Glyoxylic acid optical probe as well as preparation method and application thereof

Abstract

The invention relates to a glyoxylic acid optical probe as well as a preparation method and application thereof. In one aspect, the present invention relates to an optical probe comprising a glyoxylic acid-sensitive polypeptide or functional variant thereof and an optically active polypeptide or functional variant thereof wherein the optically active polypeptide or functional variant thereof is located within the sequence of the glyoxylic acid-sensitive polypeptide or functional variant thereof. The invention also relates to a preparation method of the probe and application of the probe in detection of glyoxylic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of optical probes, and in particular to a glyoxylic acid optical probe and a preparation method and application thereof. Background Art

[0002] Glyoxylate is an organic compound containing 2 carbon atoms, with both an aldehyde group and a carboxylic acid group. Disorders in the metabolism of glyoxylate in the body can cause oxalic acid accumulation and induce hyperoxaluria, so the rapid, sensitive and specific determination of glyoxylate is of great significance.

[0003] Tapas patras et al. found that the growth of HT-29 and HCT-116 cells was significantly inhibited by increasing the concentration of glyoxylate through MTT and colony formation assays. In vitro experimental results showed that supraphysiological concentrations of glyoxylate had an antiproliferative effect on human colon cancer cells mediated by oxidative stress, and glyoxylate can be considered as a new therapeutic strategy for colon cancer. Patel et al. demonstrated that glyoxylate can inhibit the interaction of calcitriol receptor-vitamin D response element in vitro. Glyoxylate also blocked the expression of chloramphenicol acetyltransferase activity induced by calcitriol in transiently transfected JEG cells and inhibited the activity of rat intestinal 24-hydroxylase. These findings suggest that glyoxylate can inhibit the binding of VDR to VDRE, which may alter the biological role of calcitriol in renal failure. Victoria J. et al. used metabolite profiling to describe human plasma from diabetic and non-diabetic patients, and reactive glyoxylate levels were elevated before measuring high levels of blood glucose typical of diabetes. In C57BLKS / J-Lepr db / db- / - Increased plasma glyoxylate levels were also observed in mouse models, supporting the finding that glyoxylate is associated with the diabetic phenotype. Glyoxylate, as a new diabetes biomarker, could help further develop and improve new diagnostic and therapeutic approaches.

[0004] Currently, the most commonly used method for detecting glyoxylic acid is high performance liquid chromatography, but this detection and analysis method requires professional analytical instruments and equipment, has complicated steps and is only applicable to in vitro detection, and cannot monitor the changes in glyoxylic acid concentration in living cells in real time. Therefore, it is urgent to develop new detection methods to achieve simple, fast, highly specific, real-time localization, quantitative, and high-throughput detection of glyoxylic acid inside and outside cells. Summary of the invention

[0005] The purpose of the present invention is to provide a probe and method for real-time localization, high-throughput and quantitative detection of glyoxylic acid inside and outside cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a glyoxylate binding protein variant, which:

[0008] (1) having the sequence shown in SEQ ID NO: 1 and having a mutation at one, two, three, four, five, six or seven of the following positions: E115, N116, I118, G119, T59, S139, S141, wherein the mutation comprises a modification, substitution or deletion of an amino acid,

[0009] (2) is a truncated variant of (1) having amino acids 76 to 246, or

[0010] (3) is a sequence having at least 70% sequence identity with the sequence of (1) or (2) and having the mutation described in (1) and retaining the ability to bind to glyoxylate.

[0011] In one or more embodiments, the mutation includes a mutation at any site selected from the group consisting of: E115, N116, I118, G119, and optionally, the mutation also includes a mutation at any site selected from the group consisting of: T59, S139, S141.

[0012] In one or more embodiments, the mutation includes a mutation at a site selected from any one of the following groups: (1) I118 and G119, (2) I118, (3) E115, N116, (4) I118, E115, N116; or, the mutation includes a mutation at a site selected from any one of the following groups: (5) T59, I118, E115, N116, (6) T59, E115, N116, (7) I118, E115, N116, S139, (8) E115, N116, S139, (9) I118, E115, N116, S141, (10) E115, N116, S141, (11) T133 and I118, (12) I118 and S213, (13) I118 and S215.

[0013] In one or more embodiments, I118 is mutated to G, Q, T, L, M, V, H, F, S, A, Y, or K. In one or more embodiments, G119 is mutated to N, V, or S. In one or more embodiments, E115 is mutated to N, S, P, T, or Q. In one or more embodiments, N116 is mutated to V, C, K, E, I, T, R, or W. In one or more embodiments, T59 is mutated to G or N. In one or more embodiments, S139 is mutated to N or T. In one or more embodiments, S141 is mutated to D, H, or G.

[0014] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N 116V, (11)E115N, N116E, (12)E115N, N116I, (13)E115N, N116T, (14)E115N, N116R, (15)E115Q, N116K, (16)E115N, N116W, (17)E115P, N116V, (18)I118M, E115N, N116V, (19)I118T, E115N, N11 6V, (20)I118V, E115N, N116V, (21)I118L, E115N, N116V, (22)I118H, E115N, N116V, (23)I118V, E115N, N116V, (24) I118F, E115N, N116V, (25) I118S, E115N, N116C, (26) I118L, G119V, E115N, N 116C, (27) I118A, G119S, E115N, N116C, (28) I118Y, E115S, N116K, (29) I118K, E115S, N116K; preferably, the mutation comprises a mutation selected from any one of the following groups: (6) E115N and N116C, (24) I118F, E115N, N116V, (28) I118Y, E115S, N116K. Optionally, the mutation further comprises any one or more mutations selected from the following groups: (a) T59G or T59N, (b) S139N or S139T, (c) S141D or S141G.

[0015] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (30) T59G, I118F, E115N, N116V, (31) T59N, I118F, E115N, N116V, (32) T59G, E115N, N116V, (33) T59N, E115N, N116V, (34) T59G, I118Y, E115S, N116K, (35) T59N, I118 8Y, E115S, N116K, (36) T59G, I118K, E115S, N116K, (37) T59N, I118K, E115S, N116K, (38) I118F, E11 5N, N116V, S139N, (39) I118F, E115N, N116V, S139T, (40) E115N, N116V, S139N, (41) E115N, N116V, S 139T, (42)I118Y, E115S, N116K, S139N, (43) I118Y, E115S, N116K, S139T, (44) I118K, E115S, N116K , S139N, (45)I118K, E115S, N116K, S139T, (46)I118F, E115N, N116V, S141D, (47)I118F, E115N, N11 6V, S141G, (48)E115N, N116V, S141D, (49)E115N, N116V, S141G, (50)I118Y, E115S, N116K, S141D, ( 51)I118Y, E115S, N116K, S141G, (52) I118K, E115S, N116K, S141D, (53) I118K, E115S, N116K, S141G.

[0016] Another aspect of the present invention provides a glyoxylate optical probe, comprising a glyoxylate-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is located within the sequence of the glyoxylate-sensitive polypeptide or a functional variant thereof. The glyoxylate-sensitive polypeptide or a functional variant thereof is divided into a first part and a second part by the optically active polypeptide or a functional variant thereof.

[0017] In one or more embodiments, the glyoxylic acid optical probe comprises a glyoxylic acid-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the glyoxylic acid-sensitive polypeptide B, and the glyoxylic acid-sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of the type B1-A-B2.

[0018] In one or more embodiments, the glyoxylate-sensitive polypeptide comprises a glyoxylate-binding protein or a functional variant thereof.In one or more embodiments, the glyoxylate-sensitive polypeptide is derived from Thermotoga maritima.

[0019] In one or more embodiments, the glyoxylate-sensitive polypeptide has:

[0020] (1) the sequence shown in SEQ ID NO: 1 or a truncated variant thereof having amino acids 76 to 246, or a sequence having at least 70% sequence identity therewith and retaining glyoxylate binding activity,

[0021] (2) the sequence of the glyoxylate binding protein variant described in any embodiment of the first aspect of the present invention, or

[0022] (3) A sequence having at least 70% sequence identity with the sequence described in (2) and having the mutation described in (2) and retaining sensitivity to glyoxylic acid.

[0023] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one or more embodiments, the fluorescent protein is selected from yellow fluorescent protein (such as cpYFP as shown in SEQ ID NO: 2), orange fluorescent protein (such as cpmOrange as shown in SEQ ID NO: 3), red fluorescent protein (such as mKate as shown in SEQ ID NO: 4 or 8, such as mcherry as shown in SEQ ID NO: 5), green fluorescent protein (such as cpGFP as shown in SEQ ID NO: 6), blue fluorescent protein (such as cpBFP as shown in SEQ ID NO: 7), apple red fluorescent protein (such as cpmApple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has a sequence as shown in any one of SEQ ID NO: 2-9.

[0024] In one or more embodiments, the optically active polypeptide is located between residues 17-20, 28-31, 54-59, 67-71, 107-110, 114-120, 128-133, 142-149 of the glyoxylate-sensitive polypeptide, numbered corresponding to the full length of the glyoxylate-sensitive polypeptide. Preferably, the optically active polypeptide is located at one or more of the following positions of the glyoxylate-sensitive polypeptide: 17 / 18, 17 / 19, 17 / 20, 17 / 21, 18 / 19, 18 / 20, 18 / 21, 19 / 20, 19 / 21, 20 / 21, 28 / 29, 28 / 30, 28 / 31, 28 / 32, 29 / 30, 29 / 31, 29 / 32, 30 / 31, 30 / 32, 31 / 32, 54 / 55, 54 / 56, 54 / 57, 54 / 58, 54 / 59, 54 / 60, 55 / 56, 55 / 57, 55 / 58, 55 / 59, 55 / 60, 56 / 57, 56 / 58, 56 / 59, 56 / 60, 57 / 5 8,57 / 59,57 / 60,58 / 59,58 / 60,59 / 60,67 / 68,67 / 69,67 / 70,67 / 71,67 / 72,68 / 69,68 / 70,68 / 71,68 / 72,69 / 70,69 / 71,69 / 72,70 / 71,70 / 72,71 / 72,10 7 / 108,107 / 109,107 / 110,107 / 111,108 / 109,108 / 110,108 / 111,109 / 110,109 / 111,110 / 111,114 / 115,114 / 116,114 / 117,114 / 118,114 / 119,114 / 120 114 / 121,115 / 115,115 / 116,115 / 117,115 / 118,115 / 119,115 / 120,115 / 121,116 / 115,116 / 116,116 / 117,116 / 118,116 / 119,116 / 120,116 / 121,117 / 115,117 / 116,117 / 117,117 / 118,117 / 119,117 / 120,117 / 121,118 / 115,118 / 116,118 / 117,118 / 118,118 / 119,118 / 120,118 / 121,119 / 115,119 / 116,119 / 117,119 / 118,119 / 119,119 / 120,119 / 121,120 / 115,120 / 116,120 / 117,120 / 118,120 / 119,120 / 120,120 / 121,128 / 129,128 / 130,128 / 131,128 / 132,128 / 133,128 / 134,129 / 130,129 / 131,129 / 132,129 / 133,129 / 134,130 / 131,130 / 132,130 / 133,130 / 134,131 / 132,131 / 133,131 / 134,132 / 133,132 / 134,133 / 134,142 / 143,142 / 144,142 / 145,142 / 146,142 / 147,142 / 148,142 / 149,142 / 150,143 / 144,143 / 145,143 / 146,143 / 147,143 / 148,143 / 149,143 / 150,144 / 145,144 / 146,144 / 147,144 / 148,144 / 149,144 / 150,145 / 146,145 / 147,145 / 148 More preferably, the optically active polypeptide is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide: 114 / 115, 114 / 119, 114 / 120, 117 / 115, 117 / 116, 117 / 118, 117 / 121, 118 / 118, 118 / 119, 119 / 115, 119 / 116, 120 / 115, 120 / 120.

[0025] In one or more embodiments, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linkers of the present invention can be any amino acid sequence of any length. In one or more embodiments, the optically active polypeptide flanks comprise linkers of no more than 5 amino acids, such as linkers of 0, 1, 2, 3, or 4 amino acids.

[0026] In one or more embodiments, the linker flanking the optically active polypeptide comprises amino acid Y. In one or more embodiments, the linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one or more embodiments, the optical probe is as follows: the first part B1 of the glyoxylic acid-sensitive polypeptide-Y-optically active polypeptide A-the second part B2 of the glyoxylic acid-sensitive polypeptide. In one or more embodiments, the optical probe of the present invention does not comprise a linker.

[0027] In one or more embodiments, the optical probes of the present invention further comprise a localization sequence for localizing the probe to, for example, a specific organelle of a cell.

[0028] In one or more embodiments, the optically active polypeptide is cpYFP, ​​which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide: 114 / 115, 114 / 119, 114 / 120, 117 / 115, 117 / 116, 117 / 118, 117 / 121, 118 / 118, 118 / 119, 119 / 115, 119 / 116, 120 / 115, 120 / 120, wherein the glyoxylate-sensitive polypeptide (1) is as shown in SEQ ID NO:1 or is a truncated variant thereof having amino acids 76-246, or (2) is the glyoxylate-binding protein variant described in any embodiment of the first aspect of the present invention.

[0029] In one or more embodiments, the optically active polypeptide is cpGFP, which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide shown in SEQ ID NO:1 at positions 76-246: 117 / 120, 119 / 115, 119 / 116, 120 / 115 and 120 / 117, and the glyoxylate-sensitive polypeptide (1) is as shown in SEQ ID NO:1 or is a truncated variant thereof having amino acids 76-246, or (2) is the glyoxylate-binding protein variant described in any embodiment of the first aspect of the present invention.

[0030] In one or more embodiments, the optically active polypeptide is cpBFP, which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide shown in SEQ ID NO:1 at positions 76-246: 114 / 117, 114 / 118, 119 / 119 and 119 / 120, and the glyoxylate-sensitive polypeptide (1) is as shown in SEQ ID NO:1 or is a truncated variant thereof having amino acids 76-246, or (2) is a glyoxylate-binding protein variant as described in any embodiment of the first aspect of the present invention.

[0031] In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide shown in SEQ ID NO:1 at positions 76-246: 117 / 115, 118 / 119, 118 / 120 and 120 / 121, and the glyoxylate-sensitive polypeptide (1) is as shown in SEQ ID NO:1 or is a truncated variant thereof having amino acids 76-246, or (2) is the glyoxylate-binding protein variant described in any embodiment of the first aspect of the present invention.

[0032] In one or more embodiments, the optically active polypeptide is cpYFP, ​​which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide: 114 / 115, 114 / 119, 114 / 120, 117 / 115, 117 / 116, 117 / 118, 117 / 121, 118 / 118, 118 / 119, 119 / 115, 119 / 116, 120 / 115, 120 / 120, wherein the glyoxylate-sensitive polypeptide (i) is as shown in SEQ ID NO:1 or a truncated variant thereof having amino acids 76-246, or (ii) a variant of (i) having a mutation selected from any of the following groups: (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K,

[0033] (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V, (11) E115N, N116E, (12) E115N, N116I, (13) E115N, N116T, (14) E115N, N116R, (15) E115Q, N116K, (16) E115N, N116W, (17) E115P, N116V, (18) I118M, E115N, N116V, (19) I118T, E115N, N116V, (20) I118V, E115N, N116V, (21) I118L, E115N, N116 V, (22)I118H, E115N, N116V, (23)I118V, E115N, N116V, (24)I118F, E115N , N116V, (25) I118S, E115N, N116C, (26) I118L, G119V, E115N, N116C, (27) I118A, G119S, E115N, N116C, (28) I118Y, E115S, N116K, (29) I118K, E115S , N116K, (30) T59G, I118F, E115N, N116V, (31) T59N, I118F, E115N, N116V, ( 32)T59G, E115N, N116V, (33) T59N, E115N, N116V, (34) T59G, I118Y, E115S , N116K, (35) T59N, I118Y, E115S, N116K, (36) T59G, I118K, E115S, N116K, (37)T59N, I118K, E115S, N116K, (38)I118F, E115N, N116V, S139N, (39)I1 18F, E115N, N116V, S139T, (40)E115N, N116V, S139N, (41)E115N, N116V, S 139T, (42)I118Y, E115S, N116K, S139N, (43)I118Y, E115S, N116K, S139T, (44)I118K, E115S, N116K, S139N, (45)I118K, E115S, N116K, S139T, (46)I 118F, E115N, N116V, S141D, (47)I118F, E115N, N116V, S141G, (48)E115N, N116V, S141D, (49)E115N, N116V, S141G, (50)I118Y, E115S, N116K, S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G。,

[0034] In one or more embodiments, the optically active polypeptide is cpGFP, which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide shown in SEQ ID NO: 1 at positions 76-246: 117 / 120, 119 / 115, 119 / 116, 120 / 115 and 120 / 117, wherein the glyoxylate-sensitive polypeptide (i) is as shown in SEQ ID NO:1 or a truncated variant thereof having amino acids 76-246, or (ii) a variant of (i) having a mutation selected from any of the following groups: (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V, (11) E115N, N116E, (12) E115N, N116V 116I, (13)E115N, N116T, (14)E115N, N116R, (15)E115Q, N116K, (16)E115N, N116W, (17)E115P, N116V, (18)I118M, E115N, N116V, (19)I118T, E115N, N116V, (20) I118V, E115N, N116V, (21) I118L, E115N, N116V, (22) I118H, E115N, N116V, (23) I118V, E115N, N11 6V, (24)I118F, E115N, N116V, (25) I118S, E115N, N116C, (26) I118L, G119V, E115N, N116C, (27) I118A, G119S, E115N, N116C, (28) I118Y, E115S, N116K, (29) I118K, E115S, N116K, (30) T59G, I118F, E115N, N116V, (31) T59N, I118F, E115N, N116V, (32) T59G, E115 N, N116V, (33) T59N, E115N, N116V, (34) T59G, I118Y, E115S, N116K, (35) T59N, I118Y, E115S, N116K, (36) T59G, I118K, E115S, N11 6K, (37)T59N, I118K, E115S, N116K, (38) I118F, E115N, N116V, S139N, (39) I118F, E115N, N116V, S139T, (40) E115N, N116V, S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,

[0035] (50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G。

[0036] In one or more embodiments, the optically active polypeptide is cpBFP, which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide shown in SEQ ID NO: 1 at positions 76-246: 114 / 117, 114 / 118, 119 / 119 and 119 / 120, wherein the glyoxylate-sensitive polypeptide (i) is as shown in SEQ ID NO:1 or a truncated variant thereof having amino acids 76-246, or (ii) a variant of (i) having a mutation selected from any of the following groups: (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V, (11) E115N, N116E, (12) E115N, N116V 116I, (13)E115N, N116T, (14)E115N, N116R, (15)E115Q, N116K, (16)E115N, N116W, (17)E115P, N116V, (18)I118M, E115N, N116V, (19)I118T, E115N, N116V, (20) I118V, E115N, N116V, (21) I118L, E115N, N116V, (22) I118H, E115N, N116V, (23) I118V, E115N, N11 6V, (24)I118F, E115N, N116V, (25) I118S, E115N, N116C, (26) I118L, G119V, E115N, N116C, (27) I118A, G119S, E115N, N116C, (28) I118Y, E115S, N116K, (29) I118K, E115S, N116K, (30) T59G, I118F, E115N, N116V, (31) T59N, I118F, E115N, N116V, (32) T59G, E115 N, N116V, (33) T59N, E115N, N116V, (34) T59G, I118Y, E115S, N116K, (35) T59N, I118Y, E115S, N116K, (36) T59G, I118K, E115S, N11 6K, (37)T59N, I118K, E115S, N116K, (38) I118F, E115N, N116V, S139N, (39) I118F, E115N, N116V, S139T, (40) E115N, N116V, S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G。,

[0037] In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide shown in SEQ ID NO: 1 at positions 76-246: 117 / 115, 118 / 119, 118 / 120 and 120 / 121, wherein the glyoxylate-sensitive polypeptide (i) is as shown in SEQ ID NO:1 or a truncated variant thereof having amino acids 76-246, or (ii) a variant of (i) having a mutation selected from any of the following groups: (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V, (11) E115N, N116E, (12) E115N, N116V 116I, (13)E115N, N116T, (14)E115N, N116R, (15)E115Q, N116K, (16)E115N, N116W, (17)E115P, N116V, (18)I118M, E115N, N116V, (19)I118T, E115N, N116V, (20) I118V, E115N, N116V, (21) I118L, E115N, N116V, (22) I118H, E115N, N116V, (23) I118V, E115N, N11 6V, (24)I118F, E115N, N116V, (25) I118S, E115N, N116C, (26) I118L, G119V, E115N, N116C, (27) I118A, G119S, E115N, N116C, (28) I118Y, E115S, N116K, (29) I118K, E115S, N116K, (30) T59G, I118F, E115N, N116V, (31) T59N, I118F, E115N, N116V, (32) T59G, E115 N, N116V, (33) T59N, E115N, N116V, (34) T59G, I118Y, E115S, N116K, (35) T59N, I118Y, E115S, N116K, (36) T59G, I118K, E115S, N11 6K, (37)T59N, I118K, E115S, N116K, (38) I118F, E115N, N116V, S139N, (39) I118F, E115N, N116V, S139T, (40) E115N, N116V, S139N,(41)E115N, N116V, S139T, (42)I118Y, E115S, N116K, S139N, (43)I118Y, E115S, N116K, S139T, (44)I118 K, E115S, N116K, S139N, (45) I118K, E115S, N116K, S139T, (46) I118F, E115N, N116V, S141D, (47) I118F, E 115N, N116V, S141G, (48)E115N, N116V, S141D, (49)E115N, N116V, S141G, (50)I118Y, E115S, N116K, S14 1D, (51)I118Y, E115S, N116K, S141G, (52) I118K, E115S, N116K, S141D, (53) I118K, E115S, N116K, S141G. ,

[0038] In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NO: 10-49 or a variant thereof. In one or more embodiments, the optical probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NO: 10-49. Preferably, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NO: 10-49.

[0039] Another aspect of the present invention provides a fusion polypeptide comprising the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides for locating the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting. A joint may be provided between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0040] In another aspect, the present invention also provides a nucleic acid molecule comprising: (1) a coding sequence of a polypeptide or a probe as described in any embodiment of the present invention, or (b) a complementary sequence of (a), or (c) a fragment of (a) or (b). The fragment is a primer.

[0041] In one or more embodiments, the nucleic acid sequence comprises an amino acid sequence encoding any one of SEQ ID NOs: 23-49. Preferably, the nucleic acid sequence comprises any one of the nucleotide sequences SEQ ID NOs: 23 or a variant thereof. More preferably, the nucleic acid sequence comprises a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to any one of the nucleotide sequences SEQ ID NOs: 23; or comprises a nucleotide sequence substantially similar or identical to any one of the nucleotide sequences SEQ ID NOs: 23.

[0042] The present invention also relates to the complementary sequence of the above-mentioned nucleic acid sequence or its variant, which may comprise the nucleic acid sequence encoding the fragment, analog, derivative, soluble fragment and variant of the optical probe or fusion protein of the present invention or its complementary sequence.

[0043] The present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein. The nucleic acid sequence encodes the optical probe or fusion polypeptide described in the present invention.

[0044] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0045] In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0046] In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

[0047] The present invention also provides a host cell, wherein the host cell: (1) expresses the optical probe or fusion polypeptide according to any embodiment of the present invention; (2) contains the nucleic acid molecule according to any embodiment of the present invention; or (3) contains the nucleic acid construct according to any embodiment of the present invention. The host cell is preferably Escherichia coli.

[0048] Another aspect of the present invention provides a glyoxylic acid detection kit, comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe prepared by the method described herein.

[0049] In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of a buffer, a culture medium, and a glyoxylate standard.

[0050] The present invention provides a method for preparing the optical probe described herein, comprising: providing a host cell expressing the optical probe or fusion polypeptide described herein, culturing the host cell under conditions where the cell expresses the optical probe, and isolating the optical probe or fusion polypeptide.

[0051] In one or more embodiments, the method for preparing the glyoxylate optical probe or fusion polypeptide described herein comprises the following steps: 1) transferring the expression vector encoding the glyoxylate optical probe described herein into a host cell; 2) culturing the host cell under conditions suitable for the expression of the expression vector; 3) isolating the glyoxylate optical probe.

[0052] The present invention also provides a method for detecting glyoxylic acid in a sample, comprising: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, and detecting changes in the optically active polypeptide. The detection can be performed in vivo, in vitro, subcellularly or in situ. The sample is, for example, blood.

[0053] Also provided herein is a method for quantifying glyoxylic acid in a sample, comprising: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, detecting changes in optically active polypeptides, and quantifying glyoxylic acid in the sample based on the changes in the optically active polypeptides.

[0054] The present invention also provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with a candidate compound in a system containing glyoxylic acid, detecting changes in the optically active polypeptide, and screening the compound based on the changes in the optically active polypeptide. The method can screen compounds with high throughput.

[0055] In one or more embodiments, a host cell described herein is contacted with a candidate compound in a system containing glyoxylate, and a change in the optics of the optically active polypeptide indicates whether the candidate compound is capable of modulating the uptake of glyoxylate by the cell.

[0056] Another aspect of the present invention provides a method for localizing the glyoxylic acid inside and / or outside a cell, comprising: contacting a system containing glyoxylic acid with the optical probe or the host cell, and detecting the optical change of the optically active polypeptide.

[0057] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0058] Another aspect of the present invention provides the use of the glyoxylate optical probe or fusion polypeptide or host cell described herein in detecting glyoxylate in a sample, screening compounds or glyoxylate localization inside or outside a cell. In one or more embodiments, the localization is real-time localization.

[0059] Beneficial effects of the present invention: The glyoxylate optical probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, has good specificity, and can be expressed in cells by genetic manipulation methods, and can be used for real-time localization, high-throughput, and quantitative detection of glyoxylate inside and outside cells, eliminating the time-consuming sample processing steps. Experimental results show that the maximum response of the glyoxylate optical probe provided by the present application to glyoxylate is more than 5 times that of the control, and can locate, qualitatively, and quantitatively detect cells in subcellular structures such as cytoplasm, mitochondria, nuclei, endoplasmic reticulum, lysosomes, and Golgi bodies, and can perform high-throughput compound screening and quantitative detection of glyoxylate in blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0061] Figure 1 is an SDS-PAGE image of the exemplary glyoxylic acid optical probe described in Example 1;

[0062] Figure 2 is a titration curve diagram of the response of the exemplary glyoxylate optical probe described in Example 7 to glyoxylate at different concentrations;

[0063] Figure 3 This is a bar graph showing the specific detection of four glyoxylic acid analogs by the exemplary glyoxylic acid optical probe described in Example 7;

[0064] Figure 4 This is a photograph of the subcellular organelle localization of the exemplary glyoxylate optical probe described in Example 8 in mammalian cells;

[0065] Figure 5 A schematic diagram of dynamically monitoring the glyoxylate concentration in the cytoplasm of mammalian cells using an exemplary glyoxylate optical probe as described in Example 8;

[0066] Figure 6 A dot plot of high-throughput compound screening at the living cell level using the exemplary glyoxylate optical probe described in Example 9;

[0067] Figure 7 A bar graph showing the quantification of glyoxylate in mouse and human blood using the exemplary glyoxylate optical probe described in Example 10; DETAILED DESCRIPTION

[0068] As used herein, the term "about" when giving a value or range means that the value or range is within 20%, within 10%, and within 5% of the given value or range.

[0069] As used herein, the terms "comprising", "including" and equivalent forms thereof include the meanings of "containing" as well as "consisting of", for example, a composition "comprising" X may consist of X alone or may contain other substances, such as X+Y.

[0070] As used herein, the term "glyoxylate-sensitive polypeptide" or "glyoxylate-responsive polypeptide" refers to a polypeptide that responds to glyoxylate, including any response to a chemical, biological, electrical or physiological parameter of the polypeptide associated with the interaction of the sensitive polypeptide. Responses include small changes, for example, changes in the orientation of the amino acids or peptide fragments of the polypeptide and changes in, for example, the primary, secondary or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule containing side groups in the molecule; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include transitions from α-helix to β-fold or from β-fold to α-helix. It is understood that as long as the fluorescence of the fluorescent protein portion is changed, the detectable change does not need to be a conformational change. The glyoxylate-sensitive polypeptide described herein may also include its functional variants. Functional variants of glyoxylate-sensitive polypeptides include, but are not limited to, variants that can interact with glyoxylate to undergo the same or similar changes as the parent glyoxylate-sensitive polypeptide.

[0071] The glyoxylate-sensitive polypeptides of the present invention include but are not limited to glyoxylate-binding proteins IcIR or variants thereof having more than 90% homology thereto. The exemplary glyoxylate-binding protein IcIR of the present invention is derived from Thermotoga maritima. IclR is a member of a large family of co-folded transcriptional regulatory factors, which includes a DNA binding domain that interacts with an operating sequence and a C-terminal domain (C-IclR) that binds to small molecules. The aceBAK operon encodes enzymes of the glyoxylate bypass, which are required for the growth of these enzymes on acetate. IclR inhibits the expression of the aceBAK operon and its own genes by binding to specific operating sequences in the promoter region. The term "optical probe" used herein refers to a glyoxylate-sensitive polypeptide fused to an optically active polypeptide. The inventors have found that conformational changes produced by glyoxylate-sensitive polypeptides such as glyoxylate-binding proteins specifically binding to physiological concentrations of glyoxylate can cause conformational changes in optically active polypeptides (such as fluorescent proteins), thereby causing changes in the optical properties of optically active polypeptides. By plotting a standard curve based on the fluorescence of the fluorescent protein measured at different glyoxylic acid concentrations, the presence and / or level of glyoxylic acid can be detected and analyzed. An exemplary IcIR protein is shown in SEQ ID NO: 1, and an exemplary IcIR protein truncated variant is a fragment of SEQ ID NO: 1 comprising amino acids 76-246. When describing the optical probe, glyoxylic acid-sensitive polypeptide or glyoxylic acid-binding protein of the present invention (e.g., when describing the insertion site or mutation site), the amino acid residue numbers are all referenced to SEQ ID NO: 1.

[0072] The "optical probe" of the present invention refers to a glyoxylate-sensitive polypeptide fused to an optically active polypeptide, and the optically active polypeptide (e.g., fluorescent protein) is operably inserted into the glyoxylate-sensitive polypeptide. Protein-based "optically active polypeptides" are polypeptides with the ability to emit fluorescence. Fluorescence is an optical property of optically active polypeptides, which can be used as a means of detecting the responsiveness of the optical probe of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient, fluorescence quantum efficiency, shape of the excitation spectrum or emission spectrum, excitation wavelength maximum and emission wavelength maximum, amplitude of two different wavelength excitations, emission amplitude ratio of two different wavelengths, excited state lifetime or fluorescence anisotropy at an appropriate excitation wavelength. The measurable difference of any of these properties between the active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, for example, the amount of fluorescence at a specific wavelength or the integral of fluorescence on the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are easily distinguished in unactivated and activated conformational states. The optically active polypeptide described herein may also include its functional variants. Functional variants of optically active polypeptides include, but are not limited to, variants that can undergo the same or similar changes in fluorescence properties as the parent optically active polypeptide.

[0073] "Linker" or "connector region" refers to an amino acid or nucleotide sequence that connects two parts in the polypeptide, protein or nucleic acid of the present invention. For example, the number of amino acids at the amino terminus of the connection region between the glyoxylic acid-sensitive polypeptide and the optically active polypeptide in the present invention is selected from 0 to 3, and the number of amino acids at the carboxyl terminus is selected from 0 to 2; when the recombinant optical probe is connected to the functional protein as a basic unit, it can be fused to the amino acid or carboxyl 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.

[0074] The terms "chromophore", "fluorophore" and "fluorescent protein" used herein are synonymous with proteins that emit fluorescence under excitation light. Fluorescent proteins are used as basic detection methods in the field of biological sciences, such as the green fluorescent protein GFP commonly used in the field of biotechnology and the cyclic rearranged blue fluorescent protein (cpBFP), cyclic rearranged green fluorescent protein (cpGFP), cyclic rearranged yellow fluorescent protein (cpYFP) derived from mutations of the protein; and the red fluorescent protein RFP commonly used in the field of technology, and the cyclic rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate, etc. The sequence of an exemplary fluorescent protein is shown in any one of SEQ ID NO: 2-9.

[0075] The glyoxylate optical probe of the present invention comprises a glyoxylate-sensitive polypeptide B, such as a glyoxylate-binding protein or a variant thereof, and an optically active polypeptide A, such as a fluorescent protein. The optically active polypeptide A is inserted into the glyoxylate-sensitive polypeptide B, dividing B into two parts, B1 and B2, to form a probe structure of the type B1-A-B2; the interaction between the glyoxylate-sensitive polypeptide B and glyoxylate causes the optical signal of the optically active polypeptide A to become stronger.

[0076] In the optical probe of the present invention, the optically active polypeptide can be located at any position of the glyoxylic acid-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located at any position of the glyoxylic acid-sensitive polypeptide in the NC direction in the NC direction. Specifically, the optically active polypeptide is located in the flexible region of the glyoxylic acid-sensitive polypeptide, and the flexible region refers to some specific structures such as ring domains present in the higher-order structure of the protein, which have higher mobility and flexibility compared to other higher-order structures of the protein, and the region can dynamically change the spatial structural conformation after the protein binds to the ligand. The flexible region described in the present invention mainly refers to the region where the insertion site in the glyoxylic acid binding protein is located, such as amino acid residues 17-20, 28-31, 54-59, 67-71, 107-110, 114-120, 128-133, and 142-149 regions. Illustratively, the optically active polypeptide is located at 17 / 18, 17 / 19, 17 / 20, 17 / 21, 18 / 19, 18 / 20, 18 / 21, 19 / 20, 19 / 21, 20 / 21, 28 / 29, 28 / 30, 28 / 31, 28 / 32, 29 / 30, 29 / 31, 29 / 32, 30 / 31, 30 / 32, 31 / 32, 54 / 55, 54 / 56, 54 / 57, 54 / 58, 54 / 59, 59,54 / 60,55 / 56,55 / 57,55 / 58,55 / 59,55 / 60,56 / 57,56 / 58,56 / 59,56 / 60,57 / 58,57 / 59,57 / 60,58 / 59,58 / 60,59 / 60,67 / 68,67 / 69,67 / 70,67 / 71,67 / 72,68 / 69,68 / 70,68 / 71,68 / 72,69 / 70,69 / 71,69 / 72,70 / 7 1,70 / 72,71 / 72,107 / 108,107 / 109,107 / 110,107 / 111,108 / 109,108 / 110,108 / 111,109 / 110,109 / 111,110 / 111,114 / 115,114 / 116,114 / 117,114 / 118,114 / 119,114 / 120,114 / 121,115 / 115,115 / 116,115 / 117,1 15 / 118,115 / 119,115 / 120,115 / 121,116 / 115,116 / 116,116 / 117,116 / 118,116 / 119,116 / 120,116 / 121,117 / 115,117 / 116,117 / 117,117 / 118,117 / 119,117 / 120,117 / 121,118 / 115,118 / 116,118 / 117,118 / 118,118 / 119,118 / 120,118 / 121,119 / 115,119 / 116,119 / 117,119 / 118,119 / 119,119 / 120,119 / 121,120 / 115,120 / 116,120 / 117,120 / 118,120 / 119,120 / 120,120 / 121,128 / 129,128 / 130,128 / 131,128 / 132,128 / 133,128 / 134,129 / 130,129 / 131,129 / 132,129 / 133,129 / 134,130 / 131,130 / 132,130 / 133 ,130 / 134,131 / 132,131 / 133,131 / 134,132 / 133,132 / 134,133 / 134,142 / 143,142 / 144,142 / 145,142 / 146,142 / 147,142 / 148,142 / 149,142 / 150,143 / 144,143 / 145,143 / 146,143 / 147,143 / 148,143 / 149,143 / 150,144 / 145,144 / 146,144 / 147,144 / 148,144 / 149,144 / 150,145 / 146,145 / 147,145 / 148 145 / 149,145 / 150,146 / 147,146 / 148,146 / 149,146 / 150,147 / 148,147 / 149,147 / 150,148 / 149,148 / 150,149 / 150. ,

[0077] Herein, in the position expressed in the form of "X / Y", both ends of the optically active polypeptide respectively have a portion of the glyoxylic acid-sensitive polypeptide, wherein the N-terminus of the optically active polypeptide is the starting amino acid at the N-terminus of the glyoxylic acid-sensitive polypeptide sequence (e.g., any amino acid from position 1 to position 76) to the amino acid at position X, and the C-terminus of the optically active polypeptide is the amino acid at position Y to the last amino acid at the C-terminus of the glyoxylic acid-sensitive polypeptide sequence (e.g., any amino acid from position Y to amino acid 246). Among them, if the two numbers in the position expressed 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 147 / 148 indicates that the optically active polypeptide is located between amino acids 147 and 148 of the glyoxylic acid-sensitive polypeptide; if the two numbers in the position expressed 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 174 / 185 indicates that the optically active polypeptide replaces amino acids 175-184 of the glyoxylic acid-sensitive polypeptide; if X in the position expressed in the form of "X / Y" is greater than or equal to Y, it means that the glyoxylic acid-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide is to the glyoxylic acid-sensitive polypeptide The optically active polypeptide ends at the Xth amino acid of the sequence, and the glyoxylic acid-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide starts from the Yth amino acid of the glyoxylic acid-sensitive polypeptide sequence; for example, the insertion site 119 / 115 indicates that the N-terminus of the optically active polypeptide is fused with the N-terminal starting amino acid (e.g., any amino acid from positions 1 to 76) to the 119th amino acid of the glyoxylic acid-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is fused with the 115th amino acid to the C-terminal last amino acid (e.g., the 246th amino acid) of the glyoxylic acid-sensitive polypeptide sequence, and its exemplary structure is: (any amino acid from positions (1-76) to the 119th amino acid of the glyoxylic acid-sensitive polypeptide sequence)-(optically active polypeptide)-(amino acids from positions 115 to 246 of the glyoxylic acid-sensitive polypeptide sequence).

[0078] In one or more embodiments, the optical probe comprises, from the N-terminus to the C-terminus, the ZX residue of SEQ ID NO: 1, the optically active polypeptide shown in any one of SEQ ID NO: 2-9, and the The Y-246 residue of NO: 1, wherein Z is any integer from 1 to 76 (preferably 1 or 76), and X and Y are selected from any of the following groups: (1) X is 17 and Y is 18, (2) X is 17 and Y is 19, (3) X is 17 and Y is 20, (4) X is 17 and Y is 21, (5) X is 18 and Y is 19, (6) X is 18 and Y is 20, (7) X is 18 and Y is 21, (8) X is 19 and Y is 20, (9) X is 19 and Y is 21, (10) X is 20 and Y is 21, (11) X is 28 and Y is 29, (12) X is 28 and Y is 30, (13) X is 28 and Y is 31, (14) X is 2 8, Y is 32, (15) X is 29, Y is 30, (16) X is 29, Y is 31, (17) X is 29, Y is 32, (18) X is 30, Y is 31, (19) X is 30, Y is 32, (20) X is 31, Y is 32, (21) X is 54, Y is 55, (22) X is 54, Y is 56, (23) X is 54, Y is 57, (24) X is 54, Y is 58, (25) X is 54, Y is 59, (26) X is 54, Y is 60, (27) X is 55, Y is 56, (28) X is 55, Y is 57, (29) X is 55, Y is 58, (30) X is 55, Y is 59 , (31) X is 55, Y is 60, (32) X is 56, Y is 57, (33) X is 56, Y is 58, (34) X is 56, Y is 59, (35) X is 56, Y is 60, (36) X is 57, Y is 58, (37) X is 57, Y is 59, (38) X is 57, Y is 60, (39) X is 58, Y is 59, (40) X is 58, Y is 60, (41) X is 59, Y is 60, (42) X is 67, Y is 68, (43) X is 67, Y is 69, (44) X is 67, Y is 70, (45) X is 67, Y is 71, (46) X is 67, Y is 72, (47) X is 68, Y is 69, (48) X is 68, Y is 70, (49) X is 68, Y is 71, (50) X is 68, Y is 72, (51) X is 69, Y is 70, (52) X is 69, Y is 71, (53) X is 69, Y is 72, (54) X is 70, Y is 71, (55) X is 70, Y is 72, (56) X is 71, Y is 72 (57) X is 107, Y is 108, (58) X is 107, Y is 109, (59) X is 107, Y is 110, (60) X is 107, Y is 111, (61) X is 108, Y is 109, (62) X is 108, Y is 110,(63) X is 108, Y is 111, (64) X is 109, Y is 110, (65) X is 109, Y is 111, (66) X is 110, Y is 111, (67) X is 114, Y is 115, (68) X is 114, Y is 116, (69) X is 114, Y is 117, (70) X is 114, Y is 118, (71) X is 114, Y is 119, (72) X is 114, Y is 120, (73) X is 114, Y is 121, (74) X is 115, Y is 115, (75) X is 115, Y is 116, (76) X is 115, Y is 117, (77) X is 115, Y is 118, (78) X is 115, Y is 119, (79) X is 115, Y is 120, (80) X is 11 5, Y is 121, (81) X is 116, Y is 115, (82) X is 116, Y is 116, (83) X is 116, Y is 117, (84) X is 116, Y is 118, (85) X is 116, Y is 119, (86) X is 116, Y is 120, (87) X is 116, Y is 121, (88) X is 117, Y is 115, (89) X is 117, Y is 116, (90) X is 117, Y is 117, (91) X is 117, Y is 118, (92) X is 117, Y is 119, (93) X is 117, Y is 120, (94) X is 117, Y is 121, (95) X is 118, Y is 115, (96) X is 118, Y is 116, (97) X is 118, Y is 117,

[0079] (98) X is 118, Y is 118, (99) X is 118, Y is 119, (100) X is 118, Y is 120,

[0080] (101) X is 118, Y is 121, (102) X is 119, Y is 115, (103) X is 119, Y is 116,

[0081] (104) X is 119, Y is 117, (105) X is 119, Y is 118, (106) X is 119, Y is 119,

[0082] (107) X is 119, Y is 120, (108) X is 119, Y is 121, (109) X is 120, Y is 115,

[0083] (110) X is 120, Y is 116, (111) X is 120, Y is 117, (112) X is 120, Y is 118,

[0084] (113) X is 120, Y is 119, (114) X is 120, Y is 120, (115) X is 120, Y is 121,

[0085] (116) X is 128, Y is 129, (117) X is 128, Y is 130, (118) X is 128, Y is 131,

[0086] (119) X is 128, Y is 132, (120) X is 128, Y is 133, (121) X is 128, Y is 134,

[0087] (122) X is 129, Y is 130, (123) X is 129, Y is 131, (124) X is 129, Y is 132,

[0088] (125) X is 129, Y is 133, (126) X is 129, Y is 134, (127) X is 130, Y is 131,

[0089] (128) X is 130, Y is 132, (129) X is 130, Y is 133, (130) X is 130, Y is 134,

[0090] (131) X is 131, Y is 132, (132) X is 131, Y is 133, (133) X is 131, Y is 134,

[0091] (134) X is 132, Y is 133, (135) X is 132, Y is 134, (136) X is 133, Y is 134,

[0092] (137) X is 142, Y is 143, (138) X is 142, Y is 144, (139) X is 142, Y is 145,

[0093] (140) X is 142, Y is 146, (141) X is 142, Y is 147, (142) X is 142, Y is 148,

[0094] (143) X is 142, Y is 149, (144) X is 142, Y is 150, (145) X is 143, Y is 144,

[0095] (146) X is 143, Y is 145, (147) X is 143, Y is 146, (148) X is 143, Y is 147,

[0096] (149) X is 143, Y is 148, (150) X is 143, Y is 149, (151) X is 143, Y is 150,

[0097] (152) X is 144, Y is 145, (153) X is 144, Y is 146, (154) X is 144, Y is 147,

[0098] (155) X is 144, Y is 148, (156) X is 144, Y is 149, (157) X is 144, Y is 150,

[0099] (158) X is 145, Y is 146, (159) X is 145, Y is 147, (160) X is 145, Y is 148,

[0100] (161) X is 145, Y is 149, (162) X is 145, Y is 150, (163) X is 146, Y is 147,

[0101] (164) X is 146, Y is 148, (165) X is 146, Y is 149, (166) X is 146, Y is 150,

[0102] (167) X is 147, Y is 148, (168) X is 147, Y is 149, (169) X is 147, Y is 150,

[0103] (170) X is 148, Y is 149, (171) X is 148, Y is 150, (172) X is 149, Y is 150.

[0104] Preferably, the cpYFP as an optically active polypeptide is located at 114 / 115, 114 / 119, 114 / 120, 117 / 115, 117 / 116, 117 / 118, 117 / 121, 118 / 118, 118 / 119, 119 / 115, 119 / 116, 120 / 115, 120 / 120 of the amino acid sequence of glyoxylate binding protein as shown in SEQ ID NO: 10-22.

[0105] When referring to a certain polypeptide or protein, the term "variant" or "mutant" used in the present invention includes variants having the same function of the polypeptide or protein but different sequences. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include but are not limited to: deletion, insertion and / or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1-10, and most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to its carboxyl terminal and / or amino terminal. Without wishing to be limited by theory, changes in amino acid residues without changing the overall configuration and function of the polypeptide or protein are functional conservative mutations. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the polypeptide or protein is generally not changed. In the art, amino acids with similar properties often refer to amino acid families with similar side chains, which have been clearly defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, succinic acid), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, succinic acid, histidine). For another example, adding one or more amino acids to the amino and / or carboxyl termini generally does not change the function of a polypeptide or protein. Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids. It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed polypeptide or protein, but this does not affect the activity of the target polypeptide or protein. For example, in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions within the protein of the recombinant protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants.These variants may also include polypeptides or proteins that have 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 polypeptide or protein. Exemplary truncated variants of the IcIR protein are fragments of SEQ ID NO: 1 comprising amino acids 76-246, which retain the binding function of the IcIR protein to glyoxylic acid and do not affect the changes in optical properties of the inserted optically active polypeptide in response to glyoxylic acid binding.

[0106] The optical probe of the present invention may include a glyoxylate-sensitive polypeptide with a mutation and an optically active polypeptide with a mutation. A glyoxylate-binding protein variant with a mutation at a site selected from the following sites of SEQ ID NO: 1 or its truncated variant exhibits a different binding activity with glyoxylate: E115, N116, I118, G119, T59, S139, S141. The amino acid mutation includes modification, substitution or deletion of an amino acid.

[0107] Wherein, as an example in the embodiments, in SEQ ID NO: 1 or its truncated variants, I118 mutates to G, Q, T, L, M, V, H, F, S, A, Y or K. In one or more embodiments, G119 mutates to N, V ​​or S. In one or more embodiments, E115 mutates to N, S, P, T or Q. In one or more embodiments, N116 mutates to V, C, K, E, I, T, R or W. In one or more embodiments, T59 mutates to G or N. In one or more embodiments, S139 mutates to N or T. In one or more embodiments, S141 mutates to D, H or G.

[0108] In a preferred embodiment, the mutation of the glyoxylate binding protein variant comprises a mutation selected from any one of the following groups:

[0109] (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E11 5T and N116V, (11) E115N, N116E, (12) E115N, N116I, (13) E115N, N116T, (14) E115N, N116R, (15) E115Q, N116K, (16) E115N, N116W, (17) E115P, N116V, (18 )I118M, E115N, N116V, (19) I118T, E115N, N116V, (20) I118V, E115N, N116V , (21)I118L, E115N, N116V, (22) I118H, E115N, N116V, (23) I118V, E115N, N 116V, (24)I118F, E115N, N116V, (25)I118S, E115N, N116C, (26)I118L, G11 9V, E115N, N116C, (27) I118A, G119S, E115N, N116C, (28) I118Y, E115S, N11 6K, (29)I118K, E115S, N116K, (30) T59G, I118F, E115N, N116V, (31) T59N, I 118F, E115N, N116V, (32) T59G, E115N, N116V, (33) T59N, E115N, N116V, (34 )T59G, I118Y, E115S, N116K, (35) T59N, I118Y, E115S, N116K, (36) T59G, I1 18K, E115S, N116K, (37) T59N, I118K, E115S, N116K, (38) I118F, E115N, N11 6V, S139N, (39) I118F, E115N, N116V, S139T, (40) E115N, N116V, S139N, (41 )E115N, N116V, S139T, (42) I118Y, E115S, N116K, S139N, (43) I118Y, E115S , N116K, S139T, (44)I118K, E115S, N116K, S139N, (45)I118K, E115S, N116K , S139T, (46)I118F, E115N, N116V, S141D, (47)I118F, E115N, N116V, S141G,(48)E115N、N116V、S141D、(49)E115N、N116V、S141G、

[0110] (50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G、(52)I118K、E115S、N116K、S141D、(53)I118K、E115S、N116K、S141G。

[0111] The present invention provides glyoxylate binding protein variants having these mutations and the optical probes described herein comprising such glyoxylate binding protein variants as glyoxylate-sensitive polypeptides. Thus, the present invention includes such optical probes wherein the optically active polypeptide is located at 17 / 18, 17 / 19, 17 / 20, 17 / 21, 18 / 19, 18 / 20, 18 / 21, 19 / 20, 19 / 21, 20 / 21, 28 / 29, 28 / 30, 28 / 31, 28 / 32, 29 / 30, 29 / 31, 29 / 32, 30 / 31, 30 / 32, 31 / 32, 54 / 55, 54 / 56, 54 / 57, 54 / 58, 54 / 59, 54 / 60, 55 / 56, 55 / 57, 55 / 58, 55 / 59, 55 / 60, 56 / 57, 56 / 58, 56 / 59, 56 / 60, 57 7 / 58,57 / 59,57 / 60,58 / 59,58 / 60,59 / 60,67 / 68,67 / 69,67 / 70,67 / 71,67 / 72,68 / 69,68 / 70,68 / 71,68 / 72,69 / 70,69 / 71,69 / 72,70 / 71,70 / 72,71 / 72,1 07 / 108,107 / 109,107 / 110,107 / 111,108 / 109,108 / 110,108 / 111,109 / 110,109 / 111,110 / 111,114 / 115,114 / 116,114 / 117,114 / 118,114 / 119,114 / 120 114 / 121,115 / 115,115 / 116,115 / 117,115 / 118,115 / 119,115 / 120,115 / 121,116 / 115,116 / 116,116 / 117,116 / 118,116 / 119,116 / 120,116 / 121,117 / 115,117 / 116,117 / 117,117 / 118,117 / 119,117 / 120,117 / 121,118 / 115,118 / 116,118 / 117,118 / 118,118 / 119,118 / 120,118 / 121,119 / 115,119 / 116,119 / 117,119 / 118,119 / 119,119 / 120,119 / 121,120 / 115,120 / 116,120 / 117,120 / 118,120 / 119,120 / 120,120 / 121,128 / 129,128 / 130,128 / 131,128 / 132,128 / 133,128 / 134 ,129 / 130,129 / 131,129 / 132,129 / 133,129 / 134,130 / 131,130 / 132,130 / 133,130 / 134,131 / 132,131 / 133,131 / 134,132 / 133,132 / 134,133 / 134,142 / 143,142 / 144,142 / 145,142 / 146,142 / 147,142 / 148,142 / 149,142 / 150,143 / 144,143 / 145,143 / 146,143 / 147,143 / 148,143 / 149,143 / 150,144 / 145,144 / 146,144 / 147,144 / 148,144 / 149,144 / 150,145 / 146,145 / 147,145 / 148 145 / 149,145 / 150,146 / 147,146 / 148,146 / 149,146 / 150,147 / 148,147 / 149,147 / 150,148 / 149,148 / 150,149 / 150, preferably at 119 / 115, the glyoxylate binding protein variant is as SEQ ID NO: 1 or a truncated variant thereof comprising amino acids 76-246 and comprising a mutation selected from any one of the following groups: (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V, (11) E115N, N116E, (12) E115N, N116I, (13) E115N, N116T, (14) E115N, N116R,(15)E115Q、N116K,(16)E115N、N116W,(17)E115P、N116V,(18)I118M、E115N、N116V,(19)I118T、E115N、N116V,(20)I118V、E115N、N116V,(21)I118L、E115N、N116V,(22)I118H、E115N、N116V,(23)I118V、E115N、N116V,(24)I118F、E115N、N116V,(25)I118S、E115N、N116C,(26)I118L、G119V、E115N、N116C,(27)I118A、G119S、E115N、N116C,(28)I118Y、E115S、N116K,(29)I118K、E115S、N116K,(30)T59G、I118F、E115N、N116V,(31)T59N、I118F、E115N、N116V,(32)T59G、E115N、N116V,(33)T59N、E115N、N116V,(34)T59G、I118Y、E115S、N116K,(35)T59N、I118Y、E115S、N116K,(36)T59G、I118K、E115S、N116K,(37)T59N、I118K、E115S、N116K,(38)I118F、E115N、N116V、S139N,(39)I118F、E115N、N116V、S139T,(40)E115N、N116V、S139N,(41)E115N、N116V、S139T,(42)I118Y、E115S、N116K、S139N,(43)I118Y、E115S、N116K、S139T,(44)I118K、E115S、N116K、S139N,(45)I118K、E115S、N116K、S139T,(46)I118F、E115N、N116V、S141D,(47)I118F、E115N、N116V、S141G,(48)E115N、N116V、S141D,(49)E115N、N116V、S141G,(50)I118Y、E115S、N116K、S141D,(51)I118Y、E115S、N116K、S141G,(52)I118K、E115S、N116K、S141D,(53)I118K、E115S、N116K、S141G。,

[0112] In some specific embodiments, the glyoxylate-sensitive polypeptide in the optical probe is as shown in amino acids 76-246 of SEQ ID NO:1, the optically active polypeptide is as shown in SEQ ID NO:2, the optically active polypeptide is located at position 119 / 115 of the glyoxylate-sensitive polypeptide, and the glyoxylate-sensitive polypeptide has a mutation selected from any one of the following: (1) I118G and G119N, (2) I118Q, (3) I118T, (4) I118L, (5) E115N and N116V, (6) E115N and N116C, (7) E115S and N116K, (8) E115S and N116C, (9) E115P and N116C, (10) E115T and N116V, (11) E115N, N116E, (12) E115N, N116I, (13 )E115N, N116T, (14) E115N, N116R, (15) E115Q, N116K, (16) E115N, N116W, (17) E115P, N116V, (18) I118M, E115N, N116V, (19) I118T, E115N , N116V, (20)I118V, E115N, N116V, (21)I118L, E115N, N116V, (22)I118H, E115N, N116V, (23)I118V, E115N, N116V, (24)I118F, E115N, N11 6V, (25)I118S, E115N, N116C, (26)I118L, G119V, E115N, N116C, (27)I118A, G119S, E115N, N116C, (28)I118Y, E115S, N116K, (29)I118K, E 115S, N116K, (30) T59G, I118F, E115N, N116V, (31) T59N, I118F, E115N, N116V, (32) T59G, E115N, N116V, (33) T59N, E115N, N116V, (34) T59 G, I118Y, E115S, N116K, (35) T59N, I118Y, E115S, N116K, (36) T59G, I118K, E115S, N116K, (37) T59N, I118K, E115S, N116K, (38) I118F, E11 5N, N116V, S139N, (39) I118F, E115N, N116V, S139T, (40) E115N, N116V, S139N, (41) E115N, N116V, S139T, (42) I118Y, E115S, N116K, S139N,(43)I118Y, E115S, N116K, S139T, (44) I118K, E115S, N116K, S139N, (45) I118K, E115S, N1 16K, S139T, (46)I118F, E115N, N116V, S141D, (47)I118F, E115N, N116V, S141G, (48)E115 N, N116V, S141D, (49) E115N, N116V, S141G, (50) I118Y, E115S, N116K, S141D, (51) I118Y, E115S, N116K, S141G, (52) I118K, E115S, N116K, S141D, (53) I118K, E115S, N116K, S141G. The structure of the optical probe is:

[0113] (amino acids 76 to 119 of the glyoxylic acid-sensitive polypeptide sequence)-(optically active polypeptide)-(amino acids 115 to 246 of the glyoxylic acid-sensitive polypeptide sequence). Exemplarily, the amino acid sequence of the optical probe shown in item (1) is shown in SEQ ID NO:23; the amino acid sequence of the optical probe shown in item (2) is shown in SEQ ID NO:24; the amino acid sequence of the optical probe shown in item (5) is shown in SEQ ID NO:25; the amino acid sequences of the optical probes shown in items (18) to (29) are shown in SEQ ID NOs:26-37, respectively; the amino acid sequences of the optical probes shown in items (30), (32), (34), (36), (38), (40), (42), (44), (46), (48), (50), and (52) are shown in SEQ ID NOs:38-49, respectively.

[0114] In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NO: 10-49 or a variant thereof. In one or more embodiments, the optical probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NO: 10-49. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NO: 10-49.

[0115] In the context of two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to two or more sequences or subsequences that are identical or wherein a certain percentage of amino acid residues or nucleotides are identical over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) when compared and aligned for maximum correspondence over a comparison window or specified region using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection. For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, as described in Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0116] It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed polypeptide or protein, but this does not affect the activity of the target polypeptide or protein. For example, in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions in the protein of the recombinant protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase.

[0117] The terms "functional variant", "derivative" and "analog" as used herein refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (e.g., IcIR protein or fluorescent protein). Functional variants, derivatives or analogs of the polypeptide or protein (e.g., IcIR protein or fluorescent protein) of the present invention may be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having a substituent group in one or more amino acid residues, or (iii) proteins formed by fusion of a mature protein with another compound (e.g., a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) proteins formed by fusion of an additional amino acid sequence to the protein sequence (e.g., a secretory sequence or a sequence used to purify the protein or a proprotein sequence, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these functional variants, derivatives and analogs belong to the well-known scope of those skilled in the art.

[0118] The difference between the analog and the original polypeptide or protein can be a difference in the amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, and can also be obtained by site-directed mutagenesis or other known molecular biological techniques.

[0119] The analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the glyoxylic acid-sensitive polypeptide of the present invention is not limited to the representative proteins, variants, derivatives and analogs listed above. Modifications (usually without changing the primary structure) include: chemical derivatization forms of proteins in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of proteins or in further processing steps. This modification can be completed by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes proteins that are modified to improve their anti-proteolytic properties or optimize solubility properties.

[0120] The fusion polypeptide of the present invention comprises the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probe. The other polypeptides may be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides that locate the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting. A linker may be provided between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0121] The subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosome and peroxisome, etc. In some embodiments, tags for purification or tags for immunoblotting include 6*histidine (6*His), glutathione S-transferase (GST), Flag.

[0122] The present invention also provides a method for preparing the above-mentioned glyoxylate optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the glyoxylate optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for the expression of the expression vector; and 3) isolating the glyoxylate optical probe.

[0123] The term "nucleic acid" or "nucleotide" used in the present invention can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, the term "variant" used herein 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 may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the protein encoded by it. The nucleic acid of the present invention may contain a nucleotide sequence having a sequence identity of 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% with the nucleic acid sequence. The present invention also relates to nucleic acid fragments hybridized with the above-mentioned sequences. As used herein, a "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0124] The full-length sequence or fragments of the optical probe or fusion protein of the present invention can usually be obtained by PCR amplification, artificial synthesis or recombination. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the nucleotide sequence is greater than 2500 bp, 2 to 6 PCR amplifications are preferably performed, and then the fragments amplified in each time are spliced ​​together in the correct order. The present invention does not specifically limit the PCR amplification procedure and system, and the conventional PCR amplification procedure and system in the art can be used. Recombination methods can also be used to obtain the relevant sequence in large quantities. This is usually cloned into a vector, then transferred into cells, and then separated and purified from the host cells after proliferation by conventional methods to obtain the relevant polypeptide or protein. In addition, artificial synthesis methods can also be used to synthesize the relevant sequence, especially when the fragment length is short. In the present invention, when the nucleotide sequence of the optical probe is less than 2500 bp, an artificial synthesis method can be used for synthesis. The artificial synthesis method is a conventional DNA artificial synthesis method in the art, and there are no other special requirements. Usually, by synthesizing a plurality of small fragments first and then connecting them, a very long sequence can be obtained. At present, the DNA sequence encoding the protein of the present invention (or its functional variant, derivative or analog) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of the present invention by methods such as mutation PCR or chemical synthesis.

[0125] After obtaining the nucleotide sequence encoding the optical probe, the present invention incorporates the nucleotide sequence encoding the optical probe into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" used herein are interchangeable and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors, which can replicate and stably express in the host. An important feature of these recombinant vectors is that they usually contain expression control sequences. The term "expression control sequence" used herein refers to an element that can be operably connected to the target gene to regulate the transcription, translation and expression of the target gene, which can be a replication origin, a promoter, a marker gene or a translation control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc. The selection of the expression control sequence depends on the host cell used. Recombinant vectors applicable to the present invention include but are not limited to bacterial plasmids. In the recombinant expression vector, "operable connection" refers to the connection of the target nucleotide sequence with the regulatory sequence in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the coding sequence of the fusion protein of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; λ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTR of retrovirus and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. In one or more embodiments, the expression vector can use a commercially available pCDF vector without other special requirements. Exemplarily, HindIII and XhoI are used to perform double enzyme digestion on the nucleotide sequence encoding the optical probe and the expression vector, respectively, and then the enzyme digestion products of the two are connected to obtain a recombinant expression vector. The present invention does not specifically limit the specific steps and parameters of enzyme digestion and connection, and the conventional steps and parameters in the art can be used.

[0126] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including a fusion protein. This transfer process can be carried out using conventional techniques known to those skilled in the art such as transformation or transfection. The host cell of the present invention refers to a cell that can receive and accommodate a recombinant DNA molecule, and is the site of recombinant gene amplification. The ideal recipient cell should meet the two conditions of easy acquisition and proliferation. The "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells. Specifically, it can be Escherichia coli, Streptomyces, bacterial cells of Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS, HEK293, HEK293 cells, or animal cells of Bowes melanoma cells, etc., including but not limited to those host cells mentioned above. The host cell is preferably a variety of cells that are conducive to gene product expression or fermentation production, and such cells are well known and commonly used in the art. The exemplary host cell used in the embodiment of the present invention is the Escherichia coli BL21-DE3 strain. Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0127] The method of transferring to the host cell described in the present invention 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 prokaryotic organism such as Escherichia coli, the method is preferably treated with CaCl2 or MgCl2, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0128] After the expression vector is transferred into the host cell, the host cell into which the expression vector is transferred is cultured for amplification and expression to separate and obtain the glyoxylic acid optical probe. The host cell amplification and expression culture can be carried out by conventional methods. Depending on the type of host cell used, the culture medium used in the culture can be various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell.

[0129] In the present invention, the optical probe is expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other properties. The present invention does not specifically limit the method for separating the glyoxylic acid fluorescent protein, and the conventional separation method of the 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 breaking, 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 or more embodiments, the optical probe is separated using affinity chromatography using a His tag.

[0130] The present invention also provides the application of the glyoxylic acid optical probe in real-time positioning, quantitative detection and high-throughput compound screening of glyoxylic acid. In one aspect, the glyoxylic acid optical probe is preferably connected to a signal peptide in different parts of the cell, transferred into the cell, and the real-time positioning of glyoxylic acid is performed by detecting the strength of the fluorescence signal in the cell; the corresponding glyoxylic acid is quantitatively detected by the glyoxylic acid standard drop curve. The glyoxylic acid standard drop curve of the present invention is drawn according to the fluorescence signal of the glyoxylic acid optical probe under different concentrations of glyoxylic acid. The glyoxylic acid optical probe of the present invention is directly transferred into the cell, and in the process of real-time positioning and quantitative detection of glyoxylic acid, no time-consuming sample processing process is required, which is more accurate. When the glyoxylic acid optical probe of the present invention performs high-throughput compound screening, different compounds are added to the cell culture medium, and the change in glyoxylic acid content is measured, thereby screening out compounds that affect the change in glyoxylic acid content. The application of the glyoxylic acid optical probe in real-time positioning, quantitative detection and high-throughput compound screening of glyoxylic acid described in the present invention is non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.

[0131] In this article, concentration, content, percentage and other numerical values ​​can be expressed in the form of a range. It should also be understood that the use of this range format is only for convenience and brevity, and should be flexibly interpreted as including the numerical values ​​explicitly mentioned at the upper and lower limits of the range, and all individual numerical values ​​or sub-ranges included in the range should also be included.

[0132] Example

[0133] The glyoxylic acid optical probe provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0134] I. Experimental Materials and Reagents

[0135] The examples mainly use conventional genetic engineering molecular biology cloning methods and cell culture and imaging methods, etc. These methods are well known to those of ordinary skill in the art, such as: "Molecular Biology Laboratory Reference Manual" by Jane Rosskems et al., "Molecular Cloning Laboratory Guide" (3rd edition, August 2002, Science Press, Beijing) by J. Sambrook and DW Russell, translated by Huang Peitang et al.; "Animal Cell Culture: Basic Technical Guide" (5th edition) by Frécheni et al., translated by Zhang Jingbo, Xu Cunshuan et al.; "Concise Cell Biology Laboratory Guide" by JS Bonifesnon and M. Dassault et al., translated by Zhang Jingbo et al. According to the following examples, it is not difficult for those of ordinary skill in the art to successfully implement the present invention by making slight modifications and changes according to specific circumstances, and these modifications and changes all fall within the scope of the claims of this application.

[0136] The pCDF-cpYFP, ​​pCDF-glyoxylic acid binding protein plasmid used in the embodiment was constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Huada Gene Biotechnology Co., Ltd. The expression plasmids constructed in the embodiment were sequenced, and the sequence determination was completed by Huada Gene and Jie Li Sequencing Company. The Taq DNA polymerase used in each embodiment was purchased from Dongsheng Biology, pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and primeSTAR DNA polymerase was purchased from TaKaRa. The three polymerases were purchased with corresponding polymerase buffer and dNTP. BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI and other restriction endonucleases, T4 ligase, T4 phosphorylase (T4 PNK) were purchased from Fermentas, and corresponding buffers were provided when purchased. Transfection reagent Lip2000 Kit was purchased from Invitrogen. Glyoxylic acid and other compounds were purchased from Aladdin. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin and streptomycin were purchased from Ameresco. 96-well black plate and 384-well fluorescence black plate were purchased from Grenier.

[0137] The DNA purification kit used in the examples was purchased from BBI (Canada), and the common plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and desalting column fillers were both from GE healthcare.

[0138] The main instruments used in the embodiments include: Biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Company), nucleic acid electrophoresis instrument (Shenneng Gaming Company), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell culture incubator (SANYO), inverted fluorescence microscope (Nikon, Japan).

[0139] II. Molecular Biology Methods and Cell Experimental Methods

[0140] II.1 Polymerase chain reaction (PCR):

[0141] 1. Target fragment amplification PCR:

[0142] This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The reaction system of PCR amplification is shown in Table 1, and the amplification program is shown in Table 2.

[0143] Table 1. PCR amplification reaction system

[0144]

[0145] Table 2. PCR amplification program

[0146]

[0147] 2. PCR amplification of long fragments (>2500bp):

[0148] The long fragment amplification used in the present invention is mainly a reverse PCR amplification vector, which is a technique used to obtain site-directed mutations in the following examples. Reverse PCR primers are designed at the mutation site, and the 5' end of one of the primers contains the mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The long fragment amplification PCR reaction system is shown in Table 3, and the amplification program is shown in Table 4 or Table 5.

[0149] Table 3. PCR reaction system for amplification of long fragments (>2500bp)

[0150]

[0151]

[0152] Table 4. PCR amplification program for long fragments (>2500 bp)

[0153]

[0154] Table 5. PCR amplification program for long fragments (>2500 bp)

[0155]

[0156] II.2 Endonuclease digestion reaction:

[0157] The system for double enzyme digestion of plasmid vector is shown in Table 6, wherein n represents the amount of sterile ultrapure water μL that needs to be added to make the system reach the total volume.

[0158] Table 6. Plasmid vector double restriction enzyme digestion system

[0159]

[0160] II.3 DNA fragment 5' end phosphorylation reaction

[0161] The ends of plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Therefore, the 5' end base of the PCR product needs to be subjected to a phosphate group addition reaction. Only DNA molecules with phosphate groups at the end can undergo a ligation reaction. The phosphorylation reaction system is shown in Table 7, where T4 PNK is the abbreviation of T4 polynucleotide kinase, which is used for the addition reaction of the 5' end phosphate group of the DNA molecule.

[0162] Table 7. Phosphorylation reaction system

[0163]

[0164] II.4 Ligation reaction between target fragment and vector

[0165] The connection methods between different fragments and vectors are different. Three connection methods are used in the present invention:

[0166] 1. Blunt-end ligation of blunt-end short fragments and linearized vector

[0167] 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 a recombinant plasmid. The homologous recombination connection system is shown in Table 8.

[0168] Table 8. Blunt-end fragment ligation reaction system

[0169]

[0170] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends

[0171] DNA fragments cut by restriction endonucleases usually produce protruding sticky ends, so they can be connected to vector fragments containing complementary sticky ends to form recombinant plasmids. The ligation reaction system is shown in Table 9, where the mass ratio of PCR product fragments to vector double enzyme digestion products is roughly between 2:1-6:1.

[0172] Table 9. Sticky end ligation reaction system

[0173]

[0174]

[0175] 3. The ligation reaction of the 5' phosphorylated DNA fragment product after site-directed mutation introduced by inverse PCR and self-circularization

[0176] The 5' end phosphorylated DNA fragment was connected to the 3' end and 5' end of the linearized vector by self-circularization ligation reaction to obtain a recombinant plasmid. The self-circularization ligation reaction system is shown in Table 10.

[0177] Table 10. Self-cyclization ligation reaction system

[0178]

[0179] II.5 Preparation and transformation of competent cells

[0180] Preparation of competent cells:

[0181] 1. Pick a single colony (such as Mach1) and inoculate it into 5 mL LB medium and shake it at 37°C overnight.

[0182] 2. Transfer 0.5-1 mL of overnight culture into 50 mL LB medium and culture at 37°C, 220 rpm for 3 to 5 hours until OD600 reaches 0.5.

[0183] 3. Pre-cool the cells in an ice bath for 2 hours.

[0184] Centrifuge at 4000 rpm for 10 min at 4.4°C.

[0185] 5. Discard the supernatant and resuspend the cells with 5 mL of pre-cooled buffer. After the cells are homogenized, add resuspension buffer to a final volume of 50 mL.

[0186] 6. Ice bath for 45 minutes.

[0187] Centrifuge at 4000 rpm for 10 min at 7.4°C and resuspend the bacteria in 5 mL of ice-cold storage buffer.

[0188] 8. Place 100 μL of bacterial solution in each EP tube and freeze at -80°C or in liquid nitrogen.

[0189] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

[0190] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl2 (1 M), 1 mL 10×MgCl2 (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH2O

[0191] Transformation of competent cells:

[0192] 1. Take 100 μL of competent cells and thaw them in an ice bath.

[0193] 2. Add an appropriate volume of ligation product, gently pipette to mix, and place on ice for 30 minutes. Usually the volume of ligation product added is less than 1 / 10 of the volume of competent cells.

[0194] 3. Heat shock the bacterial solution in a 42°C water bath for 90 seconds, then quickly transfer to an ice bath for 5 minutes.

[0195] 4. Add 500 μL LB and culture at 37°C on a constant temperature shaker at 200 rpm for 1 hour.

[0196] 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, retain 200 μL of supernatant, blow the bacteria evenly, and evenly spread them on the surface of an agar plate containing appropriate antibiotics. Place the plate upside down in a 37°C constant temperature incubator overnight.

[0197] II.6 Protein expression, purification and fluorescence detection

[0198] 1. Transform the expression vector (e.g., the pCDF-based glyoxylate optical probe expression vector) into BL21 (DE3) cells, invert and culture overnight, pick clones from the plate into a 250 ml conical flask, place it in a 37°C shaker, culture at 220 rpm until OD = 0.4-0.8, add 1 / 1000 (v / v) IPTG (1 M), and induce expression at 18°C ​​for 24-36 hours.

[0199] 2. After induction, centrifuge at 4000 rpm for 30 minutes to collect the bacteria, add 50 mM phosphate buffer to resuspend the bacterial precipitate, and ultrasonically disrupt the bacteria until they are clear. Centrifuge at 9600 rpm at 4°C for 20 minutes.

[0200] 3. The centrifuged supernatant was purified by a self-assembled nickel column affinity chromatography column to obtain the protein, and the protein after the nickel column affinity chromatography was then passed through a self-assembled desalting column to obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4).

[0201] 4. After the purified protein was identified by SDS-PAGE, the probe was diluted into a protein solution with a final concentration of 0.2-5 μM using an assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4). Glyoxylic acid was prepared into a stock solution with a final concentration of 50 mM using an assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).

[0202] 5. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add glyoxylic acid for titration, and measure the fluorescence intensity of the protein at 528 nm after 420 nm light excitation and 528 nm after 485 nm light excitation. The fluorescence excitation and emission measurement of the sample is completed using a multifunctional fluorescence microplate reader.

[0203] 6. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add glyoxylic acid, and measure the absorption spectrum and fluorescence spectrum of the protein. The absorption spectrum and fluorescence spectrum of the sample are measured by a spectrophotometer and a fluorescence spectrophotometer.

[0204] II.7 Transfection of mammalian cells and fluorescence detection

[0205] 1. The plvx-based glyoxylate optical probe plasmid was transfected into HEK293 cells using the transfection reagent Lipofectamine 2000 (Invitrogen) and cultured in a cell culture incubator at 37°C and 5% CO2. Fluorescence detection was performed after the exogenous gene was fully expressed for 24 to 36 hours.

[0206] 2. After the induction of expression was completed, the attached HEK293 cells were rinsed three times with PBS and placed in HBSS solution for detection by fluorescence microscopy and microplate reader.

[0207] Example 1: Glyoxylate Binding Protein Particle

[0208] The IcIR (76-246) gene in the Thermotoga maritima gene was amplified by PCR. The PCR product was recovered after gel electrophoresis and digested with BamHI and XhoI. The pCDF vector was also double-digested. After ligation with T4 DNA ligase, the product was used to transform DH5α. The transformed DH5α was coated on an LB plate (streptomycin 100ug / mL) and cultured at 37°C overnight. The grown DH5α transformants were subjected to plasmid extraction and PCR identification. The positive plasmid was sequenced correctly for subsequent plasmid construction.

[0209] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0210] In this example, the following sites were selected based on pCDF-IcIR to insert cpYFP to obtain the corresponding pCDF-IcIR-cpYFP plasmids: 17 / 18, 17 / 19, 17 / 20, 17 / 21, 18 / 19, 18 / 20, 18 / 21, 19 / 20, 19 / 21, 20 / 21, 28 / 29, 28 / 30, 28 / 31, 28 / 32, 29 / 30, 29 / 31, 29 / 32, 30 / 31, 30 / 32, 31 / 32, 54 / 55, 54 / 56, 54 / 57, 54 / 58, 54 / 59, 54 / 60, 55 / 56, 55 / 57, 55 / 58, 55 / 59, 55 / 60, 56 / 57, 56 / 58, 56 / 59 ,56 / 60,57 / 58,57 / 59,57 / 60,58 / 59,58 / 60,59 / 60,67 / 68,67 / 69,67 / 70,67 / 71,67 / 72,68 / 69,68 / 70,68 / 71,68 / 72,69 / 70,69 / 71,69 / 72,70 / 71,70 / 72,71 / 72,107 / 108,107 / 109,107 / 110,107 / 111,108 / 109,108 / 110,108 / 111,109 / 110,109 / 111,110 / 111,114 / 115,114 / 116,114 / 117,114 / 118,114 / 119,114 / 120 114 / 121,115 / 115,115 / 116,115 / 117,115 / 118,115 / 119,115 / 120,115 / 121,116 / 115,116 / 116,116 / 117,116 / 118,116 / 119,116 / 120,116 / 121,117 / 115,117 / 116,117 / 117,117 / 118,117 / 119,117 / 120,117 / 121,118 / 115,118 / 116,118 / 117,118 / 118,118 / 119,118 / 120,118 / 121,119 / 115, 119 / 116,119 / 117,119 / 118,119 / 119,119 / 120,119 / 121,120 / 115,120 / 116,120 / 117,120 / 118,120 / 119,120 / 120,120 / 121,128 / 129,128 / 130,128 / 131,128 / 132,128 / 133,128 / 134,129 / 130,129 / 131,129 / 132,129 / 133,129 / 134,130 / 131,130 / 132,130 / 133,130 / 134,131 / 132,131 / 133,131 / 134,132 / 133,132 / 134,133 / 134,142 / 143,142 / 144,142 / 145,142 / 146,142 / 147,142 / 148,142 / 149,142 / 150,143 / 144,143 / 145,143 / 146,143 / 147,143 / 148,143 / 149,143 / 150,144 / 145,144 / 146,144 / 147,144 / 148,144 / 149,144 / 150,145 / 146,145 / 147,145 / 148 145 / 149,145 / 150,146 / 147,146 / 148,146 / 149,146 / 150,147 / 148,147 / 149,147 / 150,148 / 149,148 / 150,149 / 150. ,

[0211] The DNA fragment of cpYFP and the linearized vector pCDF-IcIR were amplified by PCR technology. The 5' and 3' ends of the vector carried sequences that were completely consistent with the two ends of cpYFP (15bp to 20bp). The linearized pCDF-IcIR and cpYFP fragments were homologously recombined under the action of Hieff Clone Enzyme. The product was transformed into DH5α, and the transformed DH5α was spread on LB plates (streptomycin 100ug / mL) and cultured at 37℃ overnight. The positive clones identified by PCR were sequenced after plasmid extraction. Sequencing was completed by Jerry Sequencing Company.

[0212] After sequencing was correct, the recombinant plasmid was transformed into BL21 (DE3) to induce expression and purify the protein. The size of the protein was around 47.2Kda by SDS-PAGE electrophoresis. This size is consistent with the size of the IcIR-cpYFP fusion protein containing the His-tag purification tag expressed by pCDF-IcIR-cpYFP. The results are shown in Figure 1 shown.

[0213] The supernatant of Escherichia coli expressing IcIR-cpYFP fusion protein was used for glyoxylic acid response screening, and the detection signal of the fusion fluorescent protein containing 10mM glyoxylic acid was divided by the detection signal of the fusion fluorescent protein containing glyoxylic acid. The results are shown in Table 1. The detection results show that there are 13 optical probes whose fold change in response to glyoxylic acid is less than 0.8 or greater than 1.2, at sites 114 / 115, 114 / 119, 114 / 120, 117 / 115, 117 / 116, 117 / 118, 117 / 121, 118 / 118, 118 / 119, 119 / 115, 119 / 116, 120 / 115, and 120 / 120, and the sequences of these probes are shown in SEQ ID NOs: 10-22, respectively.

[0214] Table 1

[0215]

[0216] Example 3: Expression and detection of cpGFP optical probes at different insertion sites

[0217] According to the method in Example 2, cpYFP was replaced with cpGFP to construct a glyoxylate green fluorescent protein fluorescent probe. As shown in Table 3, the detection results show that the optical probes with a change factor of less than 0.8 or more than 1.2 in response to glyoxylate are inserted at sites 117 / 120, 119 / 115, 119 / 116, 120 / 115 and 120 / 117 or the corresponding amino acid sites of their family proteins.

[0218] Table 2

[0219]

[0220] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0221] According to the method in Example 2, cpYFP was replaced with cpBFP to construct a glyoxylate blue fluorescent protein fluorescent probe. As shown in Table 3, the detection results show that the optical probes with a change factor of response to glyoxylate lower than 0.8 or higher than 1.2 are inserted at sites 114 / 117, 114 / 118, 119 / 119 and 119 / 120 or the corresponding amino acid sites of their family proteins.

[0222] Table 3

[0223]

[0224] Example 5: Expression and detection of cpmApple optical probes at different insertion sites

[0225] According to the method in Example 2, cpYFP was replaced with cpmApple to construct a glyoxylate red fluorescent protein fluorescent probe. As shown in Table 4, the detection results show that the optical probes with a change factor of less than 0.8 or more than 1.2 in response to glyoxylate are inserted at sites 117 / 115, 118 / 119, 118 / 120 and 120 / 121 or the corresponding amino acid sites of their family proteins.

[0226] Table 4

[0227]

[0228] Example 6: Expression and detection of mutant cpYFP optical probes

[0229] For the optical probes obtained in Example 2 whose change fold in response to glyoxylic acid is less than 0.8 or greater than 1.2, that is, based on the 13 optical probes inserted at sites 114 / 115, 114 / 119, 114 / 120, 117 / 115, 117 / 116, 117 / 118, 117 / 121, 118 / 118, 118 / 119, 119 / 115, 119 / 116, 120 / 115 and 120 / 120, the probes are linearized by inverse PCR, the sequences of the mutation sites are introduced into the primers, and the obtained PCR products are homologously recombined under the action of Hieff Clone Enzyme to establish a mutation library. The mutant library recombinant plasmid was transformed into BL21 (DE3) to induce expression, and the supernatant of E. coli expressing the probe protein was used for glyoxylic acid response screening, and the detection signal of the fusion fluorescent protein containing 10mM glyoxylic acid was divided by the detection signal of the fusion fluorescent protein without glyoxylic acid. The results are shown in Table 5. The detection results show that the optical probes with a response to glyoxylic acid greater than or equal to 3 times are as follows.

[0230] Table 5

[0231] sequence Insertion site mutation <![CDATA[R 420 / 485 ]]> SEQ ID NO:23 119 / 115 I118G / G119N 3.03 SEQ ID NO:24 119 / 115 I118Q 3.02 119 / 115 I118T 3.07 119 / 115 I118L 3.11 SEQ ID NO:25 119 / 115 E115N / N116V 3.27 119 / 115 E115N / N116C 3.24 119 / 115 E115S / N116K 3.46 119 / 115 E115S / N116C 3.04 119 / 115 E115P / N116C 3.18 119 / 115 E115T / N116V 3.12 119 / 115 E115N / N116E 3.78 119 / 115 E115N / N116I 3.11 119 / 115 E115N / N116T 3.02 119 / 115 E115N / N116R 3.17 119 / 115 E115Q / N116K 3.21 119 / 115 E115N / N116W 3.06 119 / 115 E115P / N116V 3.12 SEQ ID NO:26 119 / 115 I118M / E115N / N116V 5.74 SEQ ID NO:27 119 / 115 I118T / E115N / N116V 5.70 SEQ ID NO:28 119 / 115 I118V / E115N / N116V 5.80 SEQ ID NO:29 119 / 115 I118L / E115N / N116V 5.26 SEQ ID NO:30 119 / 115 I118H / E115N / N116V 5.01 SEQ ID NO:31 119 / 115 I118V / E115N / N116V 4.98 SEQ ID NO:32 119 / 115 I118F / E115N / N116V 5.00 SEQ ID NO:33 119 / 115 I118S / E115N / N116C 5.24 SEQ ID NO:34 119 / 115 I118L / G119V / E115N / N116C 4.97 SEQ ID NO:35 119 / 115 I118A / G119S / E115N / N116C 4.98 SEQ ID NO:36 119 / 115 I118Y / E115S / N116K 5.34 SEQ ID NO:37 119 / 115 I118K / E115S / N116K 8.6

[0232] On the basis of 118F / 115N / 116V, 115N / 116V, 118Y / 115S / 116K, I118K / E115S / N116K, the amino acids at positions 59, 139, and 141 of the IcIR protein were mutated to improve the specificity of the glyoxylic acid optical probe, and then the probe was linearized by inverse PCR, and the sequence of the mutation site was introduced into the primer. The site-directed mutation plasmid was transformed into BL21 (DE3) to induce expression, and the supernatant of Escherichia coli expressing the probe protein was used for glyoxylic acid response screening, and the detection signal of the fusion fluorescent protein containing 10mM glyoxylic acid and 10mM pyruvic acid was divided by the detection signal of the fusion fluorescent protein without glyoxylic acid and pyruvic acid. The results are shown in Table 6, and the detection results show that the optical probe with significantly improved glyoxylic acid specificity is as follows.

[0233] Table 6

[0234] sequence Mutation site mutation <![CDATA[R 420 / 485 ]]> SEQ ID NO:38 59 IcIR-T59G / I118F / E115N / N116V 3.75 59 IcIR-T59N / I118F / E115N / N116V 3.77 SEQ ID NO:39 59 IcIR-T59G / E115N / N116V 4.19 59 IcIR-T59N / E115N / N116V 3.36 SEQ ID NO:40 59 IcIR-T59G / I118Y / E115S / N116K 3.82 59 IcIR-T59N / I118Y / E115S / N116K 3.64 SEQ ID NO:41 59 IcIR-T59G / I118K / E115S / N116K 8.2 59 IcIR-T59N / I118K / E115S / N116K 7.6 SEQ ID NO:42 139 IcIR-I118F / E115N / N116V / S139N 3.69 139 IcIR-I118F / E115N / N116V / S139T 3.77 SEQ ID NO:43 139 IcIR-E115N / N116V / S139N 3.36 139 IcIR-E115N / N116V / S139T 2.05 SEQ ID NO:44 139 IcIR-I118Y / E115S / N116K / S139N 4.36 139 IcIR-I118Y / E115S / N116K / S139T 2.64 SEQ ID NO:45 139 IcIR-I118K / E115S / N116K / S139N 9.6 139 IcIR-I118K / E115S / N116K / S139T 6 SEQ ID NO:46 141 IcIR-I118F / E115N / N116V / S141D 2.97 141 IcIR-I118F / E115N / N116V / S141G 2.01 SEQ ID NO:47 141 IcIR-E115N / N116V / S141D 1.5 141 IcIR-E115N / N116V / S141G 2.87 SEQ ID NO:48 141 IcIR-I118Y / E115S / N116K / S141D 1.93 141 IcIR-I118Y / E115S / N116K / S141G 2.52 SEQ ID NO:49 141 IcIR-I118K / E115S / N116K / S141D 4.2 141 IcIR-I118K / E115S / N116K / S141G 4.42

[0235] Example 7. Performance of Optical Probe Mutants

[0236] Some of the glyoxylic acid optical probes in Table 5 and all of the glyoxylic acid optical probes in Table 6 described in Example 6 were subjected to glyoxylic acid detection at a concentration gradient (0-10 mM). After the probes were treated for 10 minutes, the changes in the fluorescence intensity at 420 nm excitation and 528 nm emission and the fluorescence intensity ratio at 485 nm excitation and 528 nm emission were detected. The probe titration results are shown in Figure 2. Figure 2 As shown, the results indicate that different mutants have different affinities for glyoxylate.

[0237] The glyoxylate probes in Tables 5 and 6 were specifically tested for reactivity with glyoxylate structural analogs, pyruvic acid, malic acid, oxaloacetic acid, α-ketoglutaric acid, fumaric acid, succinic acid, citric acid and isocitric acid, and the results showed that they had good specificity. Figure 3 shown.

[0238] Example 8: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

[0239] In this example, different localization signal peptides were used to fuse with the optical probe IcIR-T59G / E115N / N116C to localize the optical probe to different organelles. HEK293 cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, then rinsed with PBS, placed in HBSS solution, and fluorescence detection was performed under the FITC channel using an inverted fluorescence microscope. The results are shown in Figure 2. Figure 4As shown. The glyoxylic acid optical probe can be localized to the cytoplasm, mitochondria, nucleus, nuclear exclusion, outer membrane, and endoplasmic reticulum by fusing with different specific localization signal peptides. Different subcellular structures show fluorescence, and the distribution and intensity of fluorescence are different.

[0240] After transfection of HEK293 cells with the optical probe plasmid expressed in the cytoplasm for 36 hours, the cells were washed with PBS and placed in HBSS solution. The changes in the fluorescence intensity at 528 nm emission under 420 nm excitation and the fluorescence intensity ratio at 528 nm emission under 485 nm excitation were detected over a 30-min period. The results are shown in Figure 2. Figure 5 As shown. 10 mM glyoxylic acid was added and the test was continued for 30 minutes. The 485 / 420 of the sample with glyoxylic acid added gradually increased, up to 8 times the initial value, while the 485 / 420 of the control group without glyoxylic acid added remained basically unchanged.

[0241] Example 9: High-throughput compound screening based on optical probes in living cells

[0242] In this example, we used HEK293 cells expressing IcIR-T59G / E115N / N116C in the cytoplasm for high-throughput compound screening.

[0243] The transfected HEK293 cells were rinsed with PBS, placed in HBSS solution (without glyoxylic acid) for 1 hour, and then treated with 10 μM of the compound for 1 hour. Glyoxylic acid was added dropwise to each sample. The fluorescence intensity at 420 nm excitation and 528 nm emission and the ratio of the fluorescence intensity at 485 nm excitation and 528 nm emission were recorded using an ELISA reader. The sample not treated with any compound was used as a control for standardization. The results are shown in Figure 6 As shown. Among the 2000 compounds used, most of them have little effect on the entry of glyoxylic acid into cells. There are 8 compounds that can increase the cell's ability to take up glyoxylic acid, and another 7 compounds that can significantly reduce the cell's uptake of glyoxylic acid.

[0244] Example 10: Quantitative detection of glyoxylic acid in blood using optical probe

[0245] In this experiment, glyoxylate was analyzed in mouse and human blood supernatants using purified IcIR-T59G / E115N / N116C.

[0246] After IcIR-T59G / E115N / N116C was mixed with diluted blood supernatant for 10 minutes, the fluorescence intensity at 420 nm excitation and 528 nm emission and the ratio of the fluorescence intensity at 485 nm excitation and 528 nm emission were detected using an ELISA reader. Figure 7As shown, the glyoxylic acid content in mouse blood is about 30 μM, and the glyoxylic acid content in human blood is about 25 μM.

[0247] It can be seen from the above examples that the glyoxylic acid optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature, has large dynamic fluorescence changes, has good specificity, and can be expressed in cells through genetic manipulation methods, and can locate and quantitatively detect glyoxylic acid in real time inside and outside cells; and can perform high-throughput compound screening.

[0248] Other Implementations

[0249] This specification describes many embodiments. However, it should be understood that various modifications that those skilled in the art would learn from reading this specification without departing from the concept and scope of the present invention should also be included in the scope of the appended claims.

Claims

1. A glyoxylate binding protein variant, which: (1) having a sequence as shown in SEQ ID NO: 1 and having a mutation at one, two, three, four, five, six or seven positions selected from the group consisting of: E115, N116, I118, G119, T59, S139, S141, wherein the mutation comprises a modification, substitution or deletion of an amino acid, (2) is a truncated variant of (1) having amino acids 76 to 246, or (3) a sequence having at least 70% sequence identity with the sequence of (1) or (2) and having the mutation described in (1) and retaining the ability to bind to glyoxylate, Preferably, the mutation site is selected from 1, 2, 3 or 4 of any one of the following groups: E115, N116, I118, G119. Optionally, the mutation further comprises a mutation at any one of the following sites: T59, S139, S141, More preferably, the mutation includes a mutation at a site selected from any one of the following groups: (1) I118 and G119, (2) I118, (3) E115, N116, (4) I118, E115, N116; (5) T59, I118, E115, N116, (6) T59, E115, N116, (7) I118, E115, N116, S139, (8) E115, N116, S139, (9) I118, E115, N116, S141, (10) E115, N116, S141, (11) T133 and I118, (12) I118 and S213, (13) I118 and S215, Further preferably, I118 mutates to G, Q, T, L, M, V, H, F, S, A, Y or K; G119 mutates to N, V ​​or S; E115 mutates to N, S, P, T or Q; N116 mutates to V, C, K, E, I, T, R or W; T59 mutates to G or N; S139 mutates to N or T; S141 mutates to D, H or G.

2. An optical probe comprising a glyoxylic acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the glyoxylic acid-sensitive polypeptide, the glyoxylic acid-sensitive polypeptide (1) having the sequence shown in SEQ ID NO:1 or a truncate thereof having amino acids 76-246, or a sequence having at least 70% sequence identity therewith and retaining sensitivity to glyoxylic acid, or (2) being a glyoxylic acid-binding protein variant as described in claim 1, wherein the optically active polypeptide is a fluorescent protein or a functional variant thereof.

3. The optical probe according to claim 2, wherein: The optically active peptides are located between residues 17-20, 28-31, 54-59, 67-71, 107-110, 114-120, 128-133, 142-149 of the glyoxylate-sensitive peptide. Preferably, the optically active polypeptide is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide: 17 / 18, 17 / 19, 17 / 20, 17 / 21, 18 / 19, 18 / 20, 18 / 21, 19 / 20, 19 / 21, 20 / 21, 28 / 29, 28 / 30, 28 / 31, 28 / 32, 29 / 30, 29 / 31, 29 / 32, 30 / 31, 30 / 32, 31 / 32, 54 / 55, 54 / 56, 54 / 57, 54 / 58, 54 / 59, 54 / 60, 55 / 56, 55 / 57, 55 / 58, 55 / 59, 55 / 60, 56 / 57, 56 / 58, 56 / 59, 56 / 60, 57 / 5 8,57 / 59,57 / 60,58 / 59,58 / 60,59 / 60,67 / 68,67 / 69,67 / 70,67 / 71,67 / 72,68 / 69,68 / 70,68 / 71,68 / 72,69 / 70,69 / 71,69 / 72,70 / 71,70 / 72,71 / 72,10 7 / 108,107 / 109,107 / 110,107 / 111,108 / 109,108 / 110,108 / 111,109 / 110,109 / 111,110 / 111,114 / 115,114 / 116,114 / 117,114 / 118,114 / 119,114 / 120 114 / 121,115 / 115,115 / 116,115 / 117,115 / 118,115 / 119,115 / 120,115 / 121,116 / 115,116 / 116,116 / 117,116 / 118,116 / 119,116 / 120,116 / 121,117 / 115,117 / 116,117 / 117,117 / 118,117 / 119,117 / 120,117 / 121,118 / 115,118 / 116,118 / 117,118 / 118,118 / 119,118 / 120,118 / 121,119 / 115,119 / 116, 119 / 117,119 / 118,119 / 119,119 / 120,119 / 121,120 / 115,120 / 116,120 / 117,120 / 118,120 / 119,120 / 120,120 / 121,128 / 129,128 / 130,128 / 131,128 / 132,128 / 133,128 / 134,129 / 130,129 / 131,129 / 132,129 / 133,129 / 134,130 / 131,130 / 132,130 / 133,130 / 134,131 / 132,131 / 133,131 / 134,132 / 133,132 / 134,133 / 134,142 / 143,142 / 144,142 / 145,142 / 146,142 / 147,142 / 148,142 / 149,142 / 150,143 / 144,143 / 145,143 / 146,143 / 147,143 / 148,143 / 149,143 / 150,144 / 145,144 / 146,144 / 147,144 / 148,144 / 149,144 / 150,145 / 146,145 / 147,145 / 148145 / 149,145 / 150,146 / 147,146 / 148,146 / 149,146 / 150,147 / 148,147 / 149,147 / 150,148 / 149,148 / 150,149 / 150，, More preferably, the optically active polypeptide is located at any one or more of the following positions of the glyoxylate-sensitive polypeptide selected from: 114 / 115, 114 / 119, 114 / 120, 117 / 115, 117 / 116, 117 / 118, 117 / 121, 118 / 118, 118 / 119, 119 / 115, 119 / 116, 120 / 115, 120 / 120.

4. The optical probe according to claim 2, characterized in that The fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, apple red fluorescent protein, Preferably, the fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2-9.

5. A nucleic acid molecule comprising: (a) a coding sequence of the glyoxylate binding protein variant according to claim 1 or the optical probe according to any one of claims 2 to 4, (b) The complementary sequence of (a).

6. A nucleic acid construct comprising the nucleic acid molecule according to claim 5, Preferably, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

7. A host cell, wherein: (1) expressing the glyoxylate binding protein variant of claim 1 or the optical probe of any one of claims 2 to 4; (2) comprising the nucleic acid molecule of claim 5; or (3) comprising the nucleic acid construct according to claim 6.

8. A detection kit comprising: (1) The optical probe according to any one of claims 2 to 4, (2) The nucleic acid sequence according to claim 5, (3) The nucleic acid construct according to claim 6, or (4) The host cell according to claim 7, The detection kit optionally further comprises other reagents required for detecting glyoxylic acid using an optical probe. Preferably, the detection kit further comprises one or more reagents selected from the following: buffer, culture medium, glyoxylic acid standard.

9. A method for preparing the optical probe according to any one of claims 2 to 4, comprising: Providing the host cell of claim 7, culturing the host cell under conditions where the optical probe is expressed, and isolating the optical probe.

10. Use of the optical probe according to any one of claims 2 to 4, the nucleic acid sequence according to claim 5, the nucleic acid construct according to claim 6 or the host cell according to claim 7 in detecting glyoxylic acid in a sample, screening compounds, and localizing glyoxylic acid inside or outside a cell. Preferably, Detecting glyoxylic acid in a sample comprises the steps of contacting the optical probe or the host cell with the sample, detecting an optical change of the optically active polypeptide, and detecting glyoxylic acid in the sample according to the optical change of the optically active polypeptide. The screening compound comprises the steps of: contacting the optical probe or host cell with a candidate compound in a system containing glyoxylic acid, detecting an optical change of an optically active polypeptide, and screening the compound according to the optical change of the optically active polypeptide; preferably, the screening compound comprises the steps of: contacting the host cell with a candidate compound in a system containing glyoxylic acid, and the optical change of the optically active polypeptide indicates whether the candidate compound regulates the uptake of glyoxylic acid by the cell, The intracellular / extracellular localization of glyoxylic acid comprises the steps of: contacting a system containing glyoxylic acid with the optical probe or the host cell, and detecting an optical change of an optically active polypeptide. More preferably, the system is a solution system, a cell system or a subcellular system.

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