Optical probes for sialic acids and methods of making and using the same

Abstract

The present application relates to a kind of sialic acid optical probe and application. Specifically, it relates to sialic acid binding protein variant, sialic acid optical probe and its application in screening compound, localization sialic acid cell.

Description

Technical Field

[0001] This invention relates to the field of optical probe technology, and in particular to a sialic acid optical probe, its preparation method, and its application. Background Technology

[0002] Sialic acid is a vital carbohydrate compound in human physiology, widely involved in key biological processes such as cell recognition, immune regulation, and neural development. As an important component of glycoproteins and glycolipids, sialic acid forms a negatively charged barrier on the cell membrane surface through its unique carboxyl group structure, regulating intercellular interactions and signal transduction. Particularly in the nervous system, sialic acid plays an irreplaceable role in synaptic plasticity and brain development, and its metabolic abnormalities are closely related to various neurological diseases.

[0003] Sialic acid exhibits a complex and highly tissue-specific metabolic network. In the liver and kidneys, sialic acid is primarily metabolized via the N-acetylneuraminic acid aldolase (NPL) pathway, producing N-acetylmannosamine and pyruvate, the latter of which enters the tricarboxylic acid cycle for energy. In brain tissue, however, sialic acid is more readily integrated into gangliosides via glycosylation, participating in myelination and neurotransmitter transport. Recent studies have revealed that sialic acid can also be recycled as a precursor via the CMP-sialic acid synthase (CMAS) pathway, a process crucial for maintaining mucosal immune barrier function. Notably, sialic acid metabolism and inflammatory responses are bidirectionally regulated: on the one hand, the inflammatory factor TNF-α can upregulate sialyltransferase expression; on the other hand, sialylation modification can regulate the immune response by inhibiting complement activation.

[0004] Clinical studies have shown that sialic acid metabolism disorders are associated with various pathological conditions. Congenital sialic acid storage disorders, due to lysosomal sialic acidase deficiency, lead to abnormal accumulation of sialic acid in tissues, causing intellectual disability and skeletal deformities. In the tumor microenvironment, the overexpression of sialic acid on the surface of tumor cells forms a "sialic acid barrier," enabling immune evasion by inhibiting NK cell activity. In Alzheimer's disease patients, the level of free sialic acid in the cerebrospinal fluid is significantly elevated, and its altered glycosylation pattern may affect the clearance efficiency of β-amyloid protein. More importantly, some pathogens, such as influenza viruses, infect by hijacking host sialic acid receptors, a mechanism that provides new targets for antiviral drug development.

[0005] Currently, the most commonly used methods for detecting sialic acid include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS) (Fang M et al. Biomedical Chromatography, 2020, 34(2), 145-147), and enzyme-linked immunosorbent assay (ELISA) (Wu L et al. Food Chemistry, 2024, 461(31), 1455-1458). HPLC and LC-MS / MS can efficiently and accurately determine changes in sialic acid content in samples, but they involve the extraction of intracellular metabolites, making the experimental steps cumbersome and time-consuming. ELISA cannot monitor changes in sialic acid in living cells in real time; therefore, there is an urgent need to develop genetically encoded fluorescent probes that can monitor the dynamic changes of sialic acid in living cells in situ. Summary of the Invention

[0006] The purpose of this invention is to provide a probe and method for real-time, high-throughput, and quantitative detection of sialic acid inside and outside cells.

[0007] This invention provides a variant of a sialic acid-binding protein, which

[0008] (a) Having the sequence shown in SEQ ID NO:1 or a functional fragment thereof with amino acids truncated from positions 1-96 and having mutations at one or more of the following sites: S115, H116, N164, Q208, A209, N214, S217, Q220, the sites being numbered according to the sequence in SEQ ID NO:1.

[0009] (b) The sequence has at least 70% sequence identity with (a) and has the mutation described in (a) and retains the ability to bind sialic acid.

[0010] In one or more embodiments, the mutation sites are selected from any one of the following groups: (1) S115 and H116, (2) Q208 and A209; (3) Q208, A209 and N164, (4) Q208, A209 and N214, (5) Q208, A209 and S217, (6) Q208, A209 and Q220.

[0011] In one or more embodiments, S115 is mutated to E, L, A, V, R, G, or W.

[0012] In one or more embodiments, H116 is mutated to A, D, F, G, H, V, or W.

[0013] In one or more implementations, N164 mutates to I.

[0014] In one or more implementations, Q208 is mutated to R, V, or K.

[0015] In one or more implementations, A209 is mutated to T, V, or S.

[0016] In one or more implementations, N214 mutates to H.

[0017] In one or more implementations, S217 mutates to T.

[0018] In one or more implementations, Q220 is mutated to D, E, G, N, or S.

[0019] In one or more embodiments, the mutation is selected from any one of the following groups: (1) S115E and H116F, (2) S115L and H116G, (3) S115A and H116F, (4) S115V and H116L, (5) S115R and H116F, (6) S115E and H116W, (7) S115A and H116A, (8) S115G and H116H, (9) S115G and H116L, (10) S115W and H116V, (11) S115A and H116D, (12) Q208R and A209T. (13) Q208V and A209V, (14) Q208K and A209S, (15) Q208R, A209T and N164I, (16) Q208R, A209T and N214H, (17) Q208R, A209T and S217T, (18) Q208R, A209T and Q220D, (19) Q208R, A209T and Q220E, (20) Q208R, A209T and Q220G, (21) Q208R, A209T and Q220N, (22) Q208R, A209T and Q220S.

[0020] This invention provides a sialic acid optical probe comprising a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the sialic acid-sensitive polypeptide. The sialic acid-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide or a functional variant thereof.

[0021] In one or more embodiments, the sialic acid optical probe includes a sialic acid-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the sialic acid-sensitive polypeptide B, and the sialic acid-sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of type B1-A-B2.

[0022] In one or more embodiments, the sialic acid-sensitive polypeptide includes a sialic acid-binding protein or a functional fragment thereof.

[0023] In one or more embodiments, the sialic acid-sensitive polypeptide is derived from Shigella dysenteriae.

[0024] In one or more embodiments, the sialic acid-sensitive polypeptide is the sialic acid-binding protein NanR.

[0025] In one or more embodiments, the sialic acid-sensitive polypeptide has the sequence shown in SEQ ID NO:1 or a functional fragment thereof after removing amino acids 1-96, or a sequence that has at least 70% sequence identity with it and retains sialic acid binding activity.

[0026] In one or more embodiments, the sialic acid-sensitive polypeptide is a variant of the sialic acid-binding protein described in any embodiment herein.

[0027] In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof, wherein the functional variant of the fluorescent protein has a mutation within 3 amino acids at the linker to the optically active polypeptide.

[0028] In one or more embodiments, the fluorescent protein is selected from yellow fluorescent protein (such as cpYFP shown in SEQ ID NO:2), orange fluorescent protein, red fluorescent protein, green fluorescent protein (such as cpGFP shown in SEQ ID NO:3), blue fluorescent protein (such as cpBFP shown in SEQ ID NO:4), or apple red fluorescent protein (such as cpmApple shown in SEQ ID NO:5). Preferably, the fluorescent protein is cpYFP.

[0029] In one or more embodiments, the functional variant of the fluorescent protein has a mutation at amino acid positions 1-3 or 244-246 of the sequence shown in SEQ ID NO:2, preferably at amino acid position 1 or 246.

[0030] In one or more embodiments, the functional variant of the fluorescent protein is mutated at position 1 of the sequence shown in SEQ ID NO:2 to V, I, S, H or C, and / or at position 246 to S or C.

[0031] In one or more embodiments, the mutation of the functional variant of the fluorescent protein is selected from any one of the following groups: (1) Y1V, (2) Y1I, (3) Y1S, (4) Y1H, (5) Y1C, (6) N246S, (7) N246C.

[0032] In one or more embodiments, the optical probe may or may not contain one or more adapters that side-connect the optically active polypeptide.

[0033] In one or more embodiments, the linker can be any amino acid sequence of any length.

[0034] In one or more embodiments, the optically active polypeptide flanking group comprises a linker of no more than 5 amino acids.

[0035] In one or more embodiments, the linker on the flanking side of the optically active peptide comprises amino acid Y.

[0036] In one or more embodiments, the connector Y is located at the N-terminus and / or C-terminus of the optically active peptide.

[0037] In one or more embodiments, the optical probes are as follows: a first portion B1 and Y of the sialic acid-sensitive polypeptide, an optically active polypeptide A, and a second portion B2 of the sialic acid-sensitive polypeptide.

[0038] In one or more embodiments, the optical probe of the present invention further includes a positioning sequence for positioning the probe to a specific organelle, such as a cell.

[0039] In one or more embodiments, the optical probe provided by the present invention comprises a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located at one or more sites of the sialic acid-sensitive polypeptide selected from the following: 113 / 114, 113 / 115, 113 / 116, 113 / 117, 113 / 118, 113 / 119, 113 / 120, 113 / 121, 113 / 122, 114 / 114, 114 / 115, 114 / 116, 114 / 117, 114 / 118, 114 / 119, 114 / 120, 114 / 121, 114 / 122, 115 / 114, 115 / 115, 115 / 116, 115 / 117, 11 5 / 118, 115 / 119, 115 / 120, 115 / 121, 115 / 122, 116 / 114, 116 / 116, 116 / 118, 116 / 120, 116 / 122, 116 / 124, 116 / 126, 116 / 128, 116 / 130, 117 / 114, 117 / 115 117 / 116, 117 / 117, 117 / 118, 117 / 119, 117 / 120, 117 / 121, 117 / 122, 118 / 114, 118 / 115, 118 / 116, 118 / 117, 118 / 118, 118 / 119, 118 / 120, 118 / 121, 118 / 122, 119 / 114, 119 / 115, 119 / 116, 119 / 117, 119 / 118, 119 / 119, 119 / 120, 119 / 121, 119 / 122, 120 / 114, 120 / 115, 120 / 116, 120 / 117, 120 / 118, 120 / 119, 1 20 / 120, 120 / 121, 120 / 122, 139 / 140, 139 / 141, 139 / 142, 139 / 143, 139 / 144, 139 / 145, 140 / 141, 140 / 142, 140 / 143, 140 / 144, 140 / 145, 141 / 142, 141 / 14 3, 141 / 144, 141 / 145, 142 / 143, 142 / 144, 142 / 145, 143 / 144, 143 / 145, 144 / 145, 159 / 160, 159 / 161, 159 / 162, 159 / 163, 159 / 164, 159 / 165, 160 / 161, 160 / 162, 160 / 163, 160 / 164, 160 / 165, 161 / 162, 161 / 163, 161 / 164, 161 / 165, 162 / 163, 162 / 164, 162 / 165, 163 / 164, 163 / 165, 164 / 165, 178 / 179, 178 / 180,178 / 181、178 / 182、178 / 183、178 / 184、178 / 185、178 / 186、179 / 180、179 / 181、179 / 182、179 / 183、179 / 184、179 / 185、179 / 186、180 / 181、180 / 182、180 / 183、180 / 184、180 / 185、180 / 186、181 / 182、181 / 183、181 / 184、181 / 185、181 / 186、182 / 183、182 / 184、182 / 185、182 / 186、183 / 184、183 / 185、183 / 186、184 / 185、184 / 186、185 / 186、200 / 201、200 / 202、200 / 203、200 / 204、200 / 205、200 / 206、200 / 207、200 / 208、200 / 209、200 / 210、200 / 211、200 / 212、201 / 201、201 / 202、201 / 203、201 / 204、201 / 205、201 / 206、201 / 207、201 / 208、201 / 209、201 / 210、201 / 211、201 / 212、202 / 201、202 / 202、202 / 203、202 / 204、202 / 205、202 / 206、202 / 207、202 / 208、202 / 209、202 / 210、202 / 211、202 / 212、203 / 201、203 / 202、203 / 203、203 / 204、203 / 205、203 / 206、203 / 207、203 / 208、203 / 209、203 / 210、203 / 211、203 / 212、204 / 201、204 / 202、204 / 203、204 / 204、204 / 205、204 / 206、204 / 207、204 / 208、204 / 209、204 / 210、204 / 211、204 / 212、205 / 201、205 / 202、205 / 203、205 / 204、205 / 205、205 / 206、205 / 207、205 / 208、205 / 209、205 / 210、205 / 211、205 / 212、206 / 201、206 / 202、206 / 203、206 / 204、206 / 205、206 / 206、206 / 207、206 / 208、206 / 209、206 / 210、206 / 211、206 / 212、207 / 201、207 / 202、207 / 203、207 / 204、207 / 205、207 / 206、207 / 207、207 / 208, 207 / 209, 207 / 210, 207 / 211, 207 / 212, 208 / 201, 208 / 202, 208 / 203, 208 / 204, 208 / 205, 208 / 206, 208 / 207, 208 / 208, 208 / 209, 208 / 210, 208 / 211, 208 / 212, 209 / 201, 209 / 202, 209 / 203, 209 / 204, 209 / 205, 209 / 206, 209 / 207, 209 / 208, 209 / 209, 209 / 210, 209 / 211, 209 / 212, 210 / 201, 210 / 202, 210 / 203, 210 / 204, 210 / 205, 210 / 206, 210 / 207, 210 / 208, 2 10 / 209, 210 / 210, 210 / 211, 210 / 212, 211 / 201, 211 / 202, 211 / 203, 211 / 204, 211 / 205, 211 / 206, 211 / 207, 211 / 208, 211 / 209, 211 / 210, 211 / 211, 211 / 212, 228 / 229, 228 / 230, 228 / 2 31, 228 / 232, 228 / 233, 228 / 234, 229 / 230, 229 / 231, 229 / 232, 229 / 233, 229 / 234, 230 / 231, 230 / 232, 230 / 233, 230 / 234, 231 / 232, 231 / 233, 231 / 234, 232 / 233, 232 / 234 and / or 233 / 234. In one or more embodiments, the optically active polypeptide is located at one or more sites of the sialic acid-sensitive polypeptide selected from the following: 115 / 117, 116 / 116, 117 / 116, 120 / 117, 205 / 202, 120 / 121, 120 / 118, 207 / 207, 211 / 208, 211 / 209, 206 / 210, 206 / 211, 208 / 210, 209 / 210, 208 / 206, 208 / 207, 211 / 207, 209 / 209, 210 / 209, 209 / 209. 8, 206 / 208, 206 / 211, 121 / 120, 208 / 208, 205 / 203, 114 / 120, 115 / 119, 115 / 122, 116 / 121, 118 / 116, 203 / 205, 205 / 210, 207 / 210, 119 / 121, 200 / 201, 206 / 204, 208 / 204, 208 / 205, 211 / 205, 210 / 206, 210 / 207, 114 / 118, 115 / 116, 205 / 212, 118 / 114,115 / 115, 209 / 202, 210 / 202.

[0040] In one or more of these embodiments, the sialic acid-sensitive polypeptide has: (i) the sequence shown in SEQ ID NO:1 or a functional fragment thereof after removing amino acids 1-96, or a sequence having at least 70% sequence identity with it and retaining sialic acid binding activity; (ii) a sequence of a variant of the sialic acid-binding protein described in any embodiment herein; or (iii) a sequence having at least 70% sequence identity with the sequence described in (ii) and having the mutation described in (ii) and retaining sialic acid sensitivity.

[0041] In one or more embodiments, the optically active polypeptide has: (a) any of the sequences shown in SEQ ID NO:2-5, (b) the sequence shown in SEQ ID NO:2 and having a mutation in Y1 and / or N246, the mutation including modification, substitution or deletion of amino acids; preferably, the mutation is selected from any group of: (1) Y1V, (2) Y1I, (3) Y1S, (4) Y1H, (5) Y1C, (6) N246S, (7) N246C, or (c) a variant sequence having at least 70% sequence identity with (a) or (b) and retaining the function of the fluorescent protein.

[0042] In one or more embodiments, the sialic acid optical probe sequence is shown in SEQ ID NO:8-13.

[0043] The present invention also provides a fusion polypeptide comprising the sialic acid optical probe described herein and other polypeptides.

[0044] In one or more embodiments, other peptides are located at the N-terminus and / or C-terminus of the optical probe.

[0045] In one or more embodiments, other peptides include tags (such as localization sequences, tags for easy purification, or tags for use in immune responses).

[0046] In one or more embodiments, the localization sequence includes, but is not limited to, peptides that localize optical probes to different organelles or sub-organelles.

[0047] In one or more embodiments, the immune response includes, but is not limited to, immunoblotting.

[0048] In one or more embodiments, the optical probe in the fusion polypeptide has / or does not have a connector between it and other polypeptides.

[0049] The present invention also provides an isolated polynucleotide comprising:

[0050] (a) The nucleotide sequence or its complementary sequence encoding the sialic acid-binding protein variant, sialic acid optical probe, fusion polypeptide, or any embodiment thereof described herein.

[0051] (b) A nucleotide sequence or its complementary sequence that has at least 80% (preferably at least 90%) identity with the sequence shown in (a), or

[0052] (c)(a) or (b) primer fragments.

[0053] In one or more embodiments, the polynucleotide further includes fragments, analogs, derivatives, soluble fragments, and variants of the above-described nucleic acid sequence, or complementary sequences thereof.

[0054] The present invention also provides a nucleic acid construct comprising the polynucleotides described herein.

[0055] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0056] In one or more embodiments, the polynucleotide is operatively linked to an expression control sequence.

[0057] In one or more embodiments, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and / or viral vectors.

[0058] The present invention also provides an engineered host cell, wherein the cell:

[0059] (1) Containing, expressing, or secreting the sialic acid optical probe, fusion peptide, or a variant thereof as described in any embodiment herein.

[0060] (2) Contains the polynucleotides described herein, or

[0061] (3) Contains the nucleic acid constructs described in this article.

[0062] In one or more embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0063] The present invention also provides the use of the sialic acid optical probe described in any embodiment herein for intracellular and / or extracellular localization of sialic acid.

[0064] In one or more embodiments, the use is to contact the sialic acid optical probe, fusion peptide, polynucleotide, nucleic acid construct or host cell described in any of the embodiments herein with the system to detect changes in the optically active peptide.

[0065] In one or more embodiments, the positioning is real-time positioning.

[0066] The present invention also provides a method for intracellular and / or extracellular localization of sialic acid, wherein the method comprises: contacting the sialic acid optical probe, fusion peptide, polynucleotide, nucleic acid construct or host cell described in any embodiment of the present invention with the system, and detecting changes in optically active peptides.

[0067] In one or more embodiments, the positioning is real-time positioning.

[0068] This invention also provides a method for preparing the sialic acid optical probe described herein, comprising the following steps:

[0069] 1) Provide the host cell as described in any of the embodiments herein;

[0070] 2) Culture the host cells under suitable conditions.

[0071] 3) Separate the sialic acid optical probe.

[0072] In one or more embodiments, a method for preparing the sialic acid optical probe or fusion polypeptide described herein includes the following steps: 1) transferring an expression vector encoding the sialic acid optical probe described herein into a host cell; 2) culturing the host cell under conditions suitable for expression of the expression vector; and 3) isolating the sialic acid optical probe.

[0073] The present invention also provides a method for quantitative detection of sialic acid, comprising the steps of: contacting the optical probe or fusion peptide described herein with a sample, and detecting changes in the optically active peptide.

[0074] In one or more embodiments, the detection is performed in vivo, in vitro, subcellular, or in situ.

[0075] In one or more embodiments, the sample is blood.

[0076] The present invention also provides a method for screening compounds based on changes in optically active peptides, comprising the steps of: contacting the sialic acid optical probe, fusion peptide, polynucleotide, nucleic acid construct or host cell described in any embodiment of the present invention with the candidate compound, and detecting changes in the optically active peptide.

[0077] In one or more embodiments, the method can perform high-throughput screening of compounds.

[0078] In one or more embodiments, the host cells described herein are contacted with the candidate compound in a sialic acid-containing system, and optical changes in the optically active peptide indicate whether the candidate compound can regulate the uptake of sialic acid by the cells.

[0079] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0080] The present invention also provides a kit comprising the sialic acid optical probe, fusion peptide, polynucleotide, nucleic acid construct or host cell as described in any embodiment herein.

[0081] In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of buffers, culture media, and sialic acid standards.

[0082] This invention also provides the application of the sialic acid optical probes, fusion peptides, or host cells described herein in the detection of sialic acid in samples, the screening of compounds, or the intracellular / extracellular localization of sialic acid. In one or more embodiments, the localization is real-time localization.

[0083] The present invention also provides the use of the sialic acid optical probes or fusion peptides or polynucleotides or nucleic acid constructs or host cells described herein in the preparation of kits for detecting sialic acid in samples, screening compounds, or intracellular and / or extracellular localization of sialic acid.

[0084] The beneficial effects of this invention are:

[0085] The sialic acid optical probe provided by this invention is easy to mature, exhibits large fluorescence dynamics, and has good specificity. Furthermore, it can be expressed in cells through gene manipulation, enabling real-time, high-throughput, and quantitative detection of sialic acid both inside and outside cells, eliminating time-consuming sample processing steps. Experimental results show that the sialic acid optical probe provided in this application achieves a response to sialic acid that is more than 12 times that of the control. It can also perform localization, qualitative, and quantitative detection of sialic acid in subcellular structures such as the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosomes, and Golgi apparatus. Additionally, it allows for high-throughput compound screening and quantitative detection of sialic acid in blood. Attached Figure Description

[0086] Figure 1 This is an example SDS-PAGE image of a sialic acid optical probe.

[0087] Figure 2 The image shows the fluorescence spectrum of an exemplary sialic acid optical probe.

[0088] Figure 3 The titration curves of sialic acid optical probes at different concentrations of sialic acid are shown as an example.

[0089] Figure 4 The bar chart illustrates the specificity of sialic acid optical probes for metabolism-related and structural analogues.

[0090] Figure 5 This is an example of a sialic acid optical probe localizing to subcellular organelles in a mammalian cell.

[0091] Figure 6This is a dot plot illustrating high-throughput compound screening at the live-cell level using an illustrative sialic acid optical probe.

[0092] Figure 7 This is a schematic diagram illustrating the dynamic monitoring of sialic acid concentration in the cytoplasm of mammalian cells using an exemplary sialic acid optical probe.

[0093] Figure 8 A bar chart illustrating the quantification of sialic acid in mouse and human blood using an optical sialic acid probe. Detailed Implementation

[0094] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0095] The sialic acid-sensitive polypeptides described in this invention include, but are not limited to, the sialic acid-binding protein NanR from Shigella dysenteriae or variants thereof with more than 90% homology. NanR is a transcriptional regulator; the NanR protein dimer exhibits a tight asymmetric structure, while Neu5Ac binding around the α4-helix compresses the NanR monomer by 18.9 and 18.9 mm at the N-terminus and C-terminus, respectively. This conformational change before and after substrate binding makes it ideal for use as a substrate recognition element in probes. An exemplary NanR protein is shown in SEQ ID NO:1. In one or more embodiments, the sialic acid-sensitive polypeptide comprises a sialic acid-binding domain of a protein, with the DNA-binding domain of amino acids truncated from positions 1-96. When describing the optical probes or sialic acid-binding proteins of the present invention (e.g., when describing insertion or mutation sites), reference is made to the amino acid residue numbers in SEQ ID NO:1.

[0096] As used herein, the term "optical probe" refers to a sialic acid-sensitive peptide fused to an optically active peptide. The inventors discovered that conformational changes resulting from the specific binding of sialic acid-sensitive peptides, such as sialic acid-binding proteins, to physiological concentrations of sialic acid induce conformational changes in optically active peptides (e.g., fluorescent proteins), thereby altering their optical properties. By plotting standard curves using fluorescence data of fluorescent proteins measured at different sialic acid concentrations, the presence and / or levels of sialic acid can be detected and analyzed.

[0097] In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operatively inserted into a sialic acid-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide capable of emitting fluorescence. Fluorescence is an optical property of the optically active polypeptide, which can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, amplitude of excitation at two different wavelengths, ratio of emission amplitudes at two different wavelengths, excited-state lifetime, or fluorescence anisotropy. A measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrates of the present invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescence property, for example, the amount of fluorescence at a specific wavelength or the integral of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that are easily distinguishable between inactive and activated conformational states. The optically active polypeptides described herein may also include their functional variants. Functional variants of optically active peptides include, but are not limited to, variants that can undergo changes in fluorescence properties that are the same as or similar to those of the parent optically active peptide.

[0098] As used herein, the term "fluorescent protein" refers to a protein that emits fluorescence under excitation light. Fluorescent proteins are fundamental detection methods in the field of bioscience. Examples include the commonly used green fluorescent protein GFP and its cyclically rearranged derivatives such as blue fluorescent protein (cpBFP), green fluorescent protein (cpGFP), and yellow fluorescent protein (cpYFP); as well as the commonly used red fluorescent protein RFP and its cyclically rearranged derivatives, such as cpmApple, cpmOrange, and cpmKate. Fluorescent proteins and their sequences that can be used in this invention are known in the art. Exemplarily, cpYFP is shown in SEQ ID NO:2; cpGFP is shown in SEQ ID NO:3; cpBFP is shown in SEQ ID NO:4; and cpmApple is shown in SEQ ID NO:5.

[0099] The fluorescent protein in the optical probe also includes functional variants with mutations, including but not limited to fluorescent proteins with mutations at amino acid position 1 and / or amino acid position 246 corresponding to SEQ ID NO:2. The mutation at position 1 is preferably V, I, S, H, or C; the mutation at position 246 is preferably S or C. In some embodiments, the functional variant of the fluorescent protein has any of the sequences shown in SEQ ID NO:2-5 and has a mutation selected from any group of the following at amino acid position 1 corresponding to SEQ ID NO:2: Y1V; Y1I; Y1S; Y1H, or Y1C. In one or more embodiments, the functional variant of the fluorescent protein has any of the sequences shown in SEQ ID NO:2-5 and has a mutation selected from any group of the following at amino acid position 246 corresponding to SEQ ID NO:2: N246S or N246C. In some embodiments, the functional variant of the fluorescent protein has any of the sequences shown in SEQ ID NO:2-5 and has mutations selected from any of the following groups at amino acid positions 1 and 246 corresponding to SEQ ID NO:2: Y1V; Y1I; Y1S; Y1H; Y1C; N246S; N246C. Y1S indicates that amino acid Y at position 1 is mutated to S, and so on.

[0100] In the optical probe of the present invention, the optically active polypeptide is located in the NC direction along the residues 113 / 114, 113 / 115, 113 / 116, 113 / 117, 113 / 118, 113 / 119, 113 / 120, 113 / 121, 113 / 122, 114 / 114, 114 / 115, 114 / 116, 114 / 117, 114 / 118, 114 / 119, 114 / 120, 114 / 121, 114 / 122, 115 / 114, 115 / 115, 115 / 116, 115 / 117, 115 / 118, 115 / 119, 115 / 120, 115 / 121, 11 5 / 122, 116 / 114, 116 / 116, 116 / 118, 116 / 120, 116 / 122, 116 / 124, 116 / 126, 116 / 128, 116 / 130, 117 / 114, 117 / 115, 117 / 116, 117 / 117, 117 / 118, 117 / 119 117 / 120, 117 / 121, 117 / 122, 118 / 114, 118 / 115, 118 / 116, 118 / 117, 118 / 118, 118 / 119, 118 / 120, 118 / 121, 118 / 122, 119 / 114, 119 / 115, 119 / 116, 119 / 117, 119 / 118, 119 / 119, 119 / 120, 119 / 121, 119 / 122, 120 / 114, 120 / 115, 120 / 116, 120 / 117, 120 / 118, 120 / 119, 120 / 120, 120 / 121, 120 / 122, 139 / 140, 1 39 / 141, 139 / 142, 139 / 143, 139 / 144, 139 / 145, 140 / 141, 140 / 142, 140 / 143, 140 / 144, 140 / 145, 141 / 142, 141 / 143, 141 / 144, 141 / 145, 142 / 143, 142 / 14 4, 142 / 145, 143 / 144, 143 / 145, 144 / 145, 159 / 160, 159 / 161, 159 / 162, 159 / 163, 159 / 164, 159 / 165, 160 / 161, 160 / 162, 160 / 163, 160 / 164, 160 / 165, 161 / 162, 161 / 163, 161 / 164, 161 / 165, 162 / 163, 162 / 164, 162 / 165, 163 / 164, 163 / 165, 164 / 165, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 178 / 184,178 / 185、178 / 186、179 / 180、179 / 181、179 / 182、179 / 183、179 / 184、179 / 185、179 / 186、180 / 181、180 / 182、180 / 183、180 / 184、180 / 185、180 / 186、181 / 182、181 / 183、181 / 184、181 / 185、181 / 186、182 / 183、182 / 184、182 / 185、182 / 186、183 / 184、183 / 185、183 / 186、184 / 185、184 / 186、185 / 186、200 / 201、200 / 202、200 / 203、200 / 204、200 / 205、200 / 206、200 / 207、200 / 208、200 / 209、200 / 210、200 / 211、200 / 212、201 / 201、201 / 202、201 / 203、201 / 204、201 / 205、201 / 206、201 / 207、201 / 208、201 / 209、201 / 210、201 / 211、201 / 212、202 / 201、202 / 202、202 / 203、202 / 204、202 / 205、202 / 206、202 / 207、202 / 208、202 / 209、202 / 210、202 / 211、202 / 212、203 / 201、203 / 202、203 / 203、203 / 204、203 / 205、203 / 206、203 / 207、203 / 208、203 / 209、203 / 210、203 / 211、203 / 212、204 / 201、204 / 202、204 / 203、204 / 204、204 / 205、204 / 206、204 / 207、204 / 208、204 / 209、204 / 210、204 / 211、204 / 212、205 / 201、205 / 202、205 / 203、205 / 204、205 / 205、205 / 206、205 / 207、205 / 208、205 / 209、205 / 210、205 / 211、205 / 212、206 / 201、206 / 202、206 / 203、206 / 204、206 / 205、206 / 206、206 / 207、206 / 208、206 / 209、206 / 210、206 / 211、206 / 212、207 / 201、207 / 202、207 / 203、207 / 204、207 / 205、207 / 206、207 / 207、207 / 208、207 / 209、207 / 210、207 / 211、207 / 212, 208 / 201, 208 / 202, 208 / 203, 208 / 204, 208 / 205, 208 / 206, 208 / 207, 208 / 208, 208 / 209, 208 / 210, 208 / 211, 208 / 212, 209 / 201, 209 / 202, 209 / 203, 209 / 204, 209 / 205, 20 9 / 206, 209 / 207, 209 / 208, 209 / 209, 209 / 210, 209 / 211, 209 / 212, 210 / 201, 210 / 202, 210 / 203, 210 / 204, 210 / 205, 210 / 206, 210 / 207, 210 / 208, 210 / 209, 210 / 210, 210 / 211, 210 / 2 12, 211 / 201, 211 / 202, 211 / 203, 211 / 204, 211 / 205, 211 / 206, 211 / 207, 211 / 208, 211 / 209, 211 / 210, 211 / 211, 211 / 212, 228 / 229, 228 / 230, 228 / 231, 228 / 232, 228 / 233, 228 / 234 Residues in or substituting from 229 / 230, 229 / 231, 229 / 232, 229 / 233, 229 / 234, 230 / 231, 230 / 232, 230 / 233, 230 / 234, 231 / 232, 231 / 233, 231 / 234, 232 / 233, 232 / 234 and / or 233 / 234 are numbered corresponding to the full length of the sialic acid-sensitive polypeptide.

[0101] In this article, "response fold" refers to the standardized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response ability to the substrate relative to the control, or the greater its responsiveness. For example, in this application, the response fold is calculated by detecting the change in the ratio of fluorescence intensity at 528nm emission from 420nm excitation to fluorescence intensity at 528nm emission from 485nm excitation (Normalized Ratio 420 / 485), as detailed below:

[0102] Fluorescence signal values ​​were corrected by subtracting the detection signal values ​​from cells that did not express the probe protein. pH-sensitive interference was eliminated by dividing the probe detection signals from parallel experimental groups by the control detection signals to obtain corrected data.

[0103] F = F sample -F BLK

[0104]

[0105] F represents fluorescence intensity. sample F represents the total fluorescence intensity of the sample expressing the fluorescent probe. BLK F represents the background fluorescence intensity of samples without expressed fluorescent probes. cpYFP This indicates the fluorescence intensity of the sample used as a pH control. F 485 F represents the fluorescence intensity of a fluorescent protein sample excited at 485 nm and emitted at 528 nm. 420 Ratio represents the fluorescence intensity of a fluorescent protein sample excited at 420 nm and emitted at 528 nm. sensor Ratio represents the ratio of fluorescence intensity of the probe. cpYFP This represents the ratio of the fluorescence intensity of the corresponding probe to the pH control fluorescent protein. (Normalized Ratio) 420 / 485 This refers to the ratio of the probe change or the ratio of the response. (Normalized Ratio) 485 / 420 The greater the deviation from 1 (whether it increases or decreases), the greater the change factor or response factor of the probe.

[0106] As used in this invention, the term "variant" or "mutant" includes variants that have the same function as the polypeptide or protein but differ in sequence. Variants of the polypeptide or protein may include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants. These variants include, but are not limited to: deletions, insertions, and / or substitutions of one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids to its carboxyl terminus and / or amino terminus. These variants may also comprise polypeptides or proteins with 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. The goal is to avoid being limited by theory, where changes to amino acid residues do not alter the overall conformation and function of the polypeptide or protein—that is, functionally conserved mutations. For example, in this field, substitution with amino acids of similar or identical properties typically does not change the function of the polypeptide or protein. In this field, amino acids of similar properties often refer to families of amino acids with similar side chains, which are well-defined. These families include amino acids with basic side chains (e.g., lysine, sialic acid, 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 nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, sialic acid, phenylalanine, methionine, sialic acid), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, sialic acid, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of a polypeptide or protein. Conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conserved substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of their genetically encoded amino acids.

[0107] The term "linker" or "connector region" refers to an amino acid or nucleotide sequence that links two parts in a polypeptide, protein, or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the linker region between the sialic acid-sensitive polypeptide and the optically active polypeptide is selected to be 0-3, and the number of amino acids at the carboxyl terminus is selected to be 0-2; when the recombinant optical probe is used as a basic unit to link with a functional protein, 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.

[0108] The inventors have discovered that sialic acid-binding protein variants with mutations at sites selected from the following: SEQ ID NO:1 or functional fragments thereof with amino acids truncated from positions 1-96, namely S115, H116, N164, Q208, A209, N214, S217, and Q220. The amino acid mutations include modifications, substitutions, or deletions of amino acids.

[0109] This invention provides sialic acid-binding protein variants having these mutations and optical probes comprising such sialic acid-binding protein variants as sialic acid-sensitive peptides. Therefore, in one or more embodiments, the sialic acid-sensitive peptide in the optical probe is a sialic acid-binding protein variant as described in any embodiment herein, and the fluorescent protein in the optical probe is as shown in SEQ ID NO:2-5 or a functional variant thereof.

[0110] In some specific embodiments, the sialic acid-sensitive polypeptide in the optical probe is as shown in SEQ ID NO:1 or its functional fragment after removing amino acids 1-96, and the optically active polypeptide is as shown in SEQ ID NO:1. As shown in NO:2, the optically active peptides are located at positions 113 / 114, 113 / 115, 113 / 116, 113 / 117, 113 / 118, 113 / 119, 113 / 120, 113 / 121, 113 / 122, 114 / 114, 114 / 115, 114 / 116, 114 / 117, 114 / 118, 114 / 119, 114 / 120, 114 / 121, 114 / 122, 115 / 114, 115 / 115, 115 / 116, 115 / 117, 115 / 118, 115 / 119, 115 / 120, and 115 / 121 of the sialic acid-sensitive peptides. 115 / 122, 116 / 114, 116 / 116, 116 / 118, 116 / 120, 116 / 122, 116 / 124, 116 / 126, 116 / 128, 116 / 130, 117 / 114, 117 / 115, 117 / 116, 117 / 117, 117 / 118, 117 / 119, 117 / 120, 117 / 121, 117 / 122, 118 / 114, 118 / 115, 118 / 116, 118 / 117, 118 / 118, 118 / 119, 118 / 120, 118 / 121, 118 / 122, 119 / 114 119 / 115, 119 / 116, 119 / 117, 119 / 118, 119 / 119, 119 / 120, 119 / 121, 119 / 122, 120 / 114, 120 / 115, 120 / 116, 120 / 117, 120 / 118, 120 / 119, 120 / 120, 120 / 121, 120 / 122, 139 / 140, 139 / 141, 139 / 142, 139 / 143, 139 / 144, 139 / 145, 140 / 141, 140 / 142, 140 / 143, 140 / 144, 140 / 145, 141 / 142 141 / 143, 141 / 144, 141 / 145, 142 / 143, 142 / 144, 142 / 145, 143 / 144, 143 / 145, 144 / 145, 159 / 160, 159 / 161, 159 / 162, 159 / 163, 159 / 164, 159 / 165, 160 / 161, 160 / 162, 160 / 163, 160 / 164, 160 / 165, 161 / 162, 161 / 163, 161 / 164, 161 / 165, 162 / 163, 162 / 164, 162 / 165, 163 / 164, 163 / 165164 / 165、178 / 179、178 / 180、178 / 181、178 / 182、178 / 183、178 / 184、178 / 185、178 / 186、179 / 180、179 / 181、179 / 182、179 / 183、179 / 184、179 / 185、179 / 186、180 / 181、180 / 182、180 / 183、180 / 184、180 / 185、180 / 186、181 / 182、181 / 183、181 / 184、181 / 185、181 / 186、182 / 183、182 / 184、182 / 185、182 / 186、183 / 184、183 / 185、183 / 186、184 / 185、184 / 186、185 / 186、200 / 201、200 / 202、200 / 203、200 / 204、200 / 205、200 / 206、200 / 207、200 / 208、200 / 209、200 / 210、200 / 211、200 / 212、201 / 201、201 / 202、201 / 203、201 / 204、201 / 205、201 / 206、201 / 207、201 / 208、201 / 209、201 / 210、201 / 211、201 / 212、202 / 201、202 / 202、202 / 203、202 / 204、202 / 205、202 / 206、202 / 207、202 / 208、202 / 209、202 / 210、202 / 211、202 / 212、203 / 201、203 / 202、203 / 203、203 / 204、203 / 205、203 / 206、203 / 207、203 / 208、203 / 209、203 / 210、203 / 211、203 / 212、204 / 201、204 / 202、204 / 203、204 / 204、204 / 205、204 / 206、204 / 207、204 / 208、204 / 209、204 / 210、204 / 211、204 / 212、205 / 201、205 / 202、205 / 203、205 / 204、205 / 205、205 / 206、205 / 207、205 / 208、205 / 209、205 / 210、205 / 211、205 / 212、206 / 201、206 / 202、206 / 203、206 / 204、206 / 205、206 / 206、206 / 207、206 / 208、206 / 209、206 / 210、206 / 211、206 / 212、207 / 201、207 / 202、207 / 203、207 / 204、207 / 205, 207 / 206, 207 / 207, 207 / 208, 207 / 209, 207 / 210, 207 / 211, 207 / 212, 208 / 201, 208 / 202, 208 / 203, 208 / 204, 208 / 205, 208 / 206, 208 / 207, 208 / 208, 208 / 209, 208 / 210, 208 / 211, 208 / 212 209 / 201, 209 / 202, 209 / 203, 209 / 204, 209 / 205, 209 / 206, 209 / 207, 209 / 208, 209 / 209, 209 / 210, 209 / 211, 209 / 212, 210 / 201, 210 / 202, 210 / 203, 210 / 204, 210 / 205, 210 / 206, 210 / 207, 210 / 208 210 / 209, 210 / 210, 210 / 211, 210 / 212, 211 / 201, 211 / 202, 211 / 203, 211 / 204, 211 / 205, 211 / 206, 211 / 207, 211 / 208, 211 / 209, 211 / 210, 211 / 211, 211 / 212, 228 / 229, 228 / 230, 228 / 231, 228 / 232, For sites 228 / 233, 228 / 234, 229 / 230, 229 / 231, 229 / 232, 229 / 233, 229 / 234, 230 / 231, 230 / 232, 230 / 233, 230 / 234, 231 / 232, 231 / 233, 231 / 234, 232 / 233, 232 / 234, and / or 233 / 234, the mutations of the optical probes are shown in any row of Tables 5 and 6.

[0111] In one or more embodiments, the optical probe provided by the present invention comprises a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein the sialic acid-sensitive polypeptide has: a functional fragment of the sequence shown in SEQ ID NO:1 after removing amino acids 1-96, or a sequence having at least 70% sequence identity with it and retaining sialic acid binding activity; and the optically active polypeptide has SEQ ID NO:1. The optically active polypeptide is located at one or more sites selected from the following for the sialic acid-sensitive polypeptide: 113 / 114, 113 / 115, 113 / 116, 113 / 117, 113 / 118, 113 / 119, 113 / 120, 113 / 121, 113 / 122, 114 / 114, 114 / 115, 114 / 116, 114 / 117, 114 / 118, 114 / 119, 114 / 120, 114 / 121, 114 / 122, 115 / 114, 115 / 115, 115 / 116. 115 / 117, 115 / 118, 115 / 119, 115 / 120, 115 / 121, 115 / 122, 116 / 114, 116 / 116, 116 / 118, 116 / 120, 116 / 122, 116 / 124, 116 / 126, 116 / 128, 116 / 130, 117 / 114, 117 / 115, 117 / 116, 117 / 117, 117 / 118, 117 / 119, 117 / 120, 117 / 121, 117 / 122, 118 / 114, 118 / 115, 118 / 116 118 / 117, 118 / 118, 118 / 119, 118 / 120, 118 / 121, 118 / 122, 119 / 114, 119 / 115, 119 / 116, 119 / 117, 119 / 118, 119 / 119, 119 / 120, 119 / 121, 119 / 122, 120 / 114, 120 / 115, 120 / 116, 120 / 117, 120 / 118, 120 / 119, 120 / 120, 120 / 121, 120 / 122, 139 / 140, 139 / 141, 139 / 142 139 / 143, 139 / 144, 139 / 145, 140 / 141, 140 / 142, 140 / 143, 140 / 144, 140 / 145, 141 / 142, 141 / 143, 141 / 144, 141 / 145, 142 / 143, 142 / 144, 142 / 145, 143 / 144, 143 / 145, 144 / 145, 159 / 160, 159 / 161, 159 / 162, 159 / 163, 159 / 164, 159 / 165, 160 / 161, 160 / 162, 160 / 163,160 / 164、160 / 165、161 / 162、161 / 163、161 / 164、161 / 165、162 / 163、162 / 164、162 / 165、163 / 164、163 / 165、164 / 165、178 / 179、178 / 180、178 / 181、178 / 182、178 / 183、178 / 184、178 / 185、178 / 186、179 / 180、179 / 181、179 / 182、179 / 183、179 / 184、179 / 185、179 / 186、180 / 181、180 / 182、180 / 183、180 / 184、180 / 185、180 / 186、181 / 182、181 / 183、181 / 184、181 / 185、181 / 186、182 / 183、182 / 184、182 / 185、182 / 186、183 / 184、183 / 185、183 / 186、184 / 185、184 / 186、185 / 186、200 / 201、200 / 202、200 / 203、200 / 204、200 / 205、200 / 206、200 / 207、200 / 208、200 / 209、200 / 210、200 / 211、200 / 212、201 / 201、201 / 202、201 / 203、201 / 204、201 / 205、201 / 206、201 / 207、201 / 208、201 / 209、201 / 210、201 / 211、201 / 212、202 / 201、202 / 202、202 / 203、202 / 204、202 / 205、202 / 206、202 / 207、202 / 208、202 / 209、202 / 210、202 / 211、202 / 212、203 / 201、203 / 202、203 / 203、203 / 204、203 / 205、203 / 206、203 / 207、203 / 208、203 / 209、203 / 210、203 / 211、203 / 212、204 / 201、204 / 202、204 / 203、204 / 204、204 / 205、204 / 206、204 / 207、204 / 208、204 / 209、204 / 210、204 / 211、204 / 212、205 / 201、205 / 202、205 / 203、205 / 204、205 / 205、205 / 206、205 / 207、205 / 208、205 / 209、205 / 210、205 / 211、205 / 212、206 / 201、206 / 202、206 / 203、206 / 204、206 / 205、206 / 206, 206 / 207, 206 / 208, 206 / 209, 206 / 210, 206 / 211, 206 / 212, 207 / 201, 207 / 202, 207 / 203, 207 / 204, 207 / 205, 207 / 206, 207 / 207, 207 / 208, 207 / 209, 207 / 210, 207 / 211, 207 / 212, 208 / 201, 208 / 202, 208 / 203 208 / 204, 208 / 205, 208 / 206, 208 / 207, 208 / 208, 208 / 209, 208 / 210, 208 / 211, 208 / 212, 209 / 201, 209 / 202, 209 / 203, 209 / 204, 209 / 205, 209 / 206, 209 / 207, 209 / 208, 209 / 209, 209 / 210, 209 / 211, 209 / 212, 210 / 201, 2 10 / 202, 210 / 203, 210 / 204, 210 / 205, 210 / 206, 210 / 207, 210 / 208, 210 / 209, 210 / 210, 210 / 211, 210 / 212, 211 / 201, 211 / 202, 211 / 203, 211 / 204, 211 / 205, 211 / 206, 211 / 207, 211 / 208, 211 / 209, 211 / 210, 211 / 211, 2 11 / 212, 228 / 229, 228 / 230, 228 / 231, 228 / 232, 228 / 233, 228 / 234, 229 / 230, 229 / 231, 229 / 232, 229 / 233, 229 / 234, 230 / 231, 230 / 232, 230 / 233, 230 / 234, 231 / 232, 231 / 233, 231 / 234, 232 / 233, 232 / 234 and / or 233 / 234.

[0112] In one or more embodiments, the optical probe provided by the present invention comprises a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein the sialic acid-sensitive polypeptide has: a functional fragment of the sequence shown in SEQ ID NO:1 after removing amino acids 1-96, or a sequence having at least 70% sequence identity with it and retaining sialic acid binding activity; and the optically active polypeptide has SEQ ID NO:1. The optically active polypeptide is located at one or more sites selected from the following for the sialic acid-sensitive polypeptide: 115 / 117, 116 / 116, 117 / 116, 120 / 117, 205 / 202, 120 / 121, 120 / 118, 207 / 207, 211 / 208, 211 / 209, 206 / 210, 206 / 211, 208 / 210, 209 / 210, 208 / 206, 208 / 207, 211 / 207, 209 / 209, 210 / 209, 209 / 208, 206 / 208, 2 06 / 211, 121 / 120, 208 / 208, 205 / 203, 114 / 120, 115 / 119, 115 / 122, 116 / 121, 118 / 116, 203 / 205, 205 / 210, 207 / 210, 119 / 121, 200 / 201, 206 / 204, 208 / 204, 208 / 205, 211 / 205, 210 / 206, 210 / 207, 114 / 118, 115 / 116, 205 / 212, 118 / 114, 115 / 115, 209 / 202, 210 / 202.

[0113] In one or more embodiments, the optical probe provided by the present invention comprises a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein the sialic acid-sensitive polypeptide has: a functional fragment of the sequence shown in SEQ ID NO:1 after removing amino acids 1-96, or a sequence having at least 70% sequence identity with it and retaining sialic acid binding activity; and the optically active polypeptide has SEQ ID NO:1. The optically active polypeptide is located at one or more sites selected from the following for the sialic acid-sensitive polypeptide: 115 / 117, 116 / 116, 117 / 116, 120 / 117, 205 / 202, 120 / 121, 120 / 118, 207 / 207, 211 / 208, 211 / 209, 206 / 210, 206 / 211, 208 / 210, 209 / 210, 208 / 206, 208 / 207, 211 / 207, 209 / 209, 210 / 209, 209 / 208, 206 / 208, 2 06 / 211, 121 / 120, 208 / 208, 205 / 203, 114 / 120, 115 / 119, 115 / 122, 116 / 121, 118 / 116, 203 / 205, 205 / 210, 207 / 210, 119 / 121, 200 / 201, 206 / 204, 208 / 204, 208 / 205, 211 / 205, 210 / 206, 210 / 207, 114 / 118, 115 / 116, 205 / 212, 118 / 114, 115 / 115, 209 / 202, 210 / 202.

[0114] In one or more embodiments, the optical probe provided by the present invention comprises a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein the sialic acid-sensitive polypeptide has: a functional fragment of the sequence shown in SEQ ID NO:1 after removing amino acids 1-96, or a sequence having at least 70% sequence identity with it and retaining sialic acid binding activity; and the optically active polypeptide has SEQ ID NO:1. The optically active polypeptide is located at one or more sites selected from the following for the sialic acid-sensitive polypeptide: 115 / 117, 116 / 116, 117 / 116, 120 / 117, 205 / 202, 120 / 121, 120 / 118, 207 / 207, 211 / 208, 211 / 209, 206 / 210, 206 / 211, 208 / 210, 209 / 210, 208 / 206, 208 / 207, 211 / 207, 209 / 209, 210 / 209, 209 / 208, 206 / 208, 2 06 / 211, 121 / 120, 208 / 208, 205 / 203, 114 / 120, 115 / 119, 115 / 122, 116 / 121, 118 / 116, 203 / 205, 205 / 210, 207 / 210, 119 / 121, 200 / 201, 206 / 204, 208 / 204, 208 / 205, 211 / 205, 210 / 206, 210 / 207, 114 / 118, 115 / 116, 205 / 212, 118 / 114, 115 / 115, 209 / 202, 210 / 202.

[0115] In one or more embodiments, the optical probe provided by the present invention comprises a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein the sialic acid-sensitive polypeptide has: a functional fragment of the sequence shown in SEQ ID NO:1 after removing amino acids 1-96, or a sequence having at least 70% sequence identity with it and retaining sialic acid binding activity; and the optically active polypeptide has SEQ ID NO:1. The optically active polypeptide is located at one or more sites selected from the following for the sialic acid-sensitive polypeptide: 115 / 117, 116 / 116, 117 / 116, 120 / 117, 205 / 202, 120 / 121, 120 / 118, 207 / 207, 211 / 208, 211 / 209, 206 / 210, 206 / 211, 208 / 210, 209 / 210, 208 / 206, 208 / 207, 211 / 207, 209 / 209, 210 / 209, 209 / 208, 206 / 208, 2 06 / 211, 121 / 120, 208 / 208, 205 / 203, 114 / 120, 115 / 119, 115 / 122, 116 / 121, 118 / 116, 203 / 205, 205 / 210, 207 / 210, 119 / 121, 200 / 201, 206 / 204, 208 / 204, 208 / 205, 211 / 205, 210 / 206, 210 / 207, 114 / 118, 115 / 116, 205 / 212, 118 / 114, 115 / 115, 209 / 202, 210 / 202.

[0116] In one or more embodiments, the optically active polypeptide is located between the N-terminal and C-terminal portions of the sialic acid-sensitive polypeptide, and the optical probe comprises, from the N-terminus to the C-terminus, the N-terminal portion of the sialic acid-sensitive polypeptide as shown by residues 97-X of SEQ ID NO:1, the optically active polypeptide or a variant thereof shown by any one of SEQ ID NO:2-5, and the C-terminal portion of the sialic acid-sensitive polypeptide as shown by residues Y-262 of SEQ ID NO:1, wherein...

[0117] X and Y are selected from any of the following groups: (1) X is 115, Y is 117; (2) X is 116, Y is 116; (3) X is 117, Y is 116; (4) X is 120, Y is 117; (5) X is 205, Y is 202; (6) X is 120, Y is 121; (7) X is 120, Y is 118; (8) X is 207, Y is 207; (9) X is 211, Y is 208; (10) X is 211, Y is 209; (11) X is 206, Y is 210; (12) X is 206, Y is 21 1; (13) X is 208, Y is 210; (14) X is 209, Y is 210; (15) X is 208, Y is 206; (16) X is 208, Y is 207; (17) X is 211, Y is 207; (18) X is 209, Y is 209; (19) X is 210, Y is 209; (20) X is 209, Y is 208; (21) X is 206, Y is 208; (22) X is 206, Y is 211; (23) X is 121, Y is 120; (24) X is 208, Y is 208 (25) X is 205, Y is 203; (26) X is 114, Y is 120; (27) X is 115, Y is 119; (28) X is 115, Y is 122; (29) X is 116, Y is 121; (30) X is 118, Y is 116; (31) X is 203, Y is 205; (32) X is 205, Y is 210; (33) X is 207, Y is 210; (34) X is 119, Y is 121; (35) X is 200, Y is 201; (36) X is 206, Y is 204 (37) X is 208, Y is 204; (38) X is 208, Y is 205; (39) X is 211, Y is 205; (40) X is 210, Y is 206; (41) X is 210, Y is 207; (42) X is 114, Y is 118; (43) X is 115, Y is 116; (44) X is 205, Y is 212; (45) X is 118, Y is 114; (46) X is 115, Y is 115; (47) X is 209, Y is 202; (48) X is 210, Y is 202.

[0118] and

[0119] The optical probe contains mutations selected from any of the following groups: (1) S115E and H116F of the N-terminal portion of the sialic acid-sensitive peptide, (2) S115L and H116G of the N-terminal portion of the sialic acid-sensitive peptide, (3) S115A and H116F of the N-terminal portion of the sialic acid-sensitive peptide, (4) S115V and H116L of the N-terminal portion of the sialic acid-sensitive peptide, (5) S115R and H116F of the N-terminal portion of the sialic acid-sensitive peptide, (6) S115E and H116W of the N-terminal portion of the sialic acid-sensitive peptide, (7) S115A and H116A of the N-terminal portion of the sialic acid-sensitive peptide and Y1S of the optically active peptide, (8) S115G and H116L of the N-terminal portion of the sialic acid-sensitive peptide. (9) S115G and H116A of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (10) S115W and H116V of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1C, (11) S115A and H116D of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1V, (12) S115G and H116L of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (13) S115A and H116F of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1V and N246S, (14) S115R and H116F of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1S, (9) S115G and H116A of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (15) S115R and H116F of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (16) S115G and H116L of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (17) S115G and H116F of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (18) S115G and H116F of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (19) S115G and H116A of the N-terminal portion of the sialic acid-sensitive peptide and the optically active peptide Y1H, (19) S115G and H116A (15) S115A and H116F of the N-terminal portion of sialic acid-sensitive peptides, and Y1V and N246C of optically active peptides, (16) Q208R and A209T of sialic acid-sensitive peptides and Y1V of optically active peptides, (17) Q208V and A209V of sialic acid-sensitive peptides and Y1V of optically active peptides, (18) Q208K and A209S of sialic acid-sensitive peptides and Y1I of optically active peptides, (19) Q208R, A209T and N164I of sialic acid-sensitive peptides and Y1V of optically active peptides, (20) Q208R, A209T and N214H of sialic acid-sensitive peptides and Y1V of optically active peptides, (21) Sialic acid-sensitive peptides Q208R, A209T and S217T and optically active peptide Y1V, (22) Sialic acid-sensitive peptides Q208R, A209T and Q220D and optically active peptide Y1V, (23) Sialic acid-sensitive peptides Q208R, A209T and Q220E and optically active peptide Y1V, (24) Sialic acid-sensitive peptides Q208R, A209T and Q220G and optically active peptide Y1V, (25) Sialic acid-sensitive peptides Q208R, A209T and Q220N and optically active peptide Y1V, (26) Sialic acid-sensitive peptides Q208R, A209T and Q220S and optically active peptide Y1V.

[0120] In one or more embodiments, the optically active polypeptide is located between the N-terminal and C-terminal portions of the sialic acid-sensitive polypeptide, and the optical probe comprises, from the N-terminus to the C-terminus, the N-terminal portion of the sialic acid-sensitive polypeptide with the sequence of residues 97-X of SEQ ID NO:1, the optically active polypeptide or a variant thereof shown in any one of SEQ ID NO:2-5, and the C-terminal portion of the sialic acid-sensitive polypeptide with the sequence of residues Y-262 of SEQ ID NO:1, wherein X is 116, Y is 116, and the optical probe comprises mutations selected from any one of the following groups: (1) S115E and H116F of the N-terminal portion of the sialic acid-sensitive polypeptide, (2) S115L and H116G of the N-terminal portion of the sialic acid-sensitive polypeptide, (3) S115A and H116F of the N-terminal portion of the sialic acid-sensitive polypeptide, (4) (5) S115V and H116L of the N-terminal portion of the sialic acid-sensitive peptide, (6) S115E and H116W of the N-terminal portion of the sialic acid-sensitive peptide, (7) S115A and H116A of the N-terminal portion of the sialic acid-sensitive peptide and Y1S of the optically active peptide, (8) S115G and H116L of the N-terminal portion of the sialic acid-sensitive peptide and Y1S of the optically active peptide. (9) S115G and H116A of the N-terminal portion of the sialic acid-sensitive peptide and Y1H of the optically active peptide, (10) S115W and H116V of the N-terminal portion of the sialic acid-sensitive peptide and Y1C of the optically active peptide, (11) S115A and H116D of the N-terminal portion of the sialic acid-sensitive peptide and Y1V of the optically active peptide, (12) S115G and H116L of the N-terminal portion of the sialic acid-sensitive peptide (13) S115A and H116F of the N-terminal portion of the sialic acid-sensitive peptide, and (14) S115R and H116F of the N-terminal portion of the sialic acid-sensitive peptide, and (15) S115A and H116F of the N-terminal portion of the sialic acid-sensitive peptide, and (16) Y1V and N246C of the optically active peptide.

[0121] In one or more embodiments, the optically active polypeptide is located between the N-terminal and C-terminal portions of the sialic acid-sensitive polypeptide, and the optical probe comprises, from the N-terminus to the C-terminus, the N-terminal portion of the sialic acid-sensitive polypeptide as shown by residues 97-X of SEQ ID NO:1, the optically active polypeptide or a variant thereof shown by any one of SEQ ID NO:2-5, and the sequence as shown in SEQ ID NO:1. The C-terminal portion of the sialic acid-sensitive polypeptide represented by residue Y-262 of NO:1, where X is 209 and Y is 210, the optical probe contains mutations selected from any of the following groups: (16) Q208R and A209T of the sialic acid-sensitive polypeptide and Y1V of the optically active polypeptide, (17) Q208V and A209V of the sialic acid-sensitive polypeptide and Y1V of the optically active polypeptide, (18) Q208K and A209S of the sialic acid-sensitive polypeptide and Y1I of the optically active polypeptide, (19) Q208R, A209T and N164I of the sialic acid-sensitive polypeptide and Y1V of the optically active polypeptide, (20) Q208R, A209T and N214H of the sialic acid-sensitive polypeptide and Y1V of the optically active polypeptide. V, (21) Sialic acid sensitive peptides Q208R, A209T and S217T and optically active peptide Y1V, (22) Sialic acid sensitive peptides Q208R, A209T and Q220D and optically active peptide Y1V, (23) Sialic acid sensitive peptides Q208R, A209T and Q220E and optically active peptide Y1V, (24) Sialic acid sensitive peptides Q208R, A209T and Q220G and optically active peptide Y1V, (25) Sialic acid sensitive peptides Q208R, A209T and Q220N and optically active peptide Y1V, (26) Sialic acid sensitive peptides Q208R, A209T and Q220S and optically active peptide Y1V.

[0122] In this paper, at sites represented in the form of "X / Y", the optically active polypeptide has partial sialic acid-sensitive polypeptides at both ends. The N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any amino acid from position 1 to 97) to the Xth amino acid of the sialic acid-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is the Yth amino acid to the C-terminal ending amino acid (e.g., any amino acid from position Y to position 262) of the sialic acid-sensitive polypeptide sequence. In this context, if the two numbers in the "X / Y" format are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids represented by those numbers. For example, insertion site 160 / 161 indicates that the optically active polypeptide is located between amino acids 160 and 161 of the sialic acid-sensitive polypeptide. If the two numbers in the "X / Y" format are not consecutive integers and X is less than Y, it indicates that the optically active polypeptide replaces the amino acid between the amino acids represented by those numbers. For example, insertion site 204 / 206 indicates that the optically active polypeptide replaces amino acid 205 of the sialic acid-sensitive polypeptide. If X in the "X / Y" format is greater than or equal to Y, it indicates that the sialic acid-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide terminates at the Xth amino acid in the sialic acid-sensitive polypeptide sequence, while the sialic acid-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide terminates at the Xth amino acid in the sialic acid-sensitive polypeptide sequence. The insertion site begins at the Y-th amino acid position of the peptide sequence; for example, insertion site 208 / 206 indicates that the N-terminus of the optically active peptide is fused with the N-terminal starting amino acid (e.g., any amino acid from position 1 to 97) to amino acid 208 of the sialic acid-sensitive peptide sequence, and the C-terminus of the optically active peptide is fused with the 206th amino acid to the C-terminal ending amino acid (e.g., amino acid 262) of the sialic acid-sensitive peptide sequence. An exemplary structure is: (amino acids 1 to 97 of the sialic acid-sensitive peptide sequence) - (optically active peptide) - (amino acids 206 to 262 of the sialic acid-sensitive peptide sequence); if X equals Y in the site represented in the form of "X / Y", it indicates that the optically active peptide is inserted between the amino acids indicated by that number. For example, insertion site 120 / 120 indicates that the optically active peptide is inserted between amino acids 120 and 120 of the sialic acid-sensitive peptide. In an exemplary embodiment, the optically active polypeptide represented by SEQ ID NO:2, 3, 4, or 5 is located at the functional fragment of SEQ ID NO:1 or the fragment after removing amino acids 1-96, and the sialic acid-sensitive polypeptide is selected from any one or more of the following sites: 113 / 114, 113 / 115, 113 / 116, 113 / 117, 113 / 118, 113 / 119, 113 / 120, 113 / 121, 113 / 122, 114 / 114, 114 / 115, 114 / 116, 114 / 117, 114 / 118, 114 / 119, 114 / 120, 114 / 121, 114 / 122, 115 / 114, 115 / 115.115 / 116、115 / 117、115 / 118、115 / 119、115 / 120、115 / 121、115 / 122、116 / 114、116 / 116、116 / 118、116 / 120、116 / 122、116 / 124、116 / 126、116 / 128、116 / 130、117 / 114、117 / 115、117 / 116、117 / 117、117 / 118、117 / 119、117 / 120、117 / 121、117 / 122、118 / 114、118 / 115、118 / 116、118 / 117、118 / 118、118 / 119、118 / 120、118 / 121、118 / 122、119 / 114、119 / 115、119 / 116、119 / 117、119 / 118、119 / 119、119 / 120、119 / 121、119 / 122、120 / 114、120 / 115、120 / 116、120 / 117、120 / 118、120 / 119、120 / 120、120 / 121、120 / 122、139 / 140、139 / 141、139 / 142、139 / 143、139 / 144、139 / 145、140 / 141、140 / 142、140 / 143、140 / 144、140 / 145、141 / 142、141 / 143、141 / 144、141 / 145、142 / 143、142 / 144、142 / 145、143 / 144、143 / 145、144 / 145、159 / 160、159 / 161、159 / 162、159 / 163、159 / 164、159 / 165、160 / 161、160 / 162、160 / 163、160 / 164、160 / 165、161 / 162、161 / 163、161 / 164、161 / 165、162 / 163、162 / 164、162 / 165、163 / 164、163 / 165、164 / 165、178 / 179、178 / 180、178 / 181、178 / 182、178 / 183、178 / 184、178 / 185、178 / 186、179 / 180、179 / 181、179 / 182、179 / 183、179 / 184、179 / 185、179 / 186、180 / 181、180 / 182、180 / 183、180 / 184、180 / 185、180 / 186、181 / 182、181 / 183、181 / 184、181 / 185、181 / 186、182 / 183、182 / 184、182 / 185、182 / 186、183 / 184、183 / 185、183 / 186、184 / 185、184 / 186、185 / 186、200 / 201、200 / 202、200 / 203、200 / 204、200 / 205、200 / 206、200 / 207、200 / 208、200 / 209、200 / 210、200 / 211、200 / 212、201 / 201、201 / 202、201 / 203、201 / 204、201 / 205、201 / 206、201 / 207、201 / 208、201 / 209、201 / 210、201 / 211、201 / 212、202 / 201、202 / 202、202 / 203、202 / 204、202 / 205、202 / 206、202 / 207、202 / 208、202 / 209、202 / 210、202 / 211、202 / 212、203 / 201、203 / 202、203 / 203、203 / 204、203 / 205、203 / 206、203 / 207、203 / 208、203 / 209、203 / 210、203 / 211、203 / 212、204 / 201、204 / 202、204 / 203、204 / 204、204 / 205、204 / 206、204 / 207、204 / 208、204 / 209、204 / 210、204 / 211、204 / 212、205 / 201、205 / 202、205 / 203、205 / 204、205 / 205、205 / 206、205 / 207、205 / 208、205 / 209、205 / 210、205 / 211、205 / 212、206 / 201、206 / 202、206 / 203、206 / 204、206 / 205、206 / 206、206 / 207、206 / 208、206 / 209、206 / 210、206 / 211、206 / 212、207 / 201、207 / 202、207 / 203、207 / 204、207 / 205、207 / 206、207 / 207、207 / 208、207 / 209、207 / 210、207 / 211、207 / 212、208 / 201、208 / 202、208 / 203、208 / 204、208 / 205、208 / 206、208 / 207、208 / 208、208 / 209、208 / 210、208 / 211、208 / 212、209 / 201、209 / 202、209 / 203、209 / 204、209 / 205、209 / 206、209 / 207、209 / 208、209 / 209、209 / 210、209 / 211、209 / 212、210 / 201, 210 / 202, 210 / 203, 210 / 204, 210 / 205, 210 / 206, 210 / 207, 210 / 208, 210 / 209, 210 / 210, 210 / 211, 210 / 212, 211 / 201, 211 / 202, 211 / 203, 211 / 204, 211 / 205, 211 / 206, 211 / 207, 211 / 208, 211 / 209, 211 / 210, 211 / 2 11, 211 / 212, 228 / 229, 228 / 230, 228 / 231, 228 / 232, 228 / 233, 228 / 234, 229 / 230, 229 / 231, 229 / 232, 229 / 233, 229 / 234, 230 / 231, 230 / 232, 230 / 233, 230 / 234, 231 / 232, 231 / 233, 231 / 234, 232 / 233, 232 / 234 and / or 233 / 234.

[0123] In one or more embodiments, the optical probe comprises a sialic acid-sensitive polypeptide as shown in SEQ ID NO:1 or a functional fragment thereof after removing amino acids 1-96, and an optically active polypeptide as shown in SEQ ID NO:2-5 (preferably SEQ ID NO:2), wherein the optically active polypeptide is located at position 116 / 116 or 209 / 210 of the sialic acid-sensitive polypeptide, and the optical probe has the following mutations: (1) Q208R and A209T of the sialic acid-sensitive polypeptide and Y1V of the optically active polypeptide; (2) Q208V and A209V of the sialic acid-sensitive polypeptide and Y1V of the optically active polypeptide; (3) Q208K and A209S of the sialic acid-sensitive polypeptide and Y1I of the optically active polypeptide; (4) Q208R, A209T, and N164I of the sialic acid-sensitive polypeptide and Y1V of the optically active polypeptide; (5) Q208R of the sialic acid-sensitive polypeptide. (6) Sialic acid-sensitive peptides Q208R, A209T, S217T, and Y1V, (7) Sialic acid-sensitive peptides Q208R, A209T, Q220D, and Y1V, (8) Sialic acid-sensitive peptides Q208R, A209T, Q220E, and Y1V, (9) Sialic acid-sensitive peptides Q208R, A209T, Q220G, and Y1V, (10) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (11) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (12 ...3) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (14) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (15) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (16) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (17) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (18) Sialic acid-sensitive peptides Q208R, A209T, Q220N, and Y1V, (19) S209T, A209T, Q220D, and Y1 The peptide Y1V, (11) sialic acid-sensitive peptides Q208R, A209T, Q220S, and optically active peptide Y1V, (12) sialic acid-sensitive peptides S115E and H116F, (13) sialic acid-sensitive peptides S115L and H116G, (14) sialic acid-sensitive peptides S115A and H116F, (15) sialic acid-sensitive peptides S115V and H116L, (16) sialic acid-sensitive peptides S115R and H116F, (17) sialic acid-sensitive peptides S115E and H116W, (18) sialic acid-sensitive peptides S115A and H116A, and optically active peptide Y1V. 1S, (19) S115G and H116L of sialic acid-sensitive peptides and Y1S of optically active peptides, (20) S115G and H116A of sialic acid-sensitive peptides and Y1H of optically active peptides, (21) S115W and H116V of sialic acid-sensitive peptides and Y1C of optically active peptides, (22) S115A and H116D of sialic acid-sensitive peptides and Y1V of optically active peptides, (23) S115G and H116L of sialic acid-sensitive peptides and Y1H of optically active peptides, (24) S115A and H116F of sialic acid-sensitive peptides and Y1V and N246S of optically active peptides,(25) S115R and H116F of sialic acid-sensitive peptides, and Y1V and N246C of optically active peptides; (26) S115A and H116F of sialic acid-sensitive peptides, and Y1V and N246C of optically active peptides.

[0124] In one or more embodiments, the optical probe comprises a sialic acid-sensitive polypeptide as shown in SEQ ID NO:1 or a functional fragment thereof after truncating amino acids 1-96, an optically active polypeptide as shown in SEQ ID NO:2-5 (preferably SEQ ID NO:2), the optically active polypeptide being located at position 116 / 116 of the sialic acid-sensitive polypeptide, and the optical probe having the following mutations: (12) S115E and H116F of the sialic acid-sensitive polypeptide at the N-terminus (upstream) of the insertion site, (13) S115L and H116G of the sialic acid-sensitive polypeptide at the N-terminus (upstream) of the insertion site, (14) S115A and H116F of the sialic acid-sensitive polypeptide at the N-terminus (upstream) of the insertion site, and (15) the sialic acid-sensitive polypeptide. S115V and H116L, (16) S115R and H116F of sialic acid-sensitive peptides at the N-terminus (upstream) of the insertion site, (17) S115E and H116W of sialic acid-sensitive peptides, (18) S115A and H116A of sialic acid-sensitive peptides at the N-terminus (upstream) of the insertion site, and Y1S of optically active peptides, (19) S115G and H116L of sialic acid-sensitive peptides at the N-terminus (upstream) of the insertion site, and Y1S of optically active peptides, (20) insertion S115G and H116A of sialic acid-sensitive peptides at the N-terminus (upstream) of the insertion site, and Y1H of optically active peptides, (21) S115W and H116V of sialic acid-sensitive peptides at the N-terminus (upstream) of the insertion site, and Y1C of optically active peptides, (22) S115A and H116D of sialic acid-sensitive peptides at the N-terminus (upstream) of the insertion site, and Y1V of optically active peptides, (23) S115G and H116L of sialic acid-sensitive peptides at the N-terminus (upstream) of the insertion site, Y1H of the optically active peptide, (24) S115A and H116F of the sialic acid-sensitive peptide at the N-terminus (upstream) of the insertion site, and Y1V and N246S of the optically active peptide, (25) S115R and H116F of the sialic acid-sensitive peptide at the N-terminus (upstream) of the insertion site, and Y1V and N246C of the optically active peptide, (26) S115A and H116F of the sialic acid-sensitive peptide at the N-terminus (upstream) of the insertion site, and Y1V and N246C of the optically active peptide.

[0125] In one or more embodiments, the optical probe may contain a sialic acid-sensitive polypeptide as shown in SEQ ID NO:1 or a functional fragment thereof after removing amino acids 1-96, and an optically active polypeptide as shown in SEQ ID NO:2-5 (preferably SEQ ID NO:1). As shown in IDNO:2), the optically active peptide is located at position 209 / 210 of the sialic acid-sensitive peptide, and the optical probe has the following mutations: (1) Q208R and A209T of the sialic acid-sensitive peptide and Y1V of the optically active peptide; (2) Q208V and A209V of the sialic acid-sensitive peptide and Y1V of the optically active peptide; (3) Q208K and A209S of the sialic acid-sensitive peptide and Y1I of the optically active peptide; (4) Q208R, A209T, and N164I of the sialic acid-sensitive peptide and Y1V of the optically active peptide; (5) Q208R, A209T, and N214H of the sialic acid-sensitive peptide and Y1V of the optically active peptide; (6) (7) Sialic acid-sensitive peptides Q208R, A209T, S217T and optically active peptide Y1V, (8) Sialic acid-sensitive peptides Q208R, A209T, Q220D and optically active peptide Y1V, (9) Sialic acid-sensitive peptides Q208R, A209T, Q220G and optically active peptide Y1V, (10) Sialic acid-sensitive peptides Q208R, A209T, Q220N and optically active peptide Y1V, (11) Sialic acid-sensitive peptides Q208R, A209T, Q220S and optically active peptide Y1V.

[0126] In two or more polypeptide or nucleic acid sequences, the term "identity" or "percentage of identity" refers to the similarity of two or more sequences or subsequences, or the similarity of a certain percentage of amino acid residues or nucleotides in a specified region, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, through manual alignment and visual inspection (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, within a comparison window or specified region). For example, preferred algorithms suitable for determining the percentage of sequence identity and the percentage of sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0127] As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic enzyme sites such as factor Xa, thrombin, or enterokinase.

[0128] As used herein, the terms “functional fragment,” “functional variant,” “derivative,” and “analyte” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., NanR protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins (e.g., NanR protein or fluorescent protein) of the present invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by the fusion of a mature protein with another compound (e.g., a compound that extends the protein's half-life, such as polyethylene glycol); or (iv) proteins formed by the fusion of an additional amino acid sequence into the protein sequence (e.g., a secreted sequence or a sequence used to purify the protein or the original protein sequence, or a fusion polypeptide formed with an antigen IgG fragment). These functional variants, derivatives, and analogs are within the scope well known to those skilled in the art in accordance with the teachings herein. The difference between the analog and the original polypeptide or protein may be a difference in amino acid sequence, 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 through a variety of techniques, such as random mutagenesis through radiation or exposure to mutagens, or through site-directed mutagenesis or other known molecular biology techniques.

[0129] The fusion polypeptide of this invention comprises the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. These other polypeptides 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 for targeting the optical probe to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting. A linker may be present between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0130] The subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, the tags used for purification or for immunoblotting include 6-histidine (6*His), glutathione S-transferase (GST), and Flag.

[0131] The expression vector of this invention comprises a nucleic acid sequence or its complementary sequence described herein, operatively linked to an expression control sequence, which encodes the optical probe or fusion polypeptide described herein. In some embodiments, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors. In some embodiments, the prokaryotic expression vector is preferably obtained by operatively linking a plasmid pCDF to the nucleic acid sequence described herein. In some embodiments, the expression control sequence includes an origin of replication, a promoter, an enhancer, an operon, a terminator, and a ribosome binding site.

[0132] The present invention also provides a method for preparing the above-mentioned sialic acid optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the sialic acid 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 expression of the expression vector; and 4) isolating the sialic acid optical probe.

[0133] As used herein, the terms "nucleic acid," "nucleotide," "polynucleotide," or "nucleic acid sequence" can be in DNA or RNA form. DNA form includes 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" as 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. The nucleic acids of this invention may comprise a nucleotide sequence with at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the described nucleic acid sequence. This invention also relates to nucleic acid fragments that hybridize with the aforementioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides long, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0134] The full-length sequence or fragment of the optical probe or fusion polypeptide of this invention can generally be obtained by PCR amplification, artificial synthesis, or recombinant methods. This invention does not specifically limit the PCR amplification procedure and system; conventional PCR amplification procedures and systems in the art can be used. Recombinant methods can also be used to obtain the relevant sequence in large quantities. This typically involves cloning it into a vector, transforming it into cells, and then isolating and purifying the relevant polypeptide or protein from the proliferated host cells using conventional methods. Furthermore, the relevant sequence can be synthesized artificially, especially when the fragment length is short. Currently, the DNA sequence encoding the protein of this invention (or its functional variants, derivatives, or analogs) can be obtained entirely through chemical synthesis. This 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 this invention through methods such as mutagenic PCR or chemical synthesis.

[0135] This invention also relates to nucleic acid constructs containing the polynucleotides described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotides described herein can be manipulated in various ways to ensure the expression of the polypeptide or protein. The nucleic acid constructs can be manipulated before insertion into a vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0136] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector, such as a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. A typical expression vector contains an expression control sequence that can be used to regulate the expression of a desired nucleic acid sequence, operatively linked to the nucleic acid sequence described herein or its complement. As used herein, the term "expression control sequence" refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, a promoter, a marker gene, or a translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. In recombinant expression vectors, "operative linking" means that the target nucleotide sequence is linked to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of such promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the LTR of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector can be a commercially available pCDF vector, with no other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and EcoRI, respectively, and then the digestion products are ligated to obtain the recombinant expression vector. This invention does not specifically limit the specific steps and parameters of digestion and ligation; conventional steps and parameters in the art can be used.

[0137] 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 performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, serving as the site for recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. The host cell is preferably a variety of cells conducive to gene product expression or fermentation production, such cells being well known and commonly used in the art. Specifically, it can be bacterial cells of *Escherichia coli*, *Streptomyces*, *Salmonella typhimurium*, fungal cells such as yeast, plant cells, insect cells of *Drosophila S2* or *Sf9*, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc. The exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0138] The method for transferring DNA to host cells described in this invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipid transfection, native competent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, the preferred method is the CaCl2 or MgCl2 method, 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 used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0139] This invention involves transferring an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate sialic acid optical probes. The host cell amplification and expression culture can be performed using conventional methods. Depending on the type of host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.

[0140] In this invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the sialic acid fluorescent protein; conventional methods for separating fusion proteins in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting out, centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. In one embodiment, the optical probe is separated using His-tagged affinity chromatography.

[0141] This invention also provides the application of the sialic acid optical probe in real-time localization, quantitative detection, and high-throughput compound screening of sialic acid. In one aspect, the sialic acid optical probe is preferably linked to signal peptides at different sites within the cell, transferred into the cell, and used to perform real-time localization of sialic acid by detecting the intensity of fluorescence signals within the cell; quantitative detection of the corresponding sialic acid is then performed using a sialic acid standard titration curve. The sialic acid standard titration curve described in this invention is plotted based on the fluorescence signals obtained by the sialic acid optical probe at different concentrations of sialic acid. The sialic acid optical probe of this invention is directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of sialic acid, thus improving accuracy. In high-throughput compound screening, the sialic acid optical probe of this invention adds different compounds to the cell culture medium and measures changes in sialic acid content to screen for compounds that affect changes in sialic acid content. The applications of the sialic acid optical probe in real-time localization, quantitative detection, and high-throughput compound screening described in this invention are not for diagnostic or therapeutic purposes and do not involve the diagnosis or treatment of diseases.

[0142] This invention also provides a detection kit comprising the optical probes, nucleic acid molecules, nucleic acid constructs, and / or cells described herein. The kit further comprises other reagents required for the detection of sialic acid. These other reagents are well known in the art, such as buffers, cell culture media, and sialic acid standards. Exemplary buffers include, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.

[0143] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range.

[0144] Example

[0145] Experimental methods and materials

[0146] I. Experimental Materials and Reagents

[0147] The embodiments mainly employ conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, which are well known to those skilled in the art. Examples include: Jane Rothcams et al., *Molecular Cloning: A Laboratory Manual* (3rd edition, August 2002, Science Press, Beijing), J. Sambrook and DW Russell, translated by Huang Peitang et al.; Fereschney et al., *Animal Cell Culture: A Basic Technical Guide* (5th edition), translated by Zhang Jingbo and Xu Cunshuan et al.; and J.S. Bonnie Fesnon, M. Dassault et al., *A Concise Laboratory Manual of Cell Biology*, translated by Zhang Jingbo et al.

[0148] The pCDF-cpYFP and pCDF-sialic acid-binding protein plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vectors were purchased from Invitrogen. All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. The expression plasmids constructed in the examples were all sequenced by BGI Genomics and J. Lee Sequencing. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase from Tiangen Biotech (Beijing) Co., Ltd., and the primeSTAR DNA polymerase from TaKaRa. Corresponding polymerase buffers and dNTPs were included with each purchase. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, as well as T4 ligase and T4 phosphorylase (T4 PNK), were purchased from Fermentas, and corresponding buffers were included with each purchase. The Lip2000 transfection kit was purchased from Invitrogen. Sialic acid and other amino acids were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier.

[0149] The DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. (China). The cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.

[0150] The main instruments used in the examples include: Biotek Synergy 2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd., China), nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).

[0151] II. Molecular Biology Methods and Cellular Experimental Methods

[0152] II.1 Polymerase Chain Reaction (PCR):

[0153] 1. PCR amplification of the target fragment:

[0154] This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The PCR amplification reaction system is as follows: template sequence 0.5-1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM).

[0155] 0.5 μL, 5 μL 10× PFU buffer, 0.5 μL PFU DNA polymerase, 1 μL dNTP (10 mM), 41.5-42 μL sterile ultrapure water (ddH2O), total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 2-10 minutes, 30 cycles (94-96℃ for 30-45 seconds, 50-65℃ for 30-45 seconds, 72℃ for a certain time (600 bp / min)), extension at 72℃ for 10 minutes.

[0156] 2. Long fragment (>2500bp) amplification PCR:

[0157] The long-fragment amplification used in this invention is mainly a reverse PCR amplification vector, a technique used in the following examples to obtain site-directed mutagenesis. Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product then contains the corresponding mutation site. The long-fragment amplification PCR reaction system is as follows: template sequence (10 pg-1 ng) 1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 5×PrimerSTAR buffer 10 μL, PrimerSTAR DNA polymerase 0.5 μL, dNTP (2.5 mM) 4 μL, sterile ultrapure water (ddH2O) 33.5 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 50-68℃ for 5-15 seconds, 72℃ for a certain time (1000bp / min)), extension at 72℃ for 10 minutes; or denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 68℃ for a certain time (1000bp / min)), extension at 72℃ for 10 minutes.

[0158] II.2 Nucleotide endonuclease digestion reaction

[0159] The double enzyme digestion system for the plasmid vector is as follows: 20 μL plasmid vector (approximately 1.5 μg), 5 μL 10× buffer, 11-2 μL restriction endonuclease, 21-2 μL restriction endonuclease, and sterile ultrapure water to a total volume of 50 μL. Reaction conditions: 37℃, 1-7 h.

[0160] II.3 Phosphorylation of DNA fragments at the 5' end

[0161] Plasmids or genomes extracted from microorganisms contain phosphate groups at their ends, while PCR products do not. Therefore, a phosphate addition reaction is required at the 5' end of the PCR product. Only DNA molecules with phosphate groups at their ends can undergo ligation. The phosphorylation reaction system is as follows: 5-8 μL of PCR product DNA sequence, 1 μL of 10×T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), and 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.

[0162] II.4 Ligation reaction between target fragment and vector

[0163] The methods for connecting different fragments and carriers vary, and this invention uses three connection methods.

[0164] 1. Blunt-end junctions of blunt-ended short fragments and linearized vectors

[0165] The principle of this method is that the blunt-end product obtained by PCR is phosphorylated at the 5' end of a DNA fragment using T4 PNK, and then ligated with a linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of T4 PNK-treated DNA fragment, 4 μL of linearized vector fragment, 1 μL of PEG4000, 1 μL of 10×T4 ligase buffer, and 1 μL of T4 DNA ligase, for a total of 10 μL. The reaction conditions are 22℃ for 30 min.

[0166] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends.

[0167] DNA fragments digested with restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence-complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7 μL of the digested PCR product DNA fragment, 0.5-7 μL of the digested plasmid, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 μL. The reaction conditions are 16°C for 4-8 hours.

[0168] 3. Ligation reaction involving the self-circularization of DNA fragment products phosphorylated at the 5' end following site-directed mutagenesis using reverse PCR.

[0169] The 5' phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via a self-circularization ligation reaction to obtain a recombinant plasmid. The self-circularization ligation reaction system was as follows: 10 μL phosphorylation reaction mixture, 0.5 μL T4 ligase (5 U / μL), total volume 10.5 μL. Reaction conditions: 16℃, 4–16 h.

[0170] II.5 Preparation and Transformation of Competent Cells

[0171] Preparation of competent cells:

[0172] 1. Pick a single colony (e.g., Mach1) and inoculate it into 5 mL of LB medium. Incubate overnight at 37°C with a shaker.

[0173] 2. Take 0.5-1 mL of the overnight culture and transfer it to 50 mL of LB medium. Incubate at 37°C and 220 rpm for 3 to 5 hours until the OD600 reaches 0.5.

[0174] 3. Pre-cool the cells in an ice bath for 2 hours.

[0175] Centrifuge at 4.4℃ and 4000rpm for 10min.

[0176] 5. Discard the supernatant, resuspend the cells in 5 mL of pre-cooled buffer, and add resuspending buffer to a final volume of 50 mL after homogenization.

[0177] 6. Incubate on ice for 45 min. Centrifuge at 4000 rpm for 10 min at 4℃, and resuspend the bacteria in 5 mL of ice-cold storage buffer.

[0178] 7. Place 100 μL of bacterial culture in each EP tube and store at -80°C or in liquid nitrogen.

[0179] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

[0180] 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

[0181] Transformation of competent cells:

[0182] 1. Take 100 μL of competent cells and thaw them on an ice bath.

[0183] 2. Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1 / 10 of the competent cell volume.

[0184] 3. Place the bacterial culture in a 42℃ water bath for 90 seconds to heat shock, then quickly transfer it to an ice bath and place it for 5 minutes.

[0185] 4. Add 500 μL LB and incubate at 200 rpm for 1 h on a constant temperature shaker at 37℃.

[0186] 5. Centrifuge the bacterial culture at 4000 rpm for 3 min, and keep 200 μL of supernatant. Spread the bacterial cells evenly on the surface of an agar plate containing appropriate antibiotics. Incubate the plate upside down in a 37°C incubator overnight.

[0187] II.6 Protein Expression, Purification, and Fluorescence Detection

[0188] 1. Transform the expression vector (e.g., a pCDF-based sialic acid optical probe expression vector) into BL21(DE3) cells, incubate upside down overnight, pick clones from the plate into 250ml Erlenmeyer flasks, place them on a 37℃ shaker, and incubate at 220rpm until OD = 0.4-0.8. Add 1 / 1000 (v / v) of IPTG (1M) and induce expression at 18℃ for 24-36h.

[0189] 2. After induction of expression, centrifuge at 4000 rpm for 30 min to collect the bacteria, resuspend the bacterial pellet in 50 mM phosphate buffer, and sonicate until the bacterial cells are clear. Centrifuge at 9600 rpm at 4℃ for 20 min.

[0190] 3. The supernatant from centrifugation was purified by a self-assembled nickel affinity chromatography column to obtain the protein. The protein after nickel affinity chromatography was then purified by a self-assembled desalting column to obtain the protein dissolved in 100mM HEPES buffer (pH 7.4).

[0191] 4. After SDS-PAGE identification of the purified protein, the probe was diluted with assay buffer (100mM HEPES, 100mM NaCl, pH 7.4) to a final concentration of 0.2-5μM. Sialic acid was prepared into a stock solution with a final concentration of 50mM using assay buffer (100mM HEPES, 100mM NaCl, pH 7.4).

[0192] 5. Take 100 μL of 1 μM protein solution, incubate at 37℃ for 10 min, add sialic acid for titration, and measure the fluorescence intensity of the protein at 528 nm emission after excitation by 420 nm light and at 528 nm emission after excitation by 485 nm light. The fluorescence excitation and emission measurements of the samples were performed using a multifunctional fluorescent microplate reader.

[0193] 6. Take 100 μL of 1 μM protein solution, incubate at 37℃ for 10 min, add sialic acid, and measure the absorption and fluorescence spectra of the protein. The absorption and fluorescence spectra of the samples were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0194] II.7 Transfection and Fluorescence Detection of Mammalian Cells

[0195] 1. The pCDNA3.1+-based sialic acid optical probe plasmid was transfected using a reagent.

[0196] Lipofectamine 2000 (Invitrogen) was transfected into HEK293 cells and cultured in a cell culture incubator at 37°C and 5% CO2. Fluorescence detection was performed 24–36 h after the exogenous gene was fully expressed.

[0197] 2. After the expression was induced, the adherent HEK293 cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection.

[0198] Example 1, Sialic acid binding protein particles

[0199] The domains 1-96 of the NanR gene (SEQ ID NO:1) from Shigella dysenteriae were truncated, and the remaining gene was amplified. The PCR product was subjected to gel electrophoresis, and the pCDF vector was simultaneously digested with the corresponding enzymes. After ligation with T4 DNA ligase, the product was used to transform DH5α cells. The transformed DH5α cells were plated on LB agar plates (streptomycin 100 μg / mL) and incubated overnight at 37°C. Plasmids were extracted from the grown DH5α transformants and identified by PCR. Positive plasmids, after being correctly sequenced, were used for subsequent plasmid construction.

[0200] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0201] In this embodiment, the following sites were selected for insertion into cpYFP based on pCDF-NanR to obtain the corresponding pCDF-NanR-cpYFP plasmids: 113 / 114, 113 / 115, 113 / 116, 113 / 117, 113 / 118, 113 / 119, 113 / 120, 113 / 121, 113 / 122, 114 / 114, 114 / 115, 114 / 116, 114 / 117, 114 / 118, 114 / 119, 114 / 120, 114 / 121, 114 / 122, 115 / 114, 115 / 115, 115 / 116, 115 / 117, 115 / 118, 115 / 119, 11 5 / 120, 115 / 121, 115 / 122, 116 / 114, 116 / 116, 116 / 118, 116 / 120, 116 / 122, 116 / 124, 116 / 126, 116 / 128, 116 / 130, 117 / 114, 117 / 115, 117 / 116, 117 / 117 117 / 118, 117 / 119, 117 / 120, 117 / 121, 117 / 122, 118 / 114, 118 / 115, 118 / 116, 118 / 117, 118 / 118, 118 / 119, 118 / 120, 118 / 121, 118 / 122, 119 / 114, 119 / 115, 119 / 116, 119 / 117, 119 / 118, 119 / 119, 119 / 120, 119 / 121, 119 / 122, 120 / 114, 120 / 115, 120 / 116, 120 / 117, 120 / 118, 120 / 119, 120 / 120, 120 / 121, 1 20 / 122, 139 / 140, 139 / 141, 139 / 142, 139 / 143, 139 / 144, 139 / 145, 140 / 141, 140 / 142, 140 / 143, 140 / 144, 140 / 145, 141 / 142, 141 / 143, 141 / 144, 141 / 14 5, 142 / 143, 142 / 144, 142 / 145, 143 / 144, 143 / 145, 144 / 145, 159 / 160, 159 / 161, 159 / 162, 159 / 163, 159 / 164, 159 / 165, 160 / 161, 160 / 162, 160 / 163, 160 / 164, 160 / 165, 161 / 162, 161 / 163, 161 / 164, 161 / 165, 162 / 163, 162 / 164, 162 / 165, 163 / 164, 163 / 165, 164 / 165, 178 / 179, 178 / 180, 178 / 181, 178 / 182,178 / 183、178 / 184、178 / 185、178 / 186、179 / 180、179 / 181、179 / 182、179 / 183、179 / 184、179 / 185、179 / 186、180 / 181、180 / 182、180 / 183、180 / 184、180 / 185、180 / 186、181 / 182、181 / 183、181 / 184、181 / 185、181 / 186、182 / 183、182 / 184、182 / 185、182 / 186、183 / 184、183 / 185、183 / 186、184 / 185、184 / 186、185 / 186、200 / 201、200 / 202、200 / 203、200 / 204、200 / 205、200 / 206、200 / 207、200 / 208、200 / 209、200 / 210、200 / 211、200 / 212、201 / 201、201 / 202、201 / 203、201 / 204、201 / 205、201 / 206、201 / 207、201 / 208、201 / 209、201 / 210、201 / 211、201 / 212、202 / 201、202 / 202、202 / 203、202 / 204、202 / 205、202 / 206、202 / 207、202 / 208、202 / 209、202 / 210、202 / 211、202 / 212、203 / 201、203 / 202、203 / 203、203 / 204、203 / 205、203 / 206、203 / 207、203 / 208、203 / 209、203 / 210、203 / 211、203 / 212、204 / 201、204 / 202、204 / 203、204 / 204、204 / 205、204 / 206、204 / 207、204 / 208、204 / 209、204 / 210、204 / 211、204 / 212、205 / 201、205 / 202、205 / 203、205 / 204、205 / 205、205 / 206、205 / 207、205 / 208、205 / 209、205 / 210、205 / 211、205 / 212、206 / 201、206 / 202、206 / 203、206 / 204、206 / 205、206 / 206、206 / 207、206 / 208、206 / 209、206 / 210、206 / 211、206 / 212、207 / 201、207 / 202、207 / 203、207 / 204、207 / 205、207 / 206、207 / 207、207 / 208、207 / 209、207 / 210, 207 / 211, 207 / 212, 208 / 201, 208 / 202, 208 / 203, 208 / 204, 208 / 205, 208 / 206, 208 / 207, 208 / 208, 208 / 209, 208 / 210, 208 / 211, 208 / 212, 209 / 201, 209 / 202, 209 / 203 209 / 204, 209 / 205, 209 / 206, 209 / 207, 209 / 208, 209 / 209, 209 / 210, 209 / 211, 209 / 212, 210 / 201, 210 / 202, 210 / 203, 210 / 204, 210 / 205, 210 / 206, 210 / 207, 210 / 208, 210 / 209, 2 10 / 210, 210 / 211, 210 / 212, 211 / 201, 211 / 202, 211 / 203, 211 / 204, 211 / 205, 211 / 206, 211 / 207, 211 / 208, 211 / 209, 211 / 210, 211 / 211, 211 / 212, 228 / 229, 228 / 230, 228 / 231, 2 28 / 232, 228 / 233, 228 / 234, 229 / 230, 229 / 231, 229 / 232, 229 / 233, 229 / 234, 230 / 231, 230 / 232, 230 / 233, 230 / 234, 231 / 232, 231 / 233, 231 / 234, 232 / 233, 232 / 234 and / or 233 / 234. For illustrative purposes only, the amino acid sequence of 209 / 210-NanR-cpYFP is shown in SEQ ID NO:6.

[0202] The cpYFP DNA fragment was generated using PCR, and a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers. PCR amplification produced a linearized pCDF-sialic acid-binding protein vector, whose 5' and 3' ends contained sequences (15-20 bp) completely identical to those at the cpYFP terminals. The linearized pCDF-NanR and cpYFP fragments underwent homologous recombination using Hieff Clone Enzyme. The product was transformed into DH5α, and the transformed DH5α was plated on LB agar plates (streptomycin 100 μg / mL) and incubated overnight at 37°C. Positive clones identified by PCR were subjected to plasmid extraction and sequencing. Sequencing was performed by J. Lee Sequencing.

[0203] After successful sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis showed a size around 56 kDa. This size is consistent with the size of the pCDF-NanR-cpYFP fusion protein containing the His-tag purified label. Results are as follows... Figure 1 As shown.

[0204] Sialic acid response screening was performed using the supernatant of fragmented E. coli expressing the NanR-cpYFP fusion protein. The detection signal of the fusion fluorescent protein containing 10 mM sialic acid was divided by the detection signal of the fusion fluorescent protein without sialic acid. As shown in Table 1, the detection results indicate that the optical probes expressing the NanR-cpYFP fusion protein in the fragmented supernatant showed a 1.3-fold response to sialic acid, including probes 115 / 117, 116 / 116, 117 / 116, 120 / 117, 205 / 202, 120 / 121, 120 / 118, 207 / 207, 211 / 208, 211 / 209, 206 / 210, 206 / 211, 208 / 210, 209 / 210, 208 / 206, 208 / 207, 211 / 207, 209 / 209, 210 / 209, 209 / 208, 206 / 208, 206 / 211, 121 / 120, 208 / 208, and 205. Optical probes were inserted at sites / 203, 114 / 120, 115 / 119, 115 / 122, 116 / 121, 118 / 116, 203 / 205, 205 / 210, 207 / 210, 119 / 121, 200 / 201, 206 / 204, 208 / 204, 208 / 205, 211 / 205, 210 / 206, 210 / 207, 114 / 118, 115 / 116, 205 / 212, 118 / 114, 115 / 115, 209 / 202, and 210 / 202. Among these, sites 116 / 116 and 209 / 210 showed a response to sialic acid more than twice that of other NanR binding substrates.

[0205] Table 1

[0206]

[0207] Example 3: Expression and detection of cpGFP optical probes at different insertion sites

[0208] Following the method described in Example 2, cpYFP at the above insertion site was replaced with cpGFP to construct a sialic acid green fluorescent protein fluorescent probe. As shown in Table 2, sites such as 116 / 116 and 209 / 210 showed a response to sialic acid exceeding 2-fold and were unaffected by the binding of other NanR substrates.

[0209] Table 2

[0210]

[0211] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0212] Following the method described in Example 2, the cpYFP at the above insertion site was replaced with cpBFP to construct a sialic acid blue fluorescent protein probe. As shown in Table 3, sites such as 116 / 116 and 209 / 210 showed a response to sialic acid exceeding 2-fold and were unaffected by the binding of other NanR substrates.

[0213] Table 3

[0214]

[0215] Example 5: Expression and detection of cpmApple optical probes at different insertion sites

[0216] Following the method described in Example 2, cpYFP at the above insertion site was replaced with cpmApple to construct a sialic acid red fluorescent protein probe. As shown in Table 4, sites such as 116 / 116 and 209 / 210 showed a response to sialic acid exceeding 2-fold and were unaffected by the binding of other NanR substrates.

[0217] Table 4

[0218]

[0219] Example 6: Expression and detection of linker-mutated cpYFP optical probes

[0220] For the optical probes obtained in Example 2 that respond more than 2-fold to sialic acid and are unaffected by other NanR binding substrates, i.e., the optical probes inserted at the 116 / 116 and 209 / 210 sites of NanR, the probes were linearized by reverse PCR, and the linker mutation site sequence was introduced into the primers. The resulting PCR products were homologously recombinated under the action of Hieff Clone Enzyme to establish a mutant library. The recombinant plasmid of the mutant library was transformed into BL21(DE3) to induce expression, and the probe proteins were purified for sialic acid response screening. The detection signal of the fusion fluorescent protein containing 10mM sialic acid was divided by the detection signal of the fusion fluorescent protein without sialic acid. The detection results show that the optical probes that respond more than 3-fold to sialic acid are shown in Table 5. As an example, the sequence 209 / 210-NanR-Q208R / A209T-cpYFP-Y1V is shown in SEQ ID NO:7.

[0221] Table 5

[0222]

[0223]

[0224] F indicates that the mutation is located at the N-terminus (upstream) of the insertion site, and R indicates that the mutation is located at the C-terminus (downstream) of the insertion site.

[0225] Example 7: Expression and detection of cpYFP optical probes with pocket mutations

[0226] For the optical probe 209 / 210-NanR-Q208R / A209T-cpYFP-Y1V obtained in Example 6, amino acid mutations were performed at positions S115, H116, N164, Q208, A209, N214, S217, and Q220 in the sialic acid substrate protein binding pocket. The probe was linearized using reverse PCR, and the sequences of the mutation sites were introduced into the primers. Homologous recombination was performed on the resulting PCR product to obtain plasmids containing the above mutation sites. The mutant plasmids were transformed into BL21(DE3) to induce expression. The mutant probe protein was purified and subjected to a sialic acid response test. The detection signal of the fusion fluorescent protein containing 10 mM sialic acid was divided by the detection signal of the fusion fluorescent protein without sialic acid. The detection results show that optical probes with a sialic acid response greater than or equal to 5 times are listed in Table 6. As an example, the sequences of 209 / 210-NanR-Q208R / A209T / Q220G-cpYFP-Y1V are shown in SEQ ID NO:8; the sequences of 209 / 210-NanR-Q208R / A209T / Q220E-cpYFP-Y1V are shown in SEQ ID NO:9; the sequences of 209 / 210-NanR-Q208R / A209T / S217T-cpYFP-Y1V are shown in SEQ ID NO:10; the sequences of 209 / 210-NanR-Q208R / A209T / Q220N-cpYFP-Y1V are shown in SEQ ID NO:11; and the sequences of 209 / 210-NanR-Q208R / A209T / N214H-cpYFP-Y1V are shown in SEQ ID NO:8. The sequence 209 / 210-NanR-Q208R / A209T / Q220S-cpYFP-Y1V, as shown in NO:12, is shown in SEQ ID NO:13.

[0227] Table 6

[0228] Insertion site NanR cpYFP <![CDATA[R 485 / 420 ]]> <![CDATA[K d (μM)]]> 209 / 210 Q208R / A209T / N164I Y1V 5.01 264.3 209 / 210 Q208R / A209T / N214H Y1V 7.99 544.6 209 / 210 Q208R / A209T / S217T Y1V 8.79 48.54 209 / 210 Q208R / A209T / Q220D Y1V 5.68 381.3 209 / 210 Q208R / A209T / Q220E Y1V 12.35 962.6 209 / 210 Q208R / A209T / Q220G Y1V 14.01 57.68 209 / 210 Q208R / A209T / Q220N Y1V 8.43 1453 209 / 210 Q208R / A209T / Q220S Y1V 7.07 105.2

[0229] Example 8, Performance of the optical probe mutant

[0230] For example, the purified sialic acid optical probe described in Example 7 was treated with 0 mM and 10 mM sialic acid for 10 minutes, and then the fluorescence spectrum was detected using a fluorescence spectrophotometer.

[0231] Excitation spectrum determination: Excitation spectra were recorded with an excitation range of 370 nm to 510 nm and an emission wavelength of 530 nm, read every 5 nm. The results showed that the probe exhibited two excitation peaks at approximately 410 and 490 nm, as... Figure 2 As shown.

[0232] Emission spectrum determination: With fixed excitation wavelengths of 420 nm and 460 nm, emission spectra were recorded at 470-600 nm and 490-600 nm, with readings taken every 5 nm. Results are as follows: Figure 2 As shown.

[0233] The 25 purified sialic acid probes described in Examples 6 and 7 were subjected to sialic acid detection at a concentration gradient (0–50 mM). After treating the purified probes for 10 minutes, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was measured. The results are as follows: Figure 3 As shown, the K values ​​of 25 sialic acid optical probes d The (binding constants) are 843.3, 602.4, 2909, 1121, 346.5, 1521, 907.2, 773.8, 1252, 1066, 42.65, 79.78, 329.8, 136.2, 52.71, 281.5, 1268, 606.2, 264.3, 544.6, 48.54, 381.3, 962.6, 57.68, 1453, and 105.2 μM, respectively.

[0234] The following formulas, with the largest corresponding amplitude and suitable affinity, are selected: 209 / 210-NanR-Q208R / A209T / Q220G-cpYFP-Y1V, 209 / 210-NanR-Q208R / A209T / Q220E-cpYFP-Y1V, 209 / 210-NanR-Q208R / A209T / S217T-cpYFP-Y1V, and 209 / 210-NanR-Q208R / A The sialic acid optical probes 209T / Q220N-cpYFP-Y1V, 209 / 210-NanR-Q208R / A209T / N214H-cpYFP-Y1V, and 209 / 210-NanR-Q208R / A209T / Q220S-cpYFP-Y1V were used to detect the reactivity of 10 mM sialic acid metabolic precursors and products, as well as other sialic acid structural analogs. The results showed that the probes had good specificity. The detection results are as follows: Figure 4 As shown.

[0235] Example 9: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

[0236] In this embodiment, different localization signal peptides are fused with the optical probe 209 / 210-NanR-Q208R / A209T / Q220G-cpYFP-Y1V to localize the optical probe to different organelles.

[0237] After transfecting 293 cells with optical probe plasmids fused with different localization signal peptides for 36 hours, the cells were washed with PBS and placed in HBSS solution for fluorescence detection using an inverted fluorescence microscope under the FITC channel. Results are as follows: Figure 5 As shown, the sialic acid optical probe can be localized to subcellular organelles, including the cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nuclear exclusion, by fusing with different specific localization signal peptides. Fluorescence was observed in different subcellular structures, and the distribution and intensity of the fluorescence varied.

[0238] Thirty-six hours after transfecting 293 cells with a cytoplasmic optical probe plasmid, the cells were washed with PBS and placed in HBSS solution. The ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected over an 18-hour period. The results are as follows: Figure 7 As shown, the 485 / 420 of the sample with added sialic acid gradually increased in a concentration-dependent manner, reaching up to nearly twice the initial value. After treatment with the neuraminidase inhibitor N-acety-2,3-dehydro-2-deoxymeuraminic acid (DANA) for the same period of time, the 485 / 420 of the probe decreased with increasing inhibitor concentration.

[0239] Example 10: High-throughput compound screening in living cells based on optical probes

[0240] In this embodiment, we used HeLa cells with cytoplasmic expression of 209 / 210-NanR-Q208R / A209T / Q220G-cpYFP-Y1V for high-throughput compound screening.

[0241] Transfected 293 cells were washed with PBS, treated with HBSS solution (without sialic acid) for 1 hour, and then treated with 10 μM of the compound for 1 hour. Sialic acid was added to each sample. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and the ratio of fluorescence intensity at 485 nm excitation to 528 nm emission were recorded using a microplate reader. Samples not treated with any compound were used as controls for standardization. Results are as follows: Figure 6As shown in the figure, among the 2000 compounds used, the vast majority had minimal effect on sialic acid entry into cells. Twenty-two compounds were able to increase the cells' ability to take up sialic acid, while seven compounds significantly reduced cellular uptake of sialic acid.

[0242] Example 11: Quantitative Detection of Sialic Acid in Blood Using an Optical Probe

[0243] In this implementation, purified K is used. d Sialic acid in the blood supernatant of mice and humans was analyzed using 60 μM 209 / 210-NanR-Q208R / A209T / Q220G-cpYFP-Y1V.

[0244] After mixing 209 / 210-NanR-Q208R / A209T / Q220G-cpYFP-Y1V with diluted blood supernatant and treating for 10 minutes, the ratio of fluorescence intensity at 420nm excitation and 528nm emission to fluorescence intensity at 485nm excitation and 528nm emission was detected using an ELISA reader. The results are as follows: Figure 8 As shown, the sialic acid content in mouse blood is around 64 μM, while the sialic acid content in human blood is around 212 μM.

[0245] As can be seen from the above embodiments, the sialic acid optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature. It exhibits large fluorescence dynamic changes, good specificity, and can be expressed in cells through gene manipulation. It can locate and quantify sialic acid in and outside cells in real time and can also perform high-throughput compound screening.

[0246] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some basic features can be applied according to the scope of the appended claims.

[0247] Partial sequence of this article:

[0248] SEQ ID NO:1SdNanR(1-262 full length)

[0249] GLMNAFDSQTEDSSPAIGRNLRSRPLARKKLSEMVEEELEQMIRRREFGEGEQLPSERELMAFFNVGRPSVREALAALKRKGLVQINNGERARVSRPSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDQALHEHNNVSYQQHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0250] SEQ ID NO:2cpYFP

[0251] YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0252] SEQ ID NO:3cpGFP

[0253] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0254] SEQ ID NO:4cpBFP

[0255] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN

[0256] SEQ ID NO:5cpmApple

[0257] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA

[0258] SEQ ID NO:6 209 / 210-SdNanR-cpYFP

[0259] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDQAYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHNNVSYQQHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0260] SEQ ID NO:7 209 / 210-NanR-Q208R / A209T-cpYFP-Y1V

[0261] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDRTVNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHNNVSYQQHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0262] SEQ ID NO:8 209 / 210-NanR-Q208R / A209T / Q220G-cpYFP-Y1V

[0263] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDRTVNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHNNVSYQGHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0264] SEQ ID NO:9 209 / 210-NanR-Q208R / A209T / Q220E-cpYFP-Y1V

[0265] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDRTVNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHNNVSYQGHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0266] SEQ ID NO:10 209 / 210-NanR-Q208R / A209T / S217T-cpYFP-Y1V

[0267] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDRTVNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHNNVTYQGHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0268] SEQ ID NO:11 209 / 210-NanR-Q208R / A209T / Q220N-cpYFP-Y1V

[0269] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDRTVNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHNNVSYQNHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0270] SEQ ID NO:12 209 / 210-NanR-Q208R / A209T / N214H-cpYFP-Y1V

[0271] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDRTVNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHHNVSYQQHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

[0272] SEQ ID NO:13 209 / 210-NanR-Q208R / A209T / Q220S-cpYFP-Y1V

[0273] PSADTIIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQSLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDRTVNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLHEHNNVSYQSHIAIVDAIRRHDPDEADRALQSHLNSVSATWYAFGQTTNKKK

Claims

1. A variant of a sialic acid-binding protein, which (a) Having the sequence shown in SEQ ID NO:1 or a functional fragment thereof with amino acids truncated from positions 1-96 and having mutations at one or more of the following sites: S115, H116, N164, Q208, A209, N214, S217, Q220, (b) The sequence has at least 70% sequence identity with (a) and has the mutation described in (a) while retaining the ability to bind sialic acid. Preferably, The mutations are selected from any of the following groups: (1) S115E and H116F, (2) S115L and H116G, (3) S115A and H116F, (4) S115V and H116L, (5) S115R and H116F, (6) S115E and H116W, (7) S115A and H116A, (8) S115G and H116H, (9) S115G and H116L, (10) S115W and H116V, (11) S115A and H116D, (12) Q208R and A209T, (13) Q2 08V and A209V, (14)Q208K and A209S, (15)Q208R, A209T and N164I, (16)Q208R, A209T and N214H, (17)Q208R, A209T and S217T, (18)Q208R, A209T and Q220D, (19)Q208R, A209T and Q220E, (20)Q208R, A209T and Q220G, (21)Q208R, A209T and Q220N, (22)Q208R, A209T and Q220S.

2. A sialic acid optical probe comprising a sialic acid-sensitive polypeptide and an optically active polypeptide, wherein, The optically active polypeptide is located within the sequence of the sialic acid-sensitive polypeptide. Preferably, the sialic acid-sensitive polypeptide has: (i) the sequence shown in SEQ ID NO:1 or a functional fragment thereof after truncating amino acids 1-96, or a sequence having at least 70% sequence identity with it and retaining sialic acid binding activity; (ii) the sequence of the sialic acid-binding protein variant of claim 1; or (iii) a sequence having at least 70% sequence identity with the sequence of (ii) and having the mutation of (ii) and retaining sialic acid sensitivity; and / or The optically active polypeptide is a fluorescent protein or a functional variant thereof, wherein the fluorescent protein functional variant has a mutation in 3 amino acids at the link with the sialic acid-sensitive polypeptide. Preferably, the optically active polypeptide is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, or apple red fluorescent protein.

3. The sialic acid optical probe as described in claim 2, characterized in that, The optically active polypeptide is located at one or more sites of the sialic acid-sensitive polypeptide selected from the following: 113 / 114, 113 / 115, 113 / 116, 113 / 117, 113 / 118, 113 / 119, 113 / 120, 113 / 121, 113 / 122, 114 / 114, 114 / 115, 114 / 116, 114 / 117, 114 / 118, 114 / 119, 114 / 120, 114 / 121, 114 / 122, 115 / 114, 115 / 115, 115 / 116, 115 / 117, 115 / 118, 115 / 119, 115 / 120, 115 / 121, 115 / 122, 11 6 / 114, 116 / 116, 116 / 118, 116 / 120, 116 / 122, 116 / 124, 116 / 126, 116 / 128, 116 / 130, 117 / 114, 117 / 115, 117 / 116, 117 / 117, 117 / 118, 117 / 119, 117 / 120 117 / 121, 117 / 122, 118 / 114, 118 / 115, 118 / 116, 118 / 117, 118 / 118, 118 / 119, 118 / 120, 118 / 121, 118 / 122, 119 / 114, 119 / 115, 119 / 116, 119 / 117, 119 / 118, 119 / 119, 119 / 120, 119 / 121, 119 / 122, 120 / 114, 120 / 115, 120 / 116, 120 / 117, 120 / 118, 120 / 119, 120 / 120, 120 / 121, 120 / 122, 139 / 140, 139 / 141, 1 39 / 142, 139 / 143, 139 / 144, 139 / 145, 140 / 141, 140 / 142, 140 / 143, 140 / 144, 140 / 145, 141 / 142, 141 / 143, 141 / 144, 141 / 145, 142 / 143, 142 / 144, 142 / 14 5, 143 / 144, 143 / 145, 144 / 145, 159 / 160, 159 / 161, 159 / 162, 159 / 163, 159 / 164, 159 / 165, 160 / 161, 160 / 162, 160 / 163, 160 / 164, 160 / 165, 161 / 162, 161 / 163, 161 / 164, 161 / 165, 162 / 163, 162 / 164, 162 / 165, 163 / 164, 163 / 165, 164 / 165, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 178 / 184, 178 / 185,178 / 186、179 / 180、179 / 181、179 / 182、179 / 183、179 / 184、179 / 185、179 / 186、180 / 181、180 / 182、180 / 183、180 / 184、180 / 185、180 / 186、181 / 182、181 / 183、181 / 184、181 / 185、181 / 186、182 / 183、182 / 184、182 / 185、182 / 186、183 / 184、183 / 185、183 / 186、184 / 185、184 / 186、185 / 186、200 / 201、200 / 202、200 / 203、200 / 204、200 / 205、200 / 206、200 / 207、200 / 208、200 / 209、200 / 210、200 / 211、200 / 212、201 / 201、201 / 202、201 / 203、201 / 204、201 / 205、201 / 206、201 / 207、201 / 208、201 / 209、201 / 210、201 / 211、201 / 212、202 / 201、202 / 202、202 / 203、202 / 204、202 / 205、202 / 206、202 / 207、202 / 208、202 / 209、202 / 210、202 / 211、202 / 212、203 / 201、203 / 202、203 / 203、203 / 204、203 / 205、203 / 206、203 / 207、203 / 208、203 / 209、203 / 210、203 / 211、203 / 212、204 / 201、204 / 202、204 / 203、204 / 204、204 / 205、204 / 206、204 / 207、204 / 208、204 / 209、204 / 210、204 / 211、204 / 212、205 / 201、205 / 202、205 / 203、205 / 204、205 / 205、205 / 206、205 / 207、205 / 208、205 / 209、205 / 210、205 / 211、205 / 212、206 / 201、206 / 202、206 / 203、206 / 204、206 / 205、206 / 206、206 / 207、206 / 208、206 / 209、206 / 210、206 / 211、206 / 212、207 / 201、207 / 202、207 / 203、207 / 204、207 / 205、207 / 206、207 / 207、207 / 208、207 / 209、207 / 210、207 / 211、207 / 212、208 / 201, 208 / 202, 208 / 203, 208 / 204, 208 / 205, 208 / 206, 208 / 207, 208 / 208, 208 / 209, 208 / 210, 208 / 211, 208 / 212, 209 / 201, 209 / 202, 209 / 203, 209 / 204, 209 / 205, 20 9 / 206, 209 / 207, 209 / 208, 209 / 209, 209 / 210, 209 / 211, 209 / 212, 210 / 201, 210 / 202, 210 / 203, 210 / 204, 210 / 205, 210 / 206, 210 / 207, 210 / 208, 210 / 209, 210 / 210, 210 / 2 11, 210 / 212, 211 / 201, 211 / 202, 211 / 203, 211 / 204, 211 / 205, 211 / 206, 211 / 207, 211 / 208, 211 / 209, 211 / 210, 211 / 211, 211 / 212, 228 / 229, 228 / 230, 228 / 231, 228 / 232 228 / 233, 228 / 234, 229 / 230, 229 / 231, 229 / 232, 229 / 233, 229 / 234, 230 / 231, 230 / 232, 230 / 233, 230 / 234, 231 / 232, 231 / 233, 231 / 234, 232 / 233, 232 / 234 and / or 233 / 234, Preferably, the optically active polypeptide is located at one or more sites of the sialic acid-sensitive polypeptide selected from the following: 115 / 117, 116 / 116, 117 / 116, 120 / 117, 205 / 202, 120 / 121, 120 / 118, 207 / 207, 211 / 208, 211 / 209, 206 / 210, 206 / 211, 208 / 210, 209 / 210, 208 / 206, 208 / 207, 211 / 207, 209 / 209, 210 / 209, 209 / 208, 206 / 208, 206 / 21 1, 121 / 120, 208 / 208, 205 / 203, 114 / 120, 115 / 119, 115 / 122, 116 / 121, 118 / 116, 203 / 205, 205 / 210, 207 / 210, 119 / 121, 200 / 201, 206 / 204, 208 / 204, 208 / 205, 211 / 205, 210 / 206, 210 / 207, 114 / 118, 115 / 116, 205 / 212, 118 / 114, 115 / 115, 209 / 202, 210 / 202, More preferably, the optically active polypeptide is located at positions 116 / 116 and / or 209 / 210 of the sialic acid-sensitive polypeptide.

4. A fusion polypeptide comprising the sialic acid optical probe as described in claim 2 or 3, and a functional polypeptide. Preferably, the functional polypeptide is a localization sequence, a tag that is easy to purify, or a tag used for immune responses.

5. An isolated polynucleotide comprising: (a) The nucleotide sequence or its complementary sequence encoding the sialic acid binding protein variant of claim 1, the sialic acid optical probe of claim 2 or 3, the fusion polypeptide of claim 4. (b) A nucleotide sequence that has at least 80% identity with the sequence shown in (a) or its complementary sequence, or (c)(a) or (b) primer fragments.

6. A nucleic acid construct comprising the polynucleotide as described in claim 5, Preferably, the nucleic acid construct is a vector.

7. An engineered host cell, said cell: (1) Containing, expressing, or secreting the sialic acid-binding protein variant of claim 1, the sialic acid optical probe of claim 2 or 3, or the fusion polypeptide of claim 4. (2) Containing the polynucleotide of claim 5, or (3) Contains the nucleic acid construct as described in claim 6.

8. A method for preparing a sialic acid optical probe as described in claim 2 or 3, comprising the steps of: (1) Providing the host cell as described in claim 7, (2) The host cells were cultured under suitable conditions for the expression of the sialic acid optical probe. (3) Separate the sialic acid optical probe.

9. A test kit comprising: (1) The optical probe according to any one of claims 2-4, (2) The polynucleotide of claim 5, (3) The nucleic acid construct according to claim 6, or (4) The host cell according to claim 7, The test kit may optionally include other reagents required for the detection of sialic acid using an optical probe. Preferably, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, and sialic acid standard.

10. The use of the sialic acid optical probe of claim 2 or 3, the fusion polypeptide of claim 4, the polynucleotide of claim 5, the nucleic acid construct of claim 6, or the host cell of claim 7 in detecting sialic acid in a sample, screening compounds, or for intracellular and / or extracellular localization of sialic acid. Preferably, the detection of sialic acid in a sample includes the steps of: contacting the optical probe or host cell with the sample, detecting optical changes in optically active peptides, and detecting sialic acid in the sample based on the optical changes in optically active peptides. The compound screening process includes the steps of: contacting the optical probe or host cells with the candidate compound in a sialic acid-containing system, detecting optical changes in the optically active peptide, and screening compounds based on the optical changes in the optically active peptide; preferably, the compound screening process includes the steps of: contacting the host cells with the candidate compound in a sialic acid-containing system, and the optical changes in the optically active peptide indicating whether the candidate compound regulates the uptake of sialic acid by the cells. The intracellular / extracellular localization of sialic acid includes the steps of: contacting the sialic acid-containing system with the optical probe or the host cell, and detecting optical changes in the optically active peptide. More preferably, the system is a solution system, a cellular system, or a subcellular system.

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