X is 352, Y is 346, (683) X is 352, Y is 347, (684) X is 352, Y is 348, (685) X is 352, Y is 349, (686) X is 352, Y is 350, (687) X is 352, Y is 351, (688) X is 352, Y is 352, (689) X is 352, Y is 353.
[0073] In this document, "response fold" is the fluorescence ratio after normalization. The more the response fold of the probe deviates from 1 (whether it is larger or smaller), the greater the change fold of the response ability of the probe to the substrate relative to the control or the response ability. For example, the response fold is calculated by detecting the change in the fluorescence intensity ratio at 420 nm excitation and 528 nm emission to the fluorescence intensity ratio at 485 nm excitation and 528 nm emission (Normalized Ratio 420 / 485) by the embodiments of the present application, as follows:
[0074] The fluorescence signal value is corrected by deducting the detection signal value of the cell without expressing the probe protein. The probe detection signal in the parallel experimental group is divided by the control detection signal to eliminate the pH-sensitive interference to obtain the corrected data.
[0075] F = F sample F = F BLK
[0076]
[0077]
[0078]
[0079]
[0080] F represents the fluorescence intensity (Fluorescence intensity), F sample represents the total fluorescence intensity of the sample expressing the fluorescent probe, F BLK represents the background fluorescence intensity of the sample not expressing the fluorescent probe, F cpYFP represents the fluorescence intensity of the sample as a pH control. F 485 represents the fluorescence intensity of the fluorescent protein sample exciting at 485 nm and emitting at 528 nm, F 420 represents the fluorescence intensity of the fluorescent protein sample exciting at 420 nm and emitting at 528 nm. Ratiosensor represents the fluorescence intensity ratio of the probe, Ratio cpYFP represents the fluorescence intensity ratio of the pH control fluorescent protein corresponding to the probe. Normalized Ratio 420 / 485 is the fold change or response of the probe. Normalized Ratio 485 / 420 The further from 1 (whether larger or smaller) indicates the greater the fold change or response of the probe.
[0081] As used herein, the term "variant" or "mutant" with respect to a polypeptide or protein includes variants having the same function as the polypeptide or protein but having a different sequence. Variants of a polypeptide or protein can include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include, but are not limited to, sequences in which one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids are deleted, inserted, and / or substituted in the sequence of the polypeptide or protein, and sequences in which one or more (typically 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids are added at the carboxy terminus and / or amino terminus. These variants can also include polypeptides or proteins having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence of the polypeptide or protein. Without wishing to be bound by theory, amino acid residues are changed without changing the overall conformation and function of the polypeptide or protein, i.e., function-conservative mutations. For example, in the art, substitution with an amino acid having similar properties does not typically change the function of the polypeptide or protein. In the art, amino acids having similar properties tend to be defined as families of amino acids having similar side chains, which are well defined in the art. These families include amino acids having basic side chains (e.g., lysine, D-glucose, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), uncharged nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, D-glucose, phenylalanine, methionine, D-glucose), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, D-glucose, histidine). Also, for example, addition of one or more amino acids at the amino terminus and / or carboxy terminus also typically does not change the function of the polypeptide or protein. Conservative substitutions of non-genetically encoded amino acids are known in the art. Other conservative substitutions of non-encoded amino acids can be determined based on a comparison of their physical properties with those of genetically encoded amino acids.
[0082] "Linker" or "linking region" refers to an amino acid or nucleotide sequence that links two moieties in a polypeptide, protein, or nucleic acid of the application. Exemplarily, the number of amino acids at the amino terminus of the linking region between the D-glucose sensitive polypeptide and the optically active polypeptide in the application is selected to be 0-3, and the number of amino acids at the carboxy terminus is selected to be 0-2; when the recombinant optical probe is linked to a functional protein as a basic unit, it can be fused at the amino acid or carboxy terminus of the recombinant optical probe. The linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.
[0083] The inventors found that D-glucose binding protein variants having mutations at a position selected from W8, W9, E13, A42, H66, D278, K312, V347, H348 of SEQ ID NO: 1 exhibit binding activity different from D-glucose. The amino acid mutations include modification, substitution, or deletion of an amino acid.
[0084] The present application provides D-glucose binding protein variants having these mutations and optical probes comprising such D-glucose binding protein variants as D-glucose sensitive polypeptides. Thus, in one or more embodiments, the D-glucose sensitive polypeptide in the optical probe is a D-glucose binding protein variant as described in any of the embodiments herein and the fluorescent protein in the optical probe is as set forth in SEQ ID NOs: 2-9 or a functional variant thereof. Preferably, the fluorescent protein is as set forth in SEQ ID NOs: 2, 6, 7, 9 or a functional variant thereof.
[0085] In some embodiments, the D-glucose-sensitive polypeptide in the optical probe is as set forth in SEQ ID NO: 1 and the optically active polypeptide is as set forth in SEQ ID NO: 2, and the optically active polypeptide is at 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271, 271 / 272, 271 / 273, 271 / 274, 271 / 275, 271 / 276, 271 / 277, 271 / 278, 271 / 279, 271 / 280, 271 / 281, 271 / 282, 271 / 283, 271 / 284, 271 / 285, 271 / 286, 271 / 287, 271 / 288, 272 / 267, 272 / 268, 272 / 269, 272 / 270, 272 / 271, 272 / 272, 272 / 273, 272 / 274, 272 / 275, 272 / 276, 272 / 277, 272 / 278, 272 / 279, 272 / 280, 272 / 281, 272 / 282, 272 / 283, 272 / 284, 272 / 285, 272 / 286, 272 / 287, 272 / 288, 273 / 267, 273 / 268, 273 / 269, 273 / 270, 273 / 271, 273 / 272, 273 / 273, 273 / 274, 273 / 275, 273 / 276, 273 / 277, 273 / 278, 273 / 279, 273 / 280, 273 / 281, 273 / 282, 273 / 283, 273 / 284, 273 / 285, 273 / 286, 273 / 287, 273 / 288, 274 / 267, 274 / 268, 274 / 269, 274 / 270, 274 / 271, 274 / 272, 274 / 273, 274 / 274, 274 / 275, 274 / 276, 274 / 277, 274 / 278, 274 / 279, 274 / 280, 274 / 281, 274 / 282, 274 / 283, 274 / 284, 274 / 285, 274 / 286, 274 / 287, 274 / 288, 275 / 267, 275 / 268, 275 / 269, 275 / 270, 275 / 271, 275 / 272, 275 / 273, 275 / 274, 275 / 275, 275 / 276, 275 / 277, 275 / 278, 275 / 279, 275 / 280, 275 / 281, 275 / 282, 275 / 283, 275 / 284, 275 / 285, 275 / 286, 275 / 287, 275 / 288, 276 / 267, 276 / 268, 276 / 269, 276 / 270, 276 / 271, 276 / 272, 276 / 273, 276 / 274, 276 / 275, 276 / 276, 276 / 277, 276 / 278, 276 / 279, 276 / 280, 276 / 281, 276 / 282, 276 / 283, 276 / 284, 276 / 285, 276 / 286, 276 / 287, 276 / 288, 277 / 267, 277 / 268, 277 / 269, 277 / 270, 277 / 271, 277 / 272, 277 / 273, 277 / 274, 277 / 275, 277 / 276, 277 / 277, 277 / 278, 277 / 279, 277 / 280, 277 / 281, 277 / 282, 277 / 283, 277 / 284, 277 / 285, 277 / 286, 277 / 287, 277 / 288, 278 / 267, 278 / 268, 278 / 269, 278 / 270, 278 / 271, 278 / 272, 278 / 273, 278 / 274, 278 / 275, 278 / 276, 278 / 277, 278 / 278, 278 / 279, 278 / 280, 278 / 281, 278 / 282, 278 / 283, 278 / 284, 278 / 285, 278 / 286, 278 / 287, 278 / 288, 279 / 267, 279 / 268, 279 / 269, 279 / 270, 279 / 271, 279 / 272, 279 / 273, 279 / 274, 279 / 275, 279 / 276, 279 / 277, 279 / 278, 279 / 279, 279 / 280, 279 / 281, 279 / 282, 279 / 283, 279 / 284, 279 / 285, 279 / 286, 279 / 287, 279 / 288, 280 / 267, 280 / 268, 280 / 269, 280 / 270, 280 / 271, 280 / 272, 280 / 273, 280 / 274, 280 / 275, 280 / 276, 280 / 277, 280 / 278, 280 / 279, 280 / 280, 280 / 281, 280 / 282, 280 / 283, 280 / 284, 280 / 285, 280 / 286, 280 / 287, 280 / 288, 281 / 267, 281 / 268, 281 / 269, 281 / 270, 281 / 271, 281 / 272, 281 / 273, 281 / 274, 281 / 275, 281 / 276, 281 / 277, 281 / 278, 281 / 279, 281 / 280, 281 / 281, 281 / 282, 281 / 283, 281 / 284, 281 / 285, 281 / 286, 281 / 287, 281 / 288, 282 / 267, 282 / 268, 282 / 269, 282 / 270, 282 / 271, 282 / 272, 282 / 273, 282 / 274, 282 / 275, 282 / 276, 282 / 277, 282 / 278, 282 / 279, 282 / 280, 282 / 281, 282 / 282, 282 / 283, 282 / 284, 282 / 285, 282 / 286, 282 / 287, 282 / 288, 283 / 267, 283 / 268, 283 / 269, 283 / 270, 283 / 271, 283 / 272, 283 / 273, 283 / 274, 283 / 275, 283 / 276, 283 / 277, 283 / 278, 283 / 279, 283 / 280, 283 / 281, 283 / 282, 283 / 283, 283 / 284, 283 / 285, 283 / 286, 283 / 287, 283 / 288, 284 / 267, 284 / 268,271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353 sites, the mutations of the optical probes are shown in any of the rows of Table 5.
[0086] In one or more embodiments, the optical probe, wherein the D-glucose sensitive polypeptide is as set forth in SEQ ID NO: 1, the optically active polypeptide is as set forth in SEQ ID NO: 2, 6, 7, 9, the optically active polypeptide is located at the 348 / 352 site of the D-glucose sensitive polypeptide, and the optical probe has the following mutations: (1) V347W, H348T of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (2) V347W, H348G of the D-glucose sensitive polypeptide, and 1G of the optically active polypeptide, (3) V347W, H348S of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (4) V347W, H348S of the D-glucose sensitive polypeptide, and 1G of the optically active polypeptide, (5) V347W, H348T of the D-glucose sensitive polypeptide, and 1G of the optically active polypeptide, (6) V347W, H348T, H66R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (7) V347W, H348T, H66E of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (8) V347W, H348T, H66A of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (9) V347W, H348T, H66F of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (10) V347W, H348T, H66M of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (11) V347W, H348T, H66C of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (12) V347W, H348T, H66P of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (13) V347W, H348T, H66Q of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (14) V347W, H348T, H66T of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (15) V347W, H348T, W9R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (16) V347W, H348T, W9H of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (17) V347W, H348T, W9E of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (18) V347W, H348T, W9A of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (19) V347W, H348T, W9V of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (20) V347W, H348T, W9L of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (21) V347W, H348T, W9F of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (22) V347W, H348T, W9I of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide.(23) V347W, H348T, W9M of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (24) V347W, H348T, W9C of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (25) V347W, H348T, W9P of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (26) V347W, H348T, W9N of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (27) V347W, H348T, W9G of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (28) V347W, H348T, W9K of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (29) V347W, H348T, W9Y of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (30) V347W, H348T, W9S of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (31) V347W, H348T, W9T of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (32) V347W, H348T, W9D of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (33) V347W, H348T, W8R of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (34) V347W, H348T, W8H of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (35) V347W, H348T, W8E of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (36) V347W, H348T, W8A of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (37) V347W, H348T, W8V of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (38) V347W, H348T, W8L of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (39) V347W, H348T, W8F of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (40) V347W, H348T, W8I of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (41) V347W, H348T, W8M of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (42) V347W, H348T, W8C of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (43) V347W, H348T, W8N of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (44) V347W, H348T, W8G of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (45) V347W, H348T, W8K of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide, (46) V347W, H348T, W8T of the D-glucose sensitive polypeptide and 1A of the optically active polypeptide,(47) V347W, H348T, W8D of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (48) V347W, H348T, A42R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (49) V347W, H348T, A42E of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (50) V347W, H348T, A42W of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (51) V347W, H348T, A42F of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (52) V347W, H348T, A42M of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (53) V347W, H348T, A42C of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (54) V347W, H348T, A42P of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (55) V347W, H348T, A42N of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (56) V347W, H348T, A42G of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (57) V347W, H348T, A42K of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (58) V347W, H348T, A42Y of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (59) V347W, H348T, A42T of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (60) V347W, H348T, K312R of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (61) V347W, H348T, K312A of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (62) V347W, H348T, K312Q of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (63) V347W, H348T, K312G of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (64) V347W, H348T, K312H of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide, (65) V347W, H348T, K312S of the D-glucose sensitive polypeptide, and 1A of the optically active polypeptide.
[0087] The terms "identity" or "percent identity," in the context of two or more polypeptide or nucleic acid molecule sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm known in the art, such as by manual alignment and visual inspection. Preferred algorithms are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.
[0088] It is well known to those skilled in the art that in the course of gene cloning manipulations, it is often necessary to engineer appropriate restriction sites, which will necessarily introduce one or more extraneous residues at the terminus of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. Also, for example, in order to construct fusion proteins, to facilitate expression of recombinant proteins, to obtain a recombinant protein that is secreted automatically outside the host cell, or to facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable region within the recombinant protein, such as, but not limited to, a suitable linker peptide, a signal peptide, a leader peptide, a terminal extension, glutathione S-transferase (GST), maltose E-binding protein, protein A, a tag such as 6His or Flag, or a proteolytic enzyme site for factor Xa or thrombin or enterokinase.
[0089] The terms "functional fragment," "derivative," and "analogue" as used herein refer to a protein that substantially maintains the same biological function or activity as the original polypeptide or protein (e.g., D-glucose binding protein or fluorescent protein). Functional variants, derivatives, or analogues of the polypeptides or proteins (e.g., D-glucose binding protein or fluorescent protein) of the present application can be (i) proteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) proteins having a substituent group at one or more amino acid residues, or (iii) proteins formed by fusing the mature protein to another compound (such as a compound that extends the half-life of the protein, e.g., polyethylene glycol), or (iv) proteins formed by fusing additional amino acid sequences to the protein sequence (such as a secretion sequence or a sequence or protein for purifying the protein or a proprotein sequence, or a fusion protein with an antigen IgG fragment). These functional variants, derivatives, and analogues are within the scope of those of ordinary skill in the art in light of the teachings herein. The analogues also include those having residues other than naturally occurring L-amino acids (e.g., D-amino acids), as well as non-naturally occurring or synthetic amino acids (e.g., beta, gamma, etc.). It is understood that the D-glucose sensitive polypeptides of the present application are not limited to the representative proteins, variants, derivatives, and analogues listed above. Modified (typically not changing the primary structure) forms include chemically derivatized forms of the protein in vivo or in vitro, such as acetylated or carboxylated. Modifications also include glycosylated, such as those produced by glycosylation modifications during or further to the synthesis and processing of the protein. Such modifications can be accomplished by exposing the protein to enzymes that glycosylate or deglycosylate (e.g., mammalian glycosylation enzymes). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their resistance to proteolysis or to optimize solubility are also included.
[0090] The fusion polypeptides of the present application comprise an optical probe as described herein and another polypeptide. In some embodiments, the optical probe as described herein further comprises another polypeptide fused thereto. The other polypeptide as described herein does not affect the properties of the optical probe. The other polypeptide can be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide comprises a polypeptide that localizes the optical probe to a different organelle or subcellular compartment, a tag for purification, or a tag for immunoblotting. There can be a linker between the optical probe and the other polypeptide in the fusion polypeptides as described herein.
[0091] Subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi, lysosome, peroxisome, and the like. In some embodiments, tags for purification or tags for immunoblotting include 6 histidines (6*His), glutathione S transferase (GST), Flag.
[0092] The present application includes nucleic acid molecules encoding the D-glucose sensitive polypeptides or optical probes described herein. The term "nucleic acid" or "nucleotide" or "polynucleotide" or "nucleic acid sequence" as used herein can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The term "variant" as used herein with respect to nucleic acid can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a nucleic acid which can result from natural or induced DNA changes or mutations. Allelic variants can have a change or changes in a single nucleotide, but can not substantially alter the functional properties of the encoded protein. A nucleic acid of the present application can comprise a nucleotide sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the sequence of the nucleic acid. The present application also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, and most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR).
[0093] The full-length sequence of the optical probes or fusion proteins of the present application, or fragments thereof, can be obtained by PCR amplification, artificial synthesis, or recombination. The steps and reagents used in conventional PCR, synthesis, and recombination are known in the art. In addition, mutations can be introduced into the sequence of the proteins of the present application by methods such as mutagenic PCR or chemical synthesis.
[0094] The present application also relates to nucleic acid constructs comprising the polynucleotides described herein, and one or more control sequences operably linked to the sequences. The polynucleotides described herein can be manipulated in a variety of ways to produce the polypeptides or proteins of the present application. Manipulation of the nucleic acid constructs prior to their insertion into a vector can be desirable for a variety of reasons, for example, to direct the expression of the polypeptides or proteins in a host. Techniques for modifying polynucleotides using recombinant DNA methods are known in the art.
[0095] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present application can be cloned into a number of types of vectors, e.g., plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
[0096] A typical expression vector contains expression control sequences operably linked to a nucleic acid sequence described herein or its complement that can be used to modulate expression of the desired nucleic acid sequence. The term "expression control sequence" as used herein refers to nucleic acid sequences that direct transcription and / or translation of a gene, and can include origins of replication, promoters, enhancers, terminators, ribosome binding sites, and the like, and will depend on the host cell used. In recombinant expression vectors, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence in a manner that allows for expression of the nucleotide sequence. One of ordinary skill in the art will readily recognize that a number of suitable expression control sequences can be used for constructing an expression vector for the fusion protein coding sequence of the present application and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination techniques. The DNA sequences described herein can be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoters of E. coli, the PL promoter of phage lambda, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the LTR from retrovirus, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for initiation of translation and a transcription terminator. In one embodiment, the expression vector can employ commercially available pCDF vector without any special requirements. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are double digested with BamHI and EcoRI, respectively, and the digested products are ligated to obtain a recombinant expression vector. The present application does not have any special limitation on the specific steps and parameters for the digestion and ligation, and the steps and parameters of the art can be used.
[0097] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including the fusion protein. Such transformation can be performed by using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell of the present application refers to a cell that can receive and accommodate a recombinant DNA molecule, and is a place for amplification of a recombinant gene. The ideal recipient cell should satisfy two conditions of easy access and proliferation. The "host cell" of the present application can include prokaryotic cells and eukaryotic cells, and specifically includes bacterial cells, yeast cells, insect cells and mammalian cells. The host cell is preferably various cells that are beneficial for expression of a gene product or fermentation production, and such cells are well known and commonly used in the art. Specifically, the cells can be bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc. An exemplary host cell used in the embodiments of the present application is Escherichia coli BL21-DE3 strain. Those skilled in the art are aware of how to select appropriate vectors, promoters, enhancers and host cells.
[0098] The method for transferring the expression vector into a host cell is a conventional method in the art, including calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer or electroporation. When the host is a prokaryote such as Escherichia coli, the method is preferably CaCl2or MgCl2treatment, and the steps used are well known in the art. When the host cell is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
[0099] After the expression vector is transferred into the host cell, the host cell into which the expression vector is transferred is subjected to amplification expression culture to isolate the D-glucose optical probe. The host cell amplification expression culture can be performed by using conventional methods. According to the type of host cell used, the culture medium used in the culture can be various conventional culture media. The culture is performed under conditions suitable for growth of the host cell.
[0100] In the present application, the optical probe is expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated or purified by various separation methods using its physical, chemical and other properties. The present application does not have special limitations on the method for isolating the D-glucose fluorescent protein, and the conventional separation method of fusion protein in the art can be used. These methods are well known to those skilled in the art, including but not limited to: conventional renaturation treatment, salting-out method, centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques, and combinations of these methods. In one embodiment, the optical probe is isolated by affinity chromatography using His tag.
[0101] The present application also provides the use of the D-glucose optical probe in real-time localization, quantitative detection of D-glucose and high-throughput compound screening. In one aspect, the D-glucose optical probe is preferably linked to a signal peptide at different parts of the cell, and is introduced into the cell. By detecting the strength of the fluorescent signal in the cell, real-time localization of D-glucose is performed; by combining the standard addition curve of D-glucose with the change of the fluorescent signal, quantitative detection of the corresponding D-glucose is performed. The change of the fluorescent signal is shown by, for example, the ratio of the normalized fluorescent signal, and in the embodiment involving cpYFP, the ratio is the ratio of the 485 nm fluorescent signal to the 420 nm fluorescent signal of the sample to the corresponding ratio of the control. The D-glucose standard addition curve of the present application is drawn according to the fluorescent signal of the D-glucose optical probe under different concentrations of D-glucose. The D-glucose optical probe of the present application is directly introduced into the cell, and in the process of real-time localization and quantitative detection of D-glucose, there is no need for time-consuming sample processing, which is more accurate. In high-throughput compound screening, different compounds are added to the cell culture medium, and the change of the D-glucose content is determined, so as to screen out compounds that have an effect on the change of the D-glucose content. The use of the D-glucose optical probe in real-time localization, quantitative detection and high-throughput compound screening of D-glucose in the present application is for non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.
[0102] The present application also provides a detection kit comprising the optical probe, nucleic acid molecule, nucleic acid construct and / or cell described herein. The kit also comprises other reagents required for detecting D-glucose. The other reagents are well known in the art, such as buffer, cell culture medium, D-glucose standard. Exemplary buffer is, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.
[0103] In this text, concentrations, amounts, percentages and other numerical values can be expressed in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
[0104] Examples
[0105] The D-glucose optical probes provided by the present application are described in detail below in connection with examples, but they should not be construed as limiting the scope of the present application.
[0106] I. Experimental materials and reagents
[0107] In the examples, the conventional genetic engineering molecular biology cloning methods and cell culture and imaging methods, etc. are mainly used, which are well known to those skilled in the art, for example: Jane Ross Kems, et al. "Molecular Biology Laboratory Reference Manual", J. Sambrook, D. W. Russell, Huang Peitang, et al. "Molecular Cloning Laboratory Guide" (3rd edition, August 2002, Science Press, Beijing); Ferechenei, et al. "Animal Cell Culture: Basic Technical Guide" (5th edition), Zhang Jingbo, Xu Cunlong, et al. "Compact Cell Biology Experiment Guide", Zhang Jingbo, et al.
[0108] The pCDF-cpYFP, pCDF-ttGBP plasmids used in the examples were constructed by the Protein Laboratory of East China University of Technology, and the pCDF plasmid vector was purchased from Invitrogen Company. All primers used for PCR were synthesized, purified and identified correctly by mass spectrometry by Shanghai Jeery Bioengineering Technology Co., Ltd. and Huada Gene. The sequence of the expression plasmid constructed in the examples was determined by Huada Gene Company and Jiali Sequencing Company. The Taq DNA polymerase used in each example was purchased from Dongsheng Bio, the pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa Company. The three kinds of polymerases purchased at the same time are attached with the corresponding polymerase buffer and dNTPs. The restriction endonucleases BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, T4 phosphorylase (T4 PNK) were purchased from Fermentas Company, and the corresponding buffer was attached when purchased. Transfection reagent Lip2000 Kit was purchased from Invitrogen Company. D-glucose and other compounds were purchased from Sigma Company. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich Company. HEPES salt, ampicillin (Amp) and puromycin were purchased from Ameresco Company. 96-well detection blackboard and 384-well fluorescence detection blackboard were purchased from Grenier Company.
[0109] The DNA purification kit used in the examples was purchased from BBI Company (Canada), and the general plasmid small extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The cloning strain Mach1 was purchased from Invitrogen Company. The nickel column affinity chromatography column and desalting column filler were from GE healthcare company.
[0110] The main instruments used in the examples include: Biotek Synergy 2 multifunctional enzyme marker (Bio-Tek Company, USA), X-15R high-speed refrigerated centrifuge (Beckman Company, USA), Microfuge22R table high-speed refrigerated centrifuge (Beckman Company, USA), PCR amplifier (Biometra Company, Germany), ultrasonic crusher (Ningbo Xinzhi Company), nucleic acid electrophoresis instrument (Shengneng Bochai Company), fluorescence spectrophotometer (Varian Company, USA), CO2 constant temperature cell incubator (SANYO), inverted fluorescence microscope (Nikon Company, Japan).
[0111] II. Molecular biology methods and cell experimental methods
[0112] II.1 Polymerase chain reaction (PCR):
[0113] 1. PCR for fragment amplification
[0114] This method is mainly used for gene fragment amplification and positive clone identification by colony PCR. The reaction system of PCR amplification is as follows: template sequence 0.5-1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 10×pfu buffer 5 μL, pfu DNA polymerase 0.5 μL, dNTP (10 mM) 1 μL, sterilized ultrapure water (ddH2O) 41.5-42 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95°C for 2-10 minutes, 30 cycles of 94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, 72°C for a certain time (600 bp / min), and extension at 72°C for 10 minutes.
[0115] 2. PCR for long fragment (>2500 bp) amplification
[0116] The long fragment amplification used in the present application is mainly reverse PCR amplification vector, which is used in the following examples to obtain a site-directed mutation technique. Reverse PCR primers are designed at the mutation site, wherein the 5' end of one primer contains a mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The reaction system of long fragment amplification PCR is as follows: template sequence (10 pg-1 ng) 1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 5×PrimerSTAR buffer 10 μL, PrimerSTAR DNA polymerase 0.5 μL, dNTP (2.5 mM) 4 μL, sterilized ultrapure water (ddH2O) 33.5 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95°C for 5 minutes, 30 cycles of 98°C for 10 seconds, 50-68°C for 5-15 seconds, and 72°C for a certain time (1000 bp / min), and extension at 72°C for 10 minutes; or denaturation at 95°C for 5 minutes, 30 cycles of 98°C for 10 seconds and 68°C for a certain time (1000 bp / min), and extension at 72°C for 10 minutes.
[0117] II.2 Endonuclease enzyme digestion reaction
[0118] The system for double enzyme digestion of plasmid vector is as follows: plasmid vector 20 μL (about 1.5 μg), 10×buffer 5 μL, restriction endonuclease 1 11-2 μL, restriction endonuclease 2 11-2 μL, and sterilized ultrapure water to a total volume of 50 μL. The reaction condition is 37°C for 1-7 hours.
[0119] II.3 DNA fragment 5' end phosphorylation reaction
[0120] The plasmid or genome extracted from microorganism contains phosphate group at the end, but the PCR product does not, so the phosphate group addition reaction is needed for the 5' end base of the PCR product, and only the DNA molecule with phosphate group at the end can have the ligation reaction. The phosphorylation reaction system is as follows: PCR product segment DNA sequence 5-8 μL, 10 x T4 ligase buffer 1 μL, T4 polynucleotide kinase (T4 PNK) 1 μL, sterilized ultrapure water 0-3 μL, total volume 10 μL. The reaction condition is 37°C for 30 minutes-2 hours, and then inactivated at 72°C for 20 minutes.
[0121] II.4 Purpose fragment and vector ligation reaction
[0122] The ligation methods between different fragments and vectors are different, and three ligation methods are used in the present application
[0123] 1. Blunt end ligation of blunt end short fragment and linearized vector
[0124] The principle of this method is that the blunt end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4 PNK, and then connected with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain the recombinant plasmid. The homologous recombination ligation system is as follows: T4 PNK treated DNA fragment 4 μL, linearized vector fragment 4 μL, PEG4000 1 μL, 10 x T4 ligase buffer 1 μL, T4 DNA ligase 1 μL, total 10 μL. The reaction condition is 22°C for 30 minutes.
[0125] 2. Ligation of DNA fragment containing sticky end and vector fragment containing sticky end
[0126] The DNA fragment cut by restriction endonuclease usually produces protruding sticky end, so it can be connected with the vector fragment containing sequence complementary sticky end to form the recombinant plasmid. The ligation reaction system is as follows: PCR product fragment DNA after enzyme cutting 1-7 μL, plasmid after enzyme cutting 0.5-7 μL, 10 x T4 ligase buffer 1 μL, T4 DNA ligase 1 μL, sterilized ultrapure water to a total volume of 10 μL. The reaction condition is 16°C for 4-8 hours.
[0127] 3. Ligation reaction of 5' end phosphorylated DNA fragment product after reverse PCR introduction of site-directed mutation
[0128] The 5' end phosphorylated DNA fragment is connected with the 3' end and 5' end of the linearized vector by self-circularization ligation reaction to obtain the recombinant plasmid. The self-circularization ligation reaction system is as follows: phosphorylation reaction system 10 μL, T4 ligase (5 U / μL) 0.5 μL, total volume 10.5 μL. The reaction condition is 16°C for 4-16 hours.
[0129] II.5 Preparation and transformation of competent cells
[0130] Preparation of competent cells:
[0131] 1. Pick a single colony (e.g. Mach 1) and inoculate into 5 mL LB medium, 37°C, overnight.
[0132] 2. Take 0.5-1 mL of the overnight culture and inoculate into 50 mL LB medium, 37°C, 220 rpm for 3-5 hours until OD 600 0.5 is reached.
[0133] 3. Pre-chill the cells in ice bath for 2 hours.
[0134] 4. Centrifuge at 4°C, 4000 rpm for 10 minutes.
[0135] 5. Discard the supernatant, resuspend the cells with 5 mL pre-chilled resuspension buffer, and add more resuspension buffer to a final volume of 50 mL after the cells are evenly suspended.
[0136] 6. Incubate in ice bath for 45 minutes.
[0137] 7. Centrifuge at 4°C, 4000 rpm for 10 minutes, and resuspend the bacteria with 5 mL ice-pre-chilled storage buffer.
[0138] 8. Put 100 μL of the bacterial solution into each EP tube, and store at -80°C or in liquid nitrogen.
[0139] Resuspension buffer: CaCl2(100 mM), MgCl2(70 mM), NaAc(40 mM)
[0140] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10x CaCl2(1 M), 1 mL 10x MgCl2(700 mM), 1 mL 10x NaAc(400 mM), 4.6 mL ddH2O
[0141] Transformation of competent cells:
[0142] 1. Thaw 100 μL of competent cells on ice.
[0143] 2. Add the appropriate volume of ligation product, mix gently, and incubate on ice for 30 minutes. Usually, the volume of ligation product added is less than 1 / 10 of the volume of competent cells.
[0144] 3. Heat shock the bacterial solution in a 42°C water bath for 90 seconds, and quickly transfer to an ice bath for 5 minutes.
[0145] 4. Add 500 μL LB, incubate at 37°C in a shaker at 200 rpm for 1 hour.
[0146] 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, take 200 μL supernatant, blow the bacterial body evenly, and evenly spread on the surface of agar plate containing appropriate antibiotic, and plate in 37°C constant temperature incubator overnight.
[0147] II.6 Expression, purification and fluorescence detection of protein
[0148] 1. Transform the expression vector (for example, the D-glucose optical probe expression vector based on pCDF) into BL21 (DE3) cells, invert and culture overnight, pick the clones from the plate into 250 ml conical flask, and place in 37°C shaker, 220 rpm, to culture until OD = 0.4-0.8, add 1 / 1000 (v / v) IPTG (1M), and induce expression at 18°C for 24-36 hours.
[0149] 2. After the induction of expression is completed, centrifuge the bacteria at 4000 rpm for 30 minutes, add 50 mM phosphate buffer to resuspend the bacterial body precipitate, and ultrasonically break until the bacterial body is clear. Centrifuge at 9600 rpm at 4°C for 20 minutes.
[0150] 3. Centrifuge the supernatant to purify the protein through a self-assembled nickel column affinity chromatography column, and then obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4) through a self-assembled desalting column after nickel column affinity chromatography.
[0151] 4. After the purified protein is identified by SDS-PAGE, dilute the probe to a final concentration of 0.2-5 μM of protein solution using the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4). Prepare a D-glucose stock solution with a final concentration of 50 mM using the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).
[0152] 5. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add D-glucose titration, and measure the fluorescence intensity of the protein at 420 nm light excitation and 528 nm emission and at 485 nm light excitation and 528 nm emission. The fluorescence excitation and emission of the sample are measured by using a multifunctional fluorescence enzyme marker.
[0153] 6. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add D-glucose, and measure the absorption spectrum and fluorescence spectrum of the protein. The absorption spectrum and fluorescence spectrum of the sample are measured by using a spectrophotometer and a fluorescence spectrophotometer.
[0154] II.7 Transfection of mammalian cells and fluorescence detection
[0155] 1. The pCDNA3.1+ based D-glucose optical probe plasmid was transfected into HEK293 by transfection reagent Lipofectamine 2000 (Invitrogen) and cultured in a cell incubator at 37°C, 5% CO2. After 24-36 hours of sufficient expression of the exogenous gene, fluorescence detection was performed.
[0156] 2. After the induction of expression was completed, the adherent HEK293 cells were washed with PBS three times and placed in HBSS solution for fluorescence microscope and microplate reader detection, respectively.
[0157] Example 1: D-glucose binding protein plasmid
[0158] The TtGBP gene (SEQ ID NO: 1) in the T-thermophilic bacteria gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with HindIII and XhoI. The pCDF vector was also subjected to the corresponding double digestion. After ligation with T4 DNA ligase, the product was used to transform DH5a, and the transformed DH5a was plated on LB plates (streptomycin 100 ug / mL) and incubated at 37°C overnight. After plasmid extraction of the growing DH5a transformant, PCR identification was performed. After the positive plasmid was sequenced correctly, subsequent plasmid construction was performed.
[0159] Example 2: Expression and detection of cpYFP optical probes with different insertion sites
[0160] In this embodiment, the following sites were selected for insertion of cpYFP based on pCDF-TtGBP: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 267 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275, 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284, 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271, 271 / 272, 271 / 273,271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345、347 / 346、347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353. By way of example only, the amino acid sequence of 348 / 352-TtGBP-cpYFP is shown in SEQ ID NO: 10.
[0161] The DNA fragment of cpYFP was generated by PCR, and the cpYFP end homologous sequences were introduced by the 5' end of the primer. The PCR amplification generated pCDF-D-glucose binding protein linearization vector, which had the same sequence (15bp-20bp) at the 5' and 3' ends as the corresponding ends of cpYFP. The linearized pCDF-TtGBP and cpYFP fragments were subjected to homologous recombination under the action of Hieff Clone Enzyme. The product was transformed into DH5a, and the transformed DH5a was coated on LB plates (streptomycin 100 ug / mL) and incubated at 37°C overnight. The positive clones identified by PCR were sequenced after plasmid extraction. Sequencing was completed by Ji Li Sequencing Company.
[0162] After sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. The size of the protein was about 70Kda by SDS-PAGE electrophoresis. The size was consistent with the size of the TtGBP-cpYFP fusion protein expressed by pCDF-TtGBP-cpYFP, which contained a His-tag purification tag. The results are shown in Figure 1
[0163] The D-glucose response screening was performed using the broken supernatant of E. coli expressing the TtGBP-cpYFP fusion protein. The detection signal of the fusion fluorescent protein containing 100 mM D-glucose was divided by the detection signal of the fusion fluorescent protein without D-glucose. As shown in Table 1, the detection results showed that the broken supernatant expressing the TtGBP-cpYFP fusion protein had more than 1.2-fold optical probes responding to D-glucose, including 273 / 279, 273 / 284, 350 / 342, 348 / 343, 349 / 343, 348 / 344, 349 / 344, 349 / 345, 348 / 349, 349 / 349, 348 / 350, 349 / 350, 346 / 351, 347 / 351, 347 / 352, 348 / 352, and 350 / 353; and 7 optical probes responding to D-glucose more than 1.25-fold, including 273 / 279, 348 / 343, 348 / 344, 349 / 345, 349 / 349, 349 / 350, and 347 / 352.
[0164] Table 1
[0165]
[0166]
[0167]
[0168] Example 3: Expression and detection of cpGFP optical probes with different insertion sites
[0169] According to the method in Example 2, cpYFP was replaced by cpGFP to construct a D-glucose green fluorescent protein fluorescent probe. As shown in Table 2, the detection results showed that the broken supernatant expressing the TtGBP-cpGFP fusion protein had more than 1.3-fold optical probes responding to D-glucose, including 348 / 344, 349 / 345, 349 / 349, 349 / 350, and 347 / 352 sites or corresponding amino acid sites of the family protein inserted at the site.
[0170] Table 2
[0171]
[0172] Example 4: Expression and detection of cpBFP optical probes with different insertion sites
[0173] The D-glucose blue fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpBFP according to the method in Example 2. As shown in Table 3, the detection results showed that the optical probes with insertion at 348 / 343, 348 / 344, 349 / 345 and 349 / 349 sites or the corresponding amino acid sites of the family protein of the optical probes had a response to D-glucose more than 1.3 times for the broken supernatant expressing TtGBP-cpBFP fusion protein.
[0174] Table 3
[0175]
[0176] Example 5: Expression and detection of cpmApple optical probes with different insertion sites
[0177] The D-glucose red fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpmApple according to the method in Example 2. As shown in Table 4, the detection results showed that the optical probes with insertion at 349 / 345 and 349 / 349 sites or the corresponding amino acid sites of the family protein of the optical probes had a response to D-glucose more than 1.3 times for the broken supernatant expressing TtGBP-cpmApple fusion protein.
[0178] Table 4
[0179]
[0180] Example 6: Performance of optical probes
[0181] The 17 optical probes with insertions at sites 273 / 279, 273 / 284, 350 / 342, 348 / 343, 349 / 343, 348 / 344, 349 / 344, 349 / 345, 348 / 349, 349 / 349, 348 / 350, 349 / 350, 346 / 351, 347 / 351, 347 / 352, 348 / 352, 350 / 353, which responded to D-glucose more than 1.2 times in Example 2, were used to detect glucose at a concentration gradient (0-100 mM) and the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to that at 485 nm excitation and 528 nm emission. The Kd (binding constant) of 14 D-glucose optical probes with insertion sites 273 / 279, 273 / 284, 350 / 342, 348 / 343, 349 / 343, 348 / 344, 348 / 349, 349 / 349, 348 / 350, 349 / 350, 346 / 351, 347 / 351, 347 / 352, 350 / 353 was too large to fit and was not suitable for detection. In addition, the Kd (binding constant) of the three D-glucose optical probes with insertion sites 349 / 344, 349 / 345, 348 / 352 was 1.2 mM, 0.2 mM and 0.008 mM, respectively.
[0182] Example 7: Expression and detection of mutant cpYFP optical probes
[0183] The optical probe mutants were constructed based on TtGBP-348 / 352-cpYFP. The plasmid pCDF-TtGBP-348 / 352-cpYFP was linearized by PCR, and the primer contained the base sequence of the desired mutation site. The obtained PCR product was subjected to homologous recombination to obtain mutant plasmids of 12 sites V347, H348, H66, D278, W9, E13, W8, A42, K312 of D-glucose sensitive polypeptide and Y1 of optical active polypeptide, and sequencing was completed by Jilei Sequencing Company. As an exemplary display, the amino acid sequence of 348 / 352-TtGBP-V347W / H348S / H66C-cpYFP-Y1A is shown in SEQ ID NO: 11.
[0184] The successfully constructed mutant plasmid was transformed into BL21(DE3) to induce expression, and the broken supernatant of E. coli expressing the probe protein was used for response screening of D-glucose and other non-specific substrates, and the detection signal of the fusion fluorescent protein containing D-glucose or other non-specific substrates was divided by the detection signal of the fusion fluorescent protein without D-glucose. The results are shown in Table 5, and the detection results show that the optical probes responding to D-glucose more than 3 times and having better specificity are as follows.
[0185] Table 5
[0186] Insertion site ttGBP mutation cpYFP mutation R 420 / 485 Kd / μM Insertion site ttGBP mutation cpYFP mutation R 420 / 485 Kd / μM 348 / 352 V347W / H348T Y1A 3.88 7.98 348 / 352 V347W / H348T / W8H Y1A 4.2 3410 348 / 352 V347W / H348G Y1G 5.58 75.85 348 / 352 V347W / H348T / W8E Y1A 10.5 747.9 348 / 352 V347W / H348S Y1A 5.78 67.74 348 / 352 V347W / H348T / W8A Y1A 4.7 950.8 348 / 352 V347W / H348S Y1G 6.65 53.37 348 / 352 V347W / H348T / W8V Y1A 10.7 1048 348 / 352 V347W / H348T Y1G 3.05 45.44 348 / 352 V347W / H348T / W8L Y1A 7.8 782.1 348 / 352 V347W / H348T / H66R Y1A 2.92 433.8 348 / 352 V347W / H348T / W8F Y1A 17.2 1825 348 / 352 V347W / H348T / H66E Y1A 4.78 27259 348 / 352 V347W / H348T / W8I Y1A 12.5 1017 348 / 352 V347W / H348T / H66A Y1A 6.66 3399 348 / 352 V347W / H348T / W8M Y1A 6.7 343.5 348 / 352 V347W / H348T / H66F Y1A 3.4 6315 348 / 352 V347W / H348T / W8C Y1A 5.0 552.6 348 / 352 V347W / H348T / H66M Y1A 6.27 5101 348 / 352 V347W / H348T / W8N Y1A 9.6 423.7 348 / 352 V347W / H348T / H66C Y1A 3.87 4792 348 / 352 V347W / H348T / W8G Y1A 5.4 503 348 / 352 V347W / H348T / H66P Y1A 4.84 1126 348 / 352 V347W / H348T / W8K Y1A 9.6 3975 348 / 352 V347W / H348T / H66Q Y1A 4.77 1429 348 / 352 V347W / H348T / W8T Y1A 12.0 1699 348 / 352 V347W / H348T / H66T Y1A 6.89 4873 348 / 352 V347W / H348T / W8D Y1A 11.8 1850 348 / 352 V347W / H348T / W9R Y1A 5.6 15893 348 / 352 V347W / H348T / A42R Y1A 9.2 268.5 348 / 352 V347W / H348T / W9H Y1A 4.26 737.1 348 / 352 V347W / H348T / A42E Y1A 7.3 317.9 348 / 352 V347W / H348T / W9E Y1A 5.21 6454 348 / 352 V347W / H348T / A42W Y1A 5.8 152 348 / 352 V347W / H348T / W9A Y1A 4.92 160.7 348 / 352 V347W / H348T / A42F Y1A 3.6 14.58 348 / 352 V347W / H348T / W9V Y1A 4.9 1474 348 / 352 V347W / H348T / A42M Y1A 1.9 9.551 348 / 352 V347W / H348T / W9L Y1A 3.01 2.113 348 / 352 V347W / H348T / A42C Y1A 2.4 43.21 348 / 352 V347W / H348T / W9F Y1A 5.03 49.69 348 / 352 V347W / H348T / A42P Y1A 15.3 509.5 348 / 352 V347W / H348T / W9I Y1A 4.03 125 348 / 352 V347W / H348T / A42N Y1A 2.4 10.35 348 / 352 V347W / H348T / W9M Y1A 1.94 16.4 348 / 352 V347W / H348T / A42G Y1A 4.5 43.74 348 / 352 V347W / H348T / W9C Y1A 8.93 783.1 348 / 352 V347W / H348T / A42K Y1A 4.4 297.2 348 / 352 V347W / H348T / W9P Y1A 8.04 14133 348 / 352 V347W / H348T / A42Y Y1A 5.4 76.46 348 / 352 V347W / H348T / W9N Y1A 5.51 1405 348 / 352 V347W / H348T / A42T Y1A 2.7 124.1 348 / 352 V347W / H348T / K312R Y1A 5.05 1920 348 / 352 V347W / H348T / K312A Y1A 2.7 83.77 348 / 352 V347W / H348T / K3120 Y1A 5.54 14534 348 / 352 V347W / H348T / K312G Y1A 3.7 49136 348 / 352 V347W / H348T / K312S Y1A 1.47 173.3 348 / 352 V347W / H348T / K312H Y1A 4.7 955.5 348 / 352 V347W / H348T / K312R Y1A 4.43 776.5 348 / 352 V347W / H348T / K312A Y1A 2.8 61603 348 / 352 V347W / H348T / K3120 Y1A 4.42 2132 348 / 352 V347W / H348T / K312G Y1A 2.5 1840 348 / 352 V347W / H348T / K312S Y1A 4.34 4545 348 / 352 V347W / H348T / K312H Y1A 1.9 77018 348 / 352 V347W / H348T / K312R Y1A 10.3 1530 348 / 352
[0187] Example 8: Performance of optical probe mutants
[0188] The D-glucose optical probes in Table 5 described in Example 7 were respectively subjected to D-glucose detection at a concentration gradient (0-100 mM). After treating the probes for 10 minutes, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected. The probe titration results are shown in V347W / H348T / K312A , and the results show that different mutants have different affinities for D-glucose.
[0189] The D-glucose probes in Table 5 were subjected to specificity detection, and were respectively reacted with D-glucose structural analogs, glucose-1-phosphate, sorbitol, mannitol, sodium lactate, sucrose, pyruvic acid, ribose, fructose, oxalacetic acid, ATP, 6-phosphoglucose, citric acid, D-glucose, isocitric acid, sodium gluconate, NAD+, NADH, malic acid, NADPH, arabinose, PEP, lactose, maltose, 2-deoxyglucose, galactose, and the results showed good specificity, as shown in Y1A .
[0190] Example 9: Subcellular organellar localization of optical probes and performance of optical probes in subcellular organelles
[0191] In this example, different localization signal peptides were used to fuse with the optical probe 348 / 352-TtGBP-V347W / H348S / H66C-cpYFP-Y1A to localize the optical probe to different organelles.
[0192] After transfecting 293 cells with optical probe plasmids fused with different localization signal peptides for 36 hours, the cells were washed with PBS, placed in HBSS solution, and subjected to fluorescence detection under the FITC channel using an inverted fluorescence microscope. The results are shown in V347W / H348T / K3120The D-glucose optical probe can be localized to subcellular organelles including cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria and nuclear exclusion by fusing with different specific localization signal peptides. The fluorescence is shown in different subcellular structures, and the distribution and intensity of fluorescence are different.
[0193] After 36 hours of transfection of HEK293 cells with cytoplasm-expressed optical probe plasmid, the cells were washed with PBS and placed in HBSS solution, and the change of the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to that at 485 nm excitation and 528 nm emission was detected within 30 minutes. The results are shown in Y1A As shown, 5 mM D-glucose was added, and the detection was continued for 30 minutes. The 420 / 485 of the sample with added D-glucose gradually increased, and the highest value was 1.8 times the initial value, while the 420 / 485 of the control group without added Oxalate remained unchanged.
[0194] Example 10: High-throughput compound screening based on optical probe in living cells
[0195] In this example, we used HeLa cells expressing cytoplasm-expressed 348 / 352-TtGBP-V347W / H348S / H66C-cpYFP-Y1A for high-throughput compound screening.
[0196] The transfected 293 cells were washed with PBS and placed in HBSS solution (without D-glucose) for 1 hour, and then treated with 10 μM of compound for 1 hour. D-glucose was added to each sample. The changes of the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to that at 485 nm excitation and 528 nm emission were recorded using a microplate reader. The sample without any compound treatment was used as a control for normalization. The results are shown in V347W / H348T / K312G Among the 2000 compounds used, most of the compounds had little effect on D-glucose entering the cells. Five compounds could improve the uptake of D-glucose by cells, and another 9 compounds could significantly reduce the uptake of D-glucose by cells.
[0197] Example 11: Quantitative detection of D-glucose in blood using optical probe
[0198] In this example, we used purified 348 / 352-TtGBP-V347W / H348S-cpYFP-Y1A with a Kd of 48.33 μM to analyze D-glucose in the supernatant of mouse and human blood.
[0199] After 348 / 352-TtGBP-V347W / H348S-cpYFP-Y1A was mixed with diluted blood supernatant for 10 minutes, the fluorescence intensity at 420nm excitation and 528nm emission and the ratio of fluorescence intensity at 485nm excitation and 528nm emission were detected by using a microplate reader. The results are shown in Figure 6. Y1A V347W / H348T / K312S Y1A V347W / H348T / K312H Y1A V347W / H348T / K312R Y1A V347W / H348T / K312A Y1A V347W / H348T / K3120 Y1A V347W / H348T / K312G Y1A V347W / H348T / K312S Y1A V347W / H348T / K312H Y1A V347W / H348T / K312R Y1A V347W / H348T / K312A Y1A V347W / H348T / K3120 Y1A V347W / H348T / K312G Y1A V347W / H348T / K312S Y1A The D-glucose content in mouse blood is about 8mM, and the D-glucose content in human blood is about 4mM.
[0200] As can be seen from the above examples, the D-glucose optical probe provided by the present application has relatively small protein molecular weight and is easy to mature, has large fluorescence dynamic change, good specificity, and can be expressed in cells by gene manipulation, and can be used for real-time positioning and quantitative detection of D-glucose in and outside cells; and can be used for high-throughput compound screening.
[0201] Other embodiments
[0202] The present specification describes many embodiments. However, it should be understood that various modifications made by those skilled in the art based on the present specification without departing from the concept and scope of the present application should also be included in the scope of the appended claims.
[0203] Sequences herein
[0204] 1> TtGBP (1-394 full length)
[0205] MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNARAVLKTRMLGGDPPDTFQVHAGMELIGTWVVANRMEDLSALFRQEGWLQAFPKGLIDLISYKGGIWSVPVNIHRSNVMWYLPAKLKEWGVNPPRTWDEFLATCQTLKQKGLEAPLALGENWTQQHLWESVALAVLGPDDWNNLWNGKLKFTDPKAVRAWEVFGRVLDCANKDAAGLSWQQAVDRVVQGKAAFNVMGDWAAGYMTTTLKLKPGTDFAWAPSPGTQGVFMMLSDSFGLPKGAKNRQNAINWLRLVGSKEGQDTFNPLKGSIAARLDSDPSKYNAYGQSAMRDWRSNRIVGSLVHGAVAPESFMSQFGTVMEIFLQTRNPQAAANAAQAIADQVGLGRLGQ
[0206] 2> cpYFP
[0207] YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN
[0208] 3>cpmOrange
[0209] VSERMYPEDGVLKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHPTGGRDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA
[0210] 4>cpmKate
[0211] MGGRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGGTGGSMVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSDMALKLVGGGHLICNLKTTYRSKK
[0212] 5>mCherry
[0213] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK
[0214] 6>cpGFP
[0215] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN
[0216] 7>cpBFP
[0217] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN
[0218] 8>mKate
[0219] MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLN
[0220] 9 > cpmApple
[0221] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA
[0222] 10 > 348 / 352-TtGBP-cpYFP
[0223] MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNARAVLKTRMLGGDPPDTFQVHAGMELIGTWVVANRMEDLSALFRQEGWLQAFPKGLIDLISYKGGIWSVPVNIHRSNVMWYLPAKLKEWGVNPPRTWDEFLATCQTLKQKGLEAPLALGENWTQQHLWESVALAVLGPDDWNNLWNGKLKFTDPKAVRAWEVFGRVLDCANKDAAGLSWQQAVDRVVQGKAAFNVMGDWAAGYMTTTLKLKPGTDFAWAPSPGTQGVFMMLSDSFGLPKGAKNRQNAINWLRLVGSKEGQDTFNPLKGSIAARLDSDPSKYNAYGQSAMRDWRSNRIVGSLVHYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNAPESFMSQFGTVMEIFLQTRNPQAAANAAQAIADQVGLGRLGQ
[0224] 11 348 / 352-TtGBP-V347W / H348S / H66C-cpYFP-Y1A
[0225] MKLEIFSWWAGDEGPALEALIRLYKQKYPGVEVINATVTGGAGVNARAVLKTRMLGGDPPDTFQVCAGMELIGTWVVANRMEDLSALFRQEGWLQAFPKGLIDLISYKGGIWSVPVNIHRSNVMWYLPAKLKEWGVNPPRTWDEFLATCQTLKQKGLEAPLALGENWTQQHLWESVALAVLGPDDWNNLWNGKLKFTDPKAVRAWEVFGRVLDCANKDAAGLSWQQAVDRVVQGKAAFNVMGDWAAGYMTTTLKLKPGTDFAWAPSPGTQGVFMMLSDSFGLPKGAKNRQNAINWLRLVGSKEGQDTFNPLKGSIAARLDSDPSKYNAYGQSAMRDWRSNRIVGSLWSANSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNAPESFMSQFGTVMEIFLQTRNPQAAANAAQAIADQVGLGRLGQ
Claims
1. An optical probe comprising a D-glucose sensitive polypeptide and an optically active polypeptide, wherein, The D-glucose-sensitive polypeptide is a TtGBP protein or a variant thereof, and the optically active polypeptide is a fluorescent protein or a variant thereof. The optically active polypeptide is located at one or more sites selected from the following: 266 / 267, 266 / 268, 266 / 269, 266 / 270, 266 / 271, 266 / 272, 266 / 273, 266 / 274, 266 / 275, 266 / 276, 266 / 277, 266 / 278, 266 / 279, 266 / 280, 266 / 281, 266 / 282, 266 / 283, 266 / 284, 266 / 285, 266 / 286, 266 / 287, 266 / 288. 8, 267 / 267, 267 / 268, 267 / 269, 267 / 270, 267 / 271, 267 / 272, 267 / 273, 267 / 274, 267 / 275, 267 / 276, 267 / 277, 267 / 278, 267 / 279, 267 / 280, 267 / 281, 26 7 / 282, 267 / 283, 267 / 284, 267 / 285, 267 / 286, 267 / 287, 267 / 288, 268 / 267, 268 / 268, 268 / 269, 268 / 270, 268 / 271, 268 / 272, 268 / 273, 268 / 274, 268 / 275 268 / 276, 268 / 277, 268 / 278, 268 / 279, 268 / 280, 268 / 281, 268 / 282, 268 / 283, 268 / 284, 268 / 285, 268 / 286, 268 / 287, 268 / 288, 269 / 267, 269 / 268, 269 / 269, 269 / 270, 269 / 271, 269 / 272, 269 / 273, 269 / 274, 269 / 275, 269 / 276, 269 / 277, 269 / 278, 269 / 279, 269 / 280, 269 / 281, 269 / 282, 269 / 283, 269 / 284 269 / 285, 269 / 286, 269 / 287, 269 / 288, 270 / 267, 270 / 268, 270 / 269, 270 / 270, 270 / 271, 270 / 272, 270 / 273, 270 / 274, 270 / 275, 270 / 276, 270 / 277, 270 / 278, 270 / 279, 270 / 280, 270 / 281, 270 / 282, 270 / 283, 270 / 284, 270 / 285, 270 / 286, 270 / 287, 270 / 288, 271 / 267, 271 / 268, 271 / 269, 271 / 270, 271 / 271271 / 272、271 / 273、271 / 274、271 / 275、271 / 276、271 / 277、271 / 278、271 / 279、271 / 280、271 / 281、271 / 282、271 / 283、271 / 284、271 / 285、271 / 286、271 / 287、271 / 288、272 / 267、272 / 268、272 / 269、272 / 270、272 / 271、272 / 272、272 / 273、272 / 274、272 / 275、272 / 276、272 / 277、272 / 278、272 / 279、272 / 280、272 / 281、272 / 282、272 / 283、272 / 284、272 / 285、272 / 286、272 / 287、272 / 288、273 / 267、273 / 268、273 / 269、273 / 270、273 / 271、273 / 272、273 / 273、273 / 274、273 / 275、273 / 276、273 / 277、273 / 278、273 / 279、273 / 280、273 / 281、273 / 282、273 / 283、273 / 284、273 / 285、273 / 286、273 / 287、273 / 288、274 / 267、274 / 268、274 / 269、274 / 270、274 / 271、274 / 272、274 / 273、274 / 274、274 / 275、274 / 276、274 / 277、274 / 278、274 / 279、274 / 280、274 / 281、274 / 282、274 / 283、274 / 284、274 / 285、274 / 286、274 / 287、274 / 288、275 / 267、275 / 268、275 / 269、275 / 270、275 / 271、275 / 272、275 / 273、275 / 274、275 / 275、275 / 276、275 / 277、275 / 278、275 / 279、275 / 280、275 / 281、275 / 282、275 / 283、275 / 284、275 / 285、275 / 286、275 / 287、275 / 288、276 / 267、276 / 268、276 / 269、276 / 270、276 / 271、276 / 272、276 / 273、276 / 274、276 / 275、276 / 276、276 / 277、276 / 278、276 / 279、276 / 280、276 / 281、276 / 282、276 / 283、276 / 284、276 / 285、276 / 286、276 / 287、276 / 288、277 / 267、277 / 268、277 / 269、277 / 270、277 / 271、277 / 272、277 / 273、277 / 274、277 / 275、277 / 276、277 / 277、277 / 278、277 / 279、277 / 280、277 / 281、277 / 282、277 / 283、277 / 284、277 / 285、277 / 286、277 / 287、277 / 288、278 / 267、278 / 268、278 / 269、278 / 270、278 / 271、278 / 272、278 / 273、278 / 274、278 / 275、278 / 276、278 / 277、278 / 278、278 / 279、278 / 280、278 / 281、278 / 282、278 / 283、278 / 284、278 / 285、278 / 286、278 / 287、278 / 288、279 / 267、279 / 268、279 / 269、279 / 270、279 / 271、279 / 272、279 / 273、279 / 274、279 / 275、279 / 276、279 / 277、279 / 278、279 / 279、279 / 280、279 / 281、279 / 282、279 / 283、279 / 284、279 / 285、279 / 286、279 / 287、279 / 288、280 / 267、280 / 268、280 / 269、280 / 270、280 / 271、280 / 272、280 / 273、280 / 274、280 / 275、280 / 276、280 / 277、280 / 278、280 / 279、280 / 280、280 / 281、280 / 282、280 / 283、280 / 284、280 / 285、280 / 286、280 / 287、280 / 288、281 / 267、281 / 268、281 / 269、281 / 270、281 / 271、281 / 272、281 / 273、281 / 274、281 / 275、281 / 276、281 / 277、281 / 278、281 / 279、281 / 280、281 / 281、281 / 282、281 / 283、281 / 284、281 / 285、281 / 286、281 / 287、281 / 288、282 / 267、282 / 268、282 / 269、282 / 270、282 / 271、282 / 272、282 / 273、282 / 274、282 / 275、282 / 276、282 / 277、282 / 278、282 / 279、282 / 280、282 / 281、282 / 282、282 / 283、282 / 284、282 / 285、282 / 286、282 / 287、282 / 288、283 / 267、283 / 268、283 / 269、283 / 270、283 / 271、283 / 272、283 / 273、283 / 274、283 / 275、283 / 276、283 / 277、283 / 278、283 / 279、283 / 280、283 / 281、283 / 282、283 / 283、283 / 284、283 / 285、283 / 286、283 / 287、283 / 288、284 / 267、284 / 268、284 / 269、284 / 270、284 / 271、284 / 272、284 / 273、284 / 274、284 / 275、284 / 276、284 / 277、284 / 278、284 / 279、284 / 280、284 / 281、284 / 282、284 / 283、284 / 284、284 / 285、284 / 286、284 / 287、284 / 288、285 / 267、285 / 268、285 / 269、285 / 270、285 / 271、285 / 272、285 / 273、285 / 274、285 / 275、285 / 276、285 / 277、285 / 278、285 / 279、285 / 280、285 / 281、285 / 282、285 / 283、285 / 284、285 / 285、285 / 286、285 / 287、285 / 288、286 / 267、286 / 268、286 / 269、286 / 270、286 / 271、286 / 272、286 / 273、286 / 274、286 / 275、286 / 276、286 / 277、286 / 278、286 / 279、286 / 280、286 / 281、286 / 282、286 / 283、286 / 284、286 / 285、286 / 286、286 / 287、286 / 288、287 / 267、287 / 268、287 / 269、287 / 270、287 / 271、287 / 272、287 / 273、287 / 274、287 / 275、287 / 276、287 / 277、287 / 278、287 / 279、287 / 280、287 / 281、287 / 282、287 / 283、287 / 284、287 / 285、287 / 286、287 / 287、287 / 288，117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、117 / 123、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、118 / 123、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、119 / 123、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、120 / 123、121 / 118、121 / 119、121 / 120、121 / 121、121 / 122、121 / 123、122 / 118、122 / 119、122 / 120、122 / 121、122 / 122、122 / 123，340 / 341、340 / 342、340 / 343、340 / 344、340 / 345、340 / 346、340 / 347、340 / 348、340 / 349、340 / 350、340 / 351、340 / 352、340 / 353、341 / 341、341 / 342、341 / 343、341 / 344、341 / 345、341 / 346、341 / 347、341 / 348、341 / 349、341 / 350、341 / 351、341 / 352、341 / 353、342 / 341、342 / 342、342 / 343、342 / 344、342 / 345、342 / 346、342 / 347、342 / 348、342 / 349、342 / 350、342 / 351、342 / 352、342 / 353、343 / 341、343 / 342、343 / 343、343 / 344、343 / 345、343 / 346、343 / 347、343 / 348、343 / 349、343 / 350、343 / 351、343 / 352、343 / 353、344 / 341、344 / 342、344 / 343、344 / 344、344 / 345、344 / 346、344 / 347、344 / 348、344 / 349、344 / 350、344 / 351、344 / 352、344 / 353、345 / 341、345 / 342、345 / 343、345 / 344、345 / 345、345 / 346、345 / 347、345 / 348、345 / 349、345 / 350、345 / 351、345 / 352、345 / 353、346 / 341、346 / 342、346 / 343、346 / 344、346 / 345、346 / 346、346 / 347、346 / 348、346 / 349、346 / 350、346 / 351、346 / 352、346 / 353、347 / 341、347 / 342、347 / 343、347 / 344、347 / 345, 347 / 346, 347 / 347, 347 / 348, 347 / 349, 347 / 350, 347 / 351, 347 / 352, 347 / 353, 348 / 341, 348 / 342, 348 / 343, 348 / 344, 348 / 345, 348 / 346, 348 / 347, 348 / 348, 348 / 349, 348 / 350, 348 / 351, 348 / 352, 348 / 353, 349 / 341, 349 / 342, 349 / 343, 349 / 344, 349 / 345, 349 / 346, 349 / 347, 349 / 348, 349 / 349, 349 / 350, 349 / 351, 349 / 352, 349 / 353, 350 / 341, 350 / 342, 350 / 343, 350 / 344, 350 / 345, 350 / 346, 350 / 347, 350 / 348, 350 / 349, 350 / 350, 350 / 351, 350 / 352, 350 / 353, 351 / 341, 351 / 342, 351 / 343, 351 / 344, 351 / 345, 351 / 346, 351 / 347, 351 / 348, 351 / 349, 351 / 350, 351 / 351, 351 / 352, 351 / 353, 352 / 341, 352 / 342, 352 / 343, 352 / 344, 352 / 345, 352 / 346, 352 / 347, 352 / 348, 352 / 349, 352 / 350, 352 / 351, 352 / 352, and 352 / 353, the TtGBP protein having the sequence set forth in SEQ ID NO: 1 or a functional fragment thereof, the variant of the TtGBP protein comprising a mutation selected from the group consisting of W8, W9, A42, H66, K312, V347, H348, the variant of the fluorescent protein comprising a mutation at the Y1 site, Preferably, the optically active polypeptide is selected from any one of cpYFP, cpGFP, cpBFP, cpmApple.
2. The optical probe of claim 1, wherein, the fluorescent protein is as set forth in any one of SEQ ID NOs: 2-9, and in a variant of the fluorescent protein, Y1 is mutated to A or G; Preferably, the fluorescent protein is as set forth in any one of SEQ ID NOs: 2, 6, 7, 9.
3. The optical probe of claim 1, wherein in a variant of the TtGBP protein, W8 is mutated to R, H, E, A, V, L, F, I, M, C, N, G, K, D or T, W9 is mutated to R, H, E, A, V, L, F, I, M, C, P, D, N, G, K, Y, S or T, A42 is mutated to R, E, W, F, M, C, P, N, G, K, Y or T, H66 is mutated to R, E, A, F, M, C, P, Q or T, K312 is mutated to R, A, Q, G, H or S, V347 is mutated to W, and H348 is mutated to T, G or S, Preferably, the mutations in the variant of the TtGBP protein comprise mutations selected from any one of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W, H348T and H66M, (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W, H348T and W9H, (15) V347W, H348T and W9E, (16) V347W, H348T and W9A, (17) V347W, H348T and W9V, (18) V347W, H348T and W9L, (19) V347W, H348T and W9F, (20) V347W, H348T and W9I, (21) V347W, H348T and W9M, (22) V347W, H348T and W9C, (23) V347W, H348T and W9P, (24) V347W, H348T and W9N, (25) V347W, H348T and W9G, (26) V347W, H348T and W9K, (27) V347W, H348T and W9Y, (28) V347W, H348T and W9S, (29) V347W, H348T and W9T, (30) V347W, H348T and W9D, (31) V347W, H348T and W8R, (32) V347W, H348T and W8H, (33) V347W, H348T and W8E, (34) V347W, H348T and W8A, (35) V347W, H348T and W8V, (36) V347W, H348T and W8L, (37) V347W, H348T and W8F, (38) V347W, H348T and W8I, (39) V347W, H348T and W8M, (40) V347W, H348T and W8C, (41) V347W, H348T and W8N, (42) V347W, H348T and W8G, (43) V347W, H348T and W8K, (44) V347W, H348T and W8T, (45) V347W, H348T and W8D, (46) V347W, H348T and A42R, (47) V347W, H348T and A42E, (48) V347W, H348T and A42W,(49) V347W, H348T and A42F, (50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H348T and A42Y, (57) V347W, H348T and A42T, (58) V347W, H348T and K312R, (59) V347W, H348T and K312A, (60) V347W, H348T and K312Q, (61) V347W, H348T and K312G, (62) V347W, H348T and K312H, (63) V347W, H348T and K312S, 4. A fusion polypeptide comprising the optical probe of any one of claims 1-3 and another polypeptide, which includes a localization sequence, a tag for facilitating purification or a tag for immunoreaction.
5. A nucleic acid molecule comprising a sequence selected from the group consisting of: (1) a polynucleotide sequence encoding the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4, and (2) a complement of (1).
6. A nucleic acid construct comprising the nucleic acid molecule of claim 5. Preferably, the nucleic acid construct is an expression vector.
7. A host cell, which: (1) comprises, expresses or secretes the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4; (2) comprises the nucleic acid molecule of claim 5; or (3) comprises the nucleic acid construct of claim 6.
8. A method of producing the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4, comprising culturing the host cell of claim 7, and isolating the optical probe from the culture.
9. Use of the optical probe of any one of claims 1-3, the fusion polypeptide of claim 4, the nucleic acid molecule of claim 5 or the nucleic acid construct of claim 6 in the manufacture of a kit for detecting D-glucose in a sample, screening a compound or intracellular and / or extracellular localization of D-glucose.
10. A detection kit, which comprises (1) the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4; (2) the nucleic acid molecule of claim 5; (3) the nucleic acid construct of claim 6; or (4) the host cell of claim 7; the detection kit optionally further comprises other reagents required for detecting D-glucose using the optical probe, Preferably, the detection kit further comprises one or more reagents selected from the group consisting of a buffer, a culture medium, a D-glucose standard.