Recombinably expressed carbohydrate binding protein and application thereof in carbohydrate analysis

Abstract

The invention discloses a recombinably expressed carbohydrate binding protein and application of the recombinably expressed carbohydrate binding protein in carbohydrate analysis, and the recombinably expressed carbohydrate binding protein is a protein which is obtained by mutating a nucleophilic attack group or an amino acid containing an acid-base group in glucoside hydrolase and has a carbohydrate binding capability. According to the embodiment of the invention, experiments prove that the RGB and the polymer thereof have better application effects in the fields of carbohydrate immunoblotting, flow labeling, affinity enrichment and immunohistochemistry; the method can be compatible with a single cell sequencing technology, and single cell conjoint analysis of various glycotypes and transcriptomes on the surface of the cell is realized.

Description

Technical Field

[0001] The present invention reports a new type of recombinantly expressed glycosyltransferase (RGB) and its application, which involves protein recombinant expression, glycosyltransferase (RGB) polymer construction and its application, including but not limited to glycosyltransferase labeling, enrichment, single cell joint analysis of cell surface glycosyltransferase and transcriptome. Background Art

[0002] There are many complex and functional carbohydrate molecules on the surface of mammalian cells, which are anchored on the cell membrane in the form of glycoproteins or glycolipids. Recent studies have found that glycans play an important role in many biological processes. For example, cancer cells can achieve immune escape by interacting with immune cells through surface sialylated carbohydrates. Compared with proteins or nucleic acids, the synthesis of carbohydrates is not regulated by templates and has complex and diverse structures. Therefore, the specific labeling of carbohydrates has become an important basis and key technology for studying the biological functions of glycans ( Figure 1 ). Among them, fluorescent group labeling enables live cell and in vivo imaging and flow cytometry analysis of carbohydrates; affinity group labeling enables the enrichment and omics analysis of glycoproteins and glycoconjugates. On the other hand, the study of the biological functions of carbohydrates has been mainly based on a large number of cell samples in the past, and our understanding of its heterogeneity and microscopic heterogeneity at the cellular level is still in its early stages. In order to promote the functional analysis of glycobiology at the single-cell level, single-cell multimodal analysis containing carbohydrate information is in the ascendant. Its essence and core strategy lies in converting carbohydrate structure and abundance information into sequence and abundance information of specific DNA through specific labeling, and integrating it with single-cell transcriptome sequencing.

[0003] At present, the main methods for labeling carbohydrates include lectin labeling, chemical labeling, chemoenzymatic labeling, and metabolic labeling. Lectins are a class of carbohydrate-binding proteins isolated from plants that can recognize and bind to carbohydrates with specific structural characteristics. Lectins are core research tools in the field of glycoscience, but due to their low specificity and affinity for sugar substrate binding and certain cytotoxicity to living cells, their applications in precise glycoform analysis and characterization of living cell carbohydrates are limited. Compared with the lectin method, although chemical labeling and chemoenzymatic labeling have higher specificity for glycoforms, their applicable glycoforms are limited. For example, chemical labeling can currently only achieve accurate labeling of sialylated carbohydrates. Metabolic labeling incorporates non-natural sugars containing bioorthogonal functional groups into the carbohydrate synthesis pathway of cells, so that specific carbohydrate structures carry bioorthogonal functional groups for labeling and analysis. However, this method requires the cultivation of cell or tissue samples, and its application range is difficult to expand to clinical samples. In addition, different cells have different abilities to take up non-natural sugars, and the labeling intensity is difficult to accurately reflect the true carbohydrate level on the cell surface.

[0004] Since the above methods have their own problems, it is necessary to develop new sugar labeling technologies. Summary of the invention

[0005] The technical problem solved by the present invention is that: the present invention solves the problems of poor specificity, high cytotoxicity, the need to culture cells or limited available labeling tools in existing carbohydrate labeling methods by developing a new carbohydrate binding protein, thereby achieving highly selective and efficient labeling of carbohydrates.

[0006] The present invention provides a recombinantly expressed carbohydrate binding protein, which is a protein with carbohydrate binding ability obtained by mutating a nucleophilic attacking group or an amino acid containing an acid-base group (including D, E, Y, etc.) in a glycoside hydrolase.

[0007] Wherein, the glycoside hydrolase is a glycoside hydrolase derived from prokaryotes.

[0008] Furthermore, the glycoside hydrolase is one of α2,3 / 6 / 8-sialyl hydrolase, α2,3-sialyl hydrolase and β-galactosidase.

[0009] The recombinantly expressible carbohydrate binding protein according to claim 2, characterized in that the carbohydrate binding protein is a protein described in any one of the following A1)-A6):

[0010] A1) The amino acid sequence is the protein shown in Sequence 3 in the sequence list;

[0011] A2) The amino acid sequence is the protein shown in Sequence 4 in the sequence list;

[0012] A3) The amino acid sequence is the protein shown in Sequence 6 in the sequence list;

[0013] A4) The amino acid sequence is the protein shown in Sequence 7 in the sequence listing;

[0014] A5) The amino acid sequence is the protein shown in Sequence 9 in the sequence listing;

[0015] A6) The amino acid sequence is the protein shown in Sequence 10 in the sequence listing;

[0016] A7) A protein whose amino acid sequence comprises at least two of the above sequences.

[0017] A method for preparing a recombinantly expressed carbohydrate binding protein comprises the following steps:

[0018] 1) Using glycosyl hydrolase, the key catalytic site in the enzyme activity pocket was selected for mutation to obtain mutant 1;

[0019] 2) The substrate binding ability and specificity of the enzyme of mutant 1 are evaluated, and mutants with good binding ability and specificity are screened as carbohydrate binding proteins.

[0020] Among them, the key sites are two amino acid residue sites containing carboxyl groups.

[0021] The present invention provides a method for preparing a carbohydrate binding protein polymer, using the carbohydrate binding protein, comprising the following steps:

[0022] 2) modifying the N-terminus of the carbohydrate binding protein with biotin to obtain a biotin-modified carbohydrate binding protein;

[0023] 2) The biotin-modified carbohydrate-binding protein is co-incubated with streptavidin to obtain carbohydrate-binding protein polymers.

[0024] The use of the recombinantly expressible carbohydrate binding protein or the preparation method in any of the following:

[0025] B1) Application in single cell polysaccharide profiling;

[0026] B2) Application in carbohydrate affinity enrichment based on recombinantly expressed carbohydrate binding proteins;

[0027] B3) Application as an antibody in immunohistochemistry;

[0028] B4) Application in the localization or quantitative detection of sugars on the surface of tissues or cells;

[0029] B5) Application in flow cytometry analysis.

[0030] The invention provides a single cell polysaccharide analysis method, using the recombinantly expressible carbohydrate binding protein.

[0031] The specific steps are:

[0032] 1) Mixing the carbohydrate binding protein with the streptavidin containing the DNA barcode to obtain a carbohydrate binding protein polymer coupled with the DNA barcode;

[0033] 2) incubating the cell suspension to be tested with the carbohydrate-binding protein polymer coupled with the DNA barcode;

[0034] 3) performing flow cytometry sorting and single-cell sequencing on the cell suspension after co-incubation in step 2);

[0035] 4) Further analyze and process the results of single-cell sequencing to obtain single-cell polysaccharide results.

[0036] The invention provides a single cell polysaccharide analysis reagent, comprising the recombinantly expressible carbohydrate binding protein.

[0037] These also include streptavidin containing DNA barcodes or fluorescent antibodies for sorting cells.

[0038] The invention provides a carbohydrate affinity enrichment method based on RGB, using the carbohydrate binding protein that can be recombinantly expressed.

[0039] The specific steps are:

[0040] 1) Co-incubating the carbohydrate-binding protein with a pairable tag with streptavidin resin to obtain Streptavidin-RGB resin;

[0041] 2) Incubate the Streptavidin-RGB resin with the mixed protein solution;

[0042] 3) Washing, non-glycoproteins are washed out with the washing solution;

[0043] 4) Competitive monosaccharide elution is used and the glycoprotein flows out with the eluent.

[0044] A method for localizing or quantifying sugars on the surface of tissues or cells, using the recombinantly expressed sugar-binding protein, specifically: using the recombinantly expressed sugar-binding protein or its polymer form as an antibody in cell imaging, or using the recombinantly expressed sugar-binding protein or its polymer form as an antibody in flow cytometry.

[0045] The technical effect achieved by the present invention is that: in order to solve some problems existing in the current sugar labeling tools, the present invention rationally designs and transforms glycosyl hydrolases from multiple bacterial sources, and successfully develops a series of novel recombinantly expressed glycosyl binding proteins (RGB), including RGB-SiaTotal, RGB-Sia3 and RGB-Gal, which can respectively recognize sialic acid-terminated glycosyl, 2,3-linked sialic acid-terminated glycosyl and galactose-terminated glycosyl. Through immunoblotting and flow cytometry, we proved that RGB-SiaTotal, RGB-Sia3 and RGB-Gal have strict substrate selectivity and strong binding ability. Further, the present invention utilizes the multivalency effect to improve the affinity of RGB, specifically, using the Escherichia coli biotin ligase system (Biotin ligase, BirA) to introduce site-specific biotinylation modification at the end of RGB and assemble it with fluorescent or DNA-modified streptavidin to obtain fluorescent or DNA-modified RGB polymers, and proves that the labeling intensity of RGB polymers is significantly improved compared with RGB monomers. In addition, it was verified that different RGB polymers can simultaneously label multiple glycoforms on the cell surface through a one-pot method. Based on RGB polymers, the present invention has developed scRGB-seq (single-cell Recombinant Glycan Binder Sequencing) to achieve a single-cell level technology for the joint analysis of multiple glycoforms and transcriptomes. In the embodiments of the present invention, it is experimentally demonstrated that RGB and its polymers have good application effects in the fields of carbohydrate immunoblotting, flow labeling, affinity enrichment, and immunohistochemistry; and can be compatible with single-cell sequencing technology to achieve single-cell joint analysis of multiple glycoforms on the cell surface and transcriptomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of carbohydrate labeling and analysis technology;

[0047] Figure 2 For the development and characterization of RGB-SiaTotal;

[0048] Figure 3 For the development and characterization of RGB-Sia3;

[0049] Figure 4 For the development and characterization of RGB-Gal;

[0050] Figure 5 To construct and characterize the binding properties of RGB polymers;

[0051] Figure 6 Optimize the labeling process and characterize the cytotoxicity of RGB polymers;

[0052] Figure 7 It is a one-pot method for simultaneous labeling of multiple glycoforms by RGB polymers;

[0053] Figure 8 RGB-based cell imaging;

[0054] Fig. 9 Development and application of scRGB-seq, a single-cell polysaccharide profiling technology based on RGB;

[0055] Fig.10 For RGB-based sugar enrichment;

[0056] Fig.11 RGB-based immunohistochemistry. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] The pET-15b-SpNanA plasmid in the following examples was kindly provided by the research group of Professor Wang Peng of Nankai University, the plasmid of RgNanH was synthesized by Beijing Xianghong Biological Co., Ltd., the plasmid of LacZ was obtained by PCR from E. coli K-12 MG1655 Escherichia coli, which was purchased from the China Industrial Microbiological Culture Collection Center CICC, the pET-Dual-His-Avi-MCS-BirA vector was purchased from Biyuntian Biotechnology Co., Ltd., and Hela cells ( CCL-2) was purchased from the American Type Culture Collection, BL21 (DE3) and Trans1-T1 Escherichia coli competent cells and rapid site-directed mutagenesis kit were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and TotalSeq TM -B0952PE Streptavidin,TotalSeq TM-B0954 PE Streptavidin, APC-anti-His flow cytometry antibody, APC-anti-mouse-CD45, Anti-mouse-CD16 / 32 were purchased from Biolegend, HRP-conjugated 6*His antibody was purchased from Proteintech, AF647-Streptavidin, AF488-Streptavidin, restriction enzymes BamHI and SalI were purchased from Thermo Fisher, 2×Phanta Max Master Mix and IIOneStep Cloning Kit homologous recombination kit was purchased from Nanjing Novezan Biotechnology Co., Ltd., IPTG, ampicillin and 5 mL nickel column were purchased from Yisheng Biotechnology Co., Ltd., biotin powder was purchased from Aladdin Biochemical Technology Co., Ltd., fetuin was purchased from Puxitang Biotechnology Co., Ltd., Neu5Ac(α2,3)-Galactose(β1,4)-GlcNAc(3'SLN) and Neu5Ac(α2,6)-Galactose(β1,4)-GlcNAc(6'SLN) were purchased from Wuhan Tangzhi Pharmaceutical Co., Ltd., px458 was purchased from Addgene Biotechnology Co., Ltd., and the high-efficiency eukaryotic transfection reagent VigoFect was purchased from Vigoras Biotechnology Co., Ltd.

[0060] Example 1 Development and characterization of RGB-SiaTotal:

[0061] The α2,3 / 6 / 8-sialyl hydrolase SpNanA from Streptococcus pneumonia was used as a substrate. The nucleotide sequence of SpNanA is shown in SEQ ID NO: 1. The key catalytic sites E647 and D372 were mutated, and SpNanA was expressed and purified. D372A 、SpNanA D372N 、SpNanA E647A and SpNanA E647Q , and characterize the binding ability and specificity of the mutants. The specific methods are as follows:

[0062] 1. Construction of mutant sequences:

[0063] All SpNanA mutants were constructed according to the procedure of the Rapid Site-Directed Mutagenesis Kit (Transgen):

[0064] (1) Design the primers required for the mutant strain, as shown in Table 1:

[0065] Table 1 Primers used to construct SpNanA mutants

[0066]

[0067] (2) PCR amplification system 25 μL: add ddH2O 10.5 μL, 10 μM forward and reverse primers 0.5 μL each, template 1 μL (containing about 5 ng wild-type pET-15b-SpNanA plasmid), 2×TransStartFastPfu PCR SuperMix 12.5 μL in sequence. After mixing, PCR amplification was performed. The PCR reaction was annealed at 55°C for 30 seconds, extended at 72°C for 2 minutes, and repeated 30 cycles. The amplified fragment was tested by 1% agarose gel electrophoresis to determine whether the target band amplified by PCR was correct.

[0068] (3) Digest the PCR product with the correct band size with DMT enzyme (37°C, 1h); transfer the digested PCR product into DMT competent cells; pick single clone colonies, extract the plasmid and sequence it; select the plasmid with the correct sequencing for subsequent research.

[0069] 2. Purification of mutant proteins and wild-type proteins:

[0070] (1) Transformation: Take 2 μL of wild-type SpNanA plasmid or mutant plasmid and transfer into 50 μL E.coli BL21 (DE3) competent cells, incubate on ice for 20 min, heat shock at 42°C for 1 min, and then immediately incubate on ice for 2 min. Add 500 μL of antibiotic-free LB medium and shake culture at 37°C for 1 h. Then spread the bacterial solution evenly on an LB plate containing 100 μg / mL ampicillin and culture in a 37°C incubator overnight.

[0071] (2) Pick-up expansion culture: Pick a single clone from the plate and place it in 4 mL of LB medium containing 100 μg / mL ampicillin, and culture it at 37°C with shaking overnight; transfer the overnight culture liquid into 1 L of LB medium (containing 100 μg / mL ampicillin) and culture it at 37°C, 220 rpm / min, for 3-5 h.

[0072] (3) Induction: When the OD600 of the bacterial solution reaches 0.6-0.8, add IPTG for induction (final concentration is 500 μM) and culture at 25°C, 180 rpm / min for 20 h.

[0073] (4) Purification: Centrifuge at 4500g, 20℃ for 10 min to remove the supernatant and collect the induced Escherichia coli; resuspend the precipitate in PBS solution; ultrasonically disrupt it in an ice-water mixed bath; centrifuge at 13000g, 4℃ for 30 min to collect the supernatant; place all the supernatant in a pre-treated nickel column (rinse with binding solution for 5 column volumes, 10mM imidazole in PBS); then rinse with 10 column volumes of cleaning solution (30mM imidazole in PBS); finally, the target protein is eluted with elution solution (250mM imidazole in PBS); finally, ultrafiltration is performed three times using a 30kDa ultrafiltration tube (4500g, 20min).

[0074] (5) Identification: The eluted target protein was subjected to polyacrylamide gel electrophoresis (10% SDS-PAGE) to detect whether the molecular weight was correct.

[0075] 3. Quantification: Use NanoDrop spectrophotometer to test the absorbance of the protein solution at 280nm to obtain the concentration of the purified protein solution. D372N 、SpNanA D372A 、SpNanA E647A and SpNanA E647Q Five proteins, WTSpNanA, SpNanA D372N 、SpNanA D372A The amino acid sequences are shown in SEQ ID NOs: 2-4, SpNanA E647A The amino acid sequence is obtained by replacing N at position 372 with D and E at position 647 with A in sequence 3; SpNanA E647Q The amino acid sequence is a sequence obtained by replacing N at position 372 with D and replacing E at position 647 with Q in sequence 3.

[0076] 4. Identification of mutant activity: Fetuin is a highly sialylated protein. When it is treated with active sialidase, a band shift of about 5 kD can be seen on gel electrophoresis.

[0077] (1) First, the wild-type SpNanA and four mutants (SpNanA D372A 、SpNanA D372N 、SpNanA E647A and SpNanA E647Q ) were incubated with fetuin (molar ratio 1:100) at 37°C for 1 h.

[0078] (2) Take an equal amount of treated or untreated fetuin sample, add an equal amount of 5× loading buffer, and heat at 95°C for 10 min; observe the position of fetuin bands by polyacrylamide gel electrophoresis (10% SDS-PAGE).

[0079] (3) The results show that Figure 2 b), the positions of the serum protein bands after treatment with the four mutants were the same as those of the untreated serum proteins, and had obvious differences in band positions compared with the serum proteins treated with the wild type, indicating that the four mutants had lost their hydrolysis activity.

[0080] 5. Screening of mutant binding ability using flow cytometry:

[0081] (1) First, the wild-type hydrolase SpNanA or mutant protein (20 μg / mL) purified in Example 1.2 was incubated with Hela cells in PBS buffer at room temperature for 1 h, and then washed twice with PBS.

[0082] (2) Incubate with anti-His fluorescent antibody conjugated to APC (20 μg / mL) at room temperature for 30 min, wash twice with PBS and detect using flow cytometry.

[0083] (3) The results show that Figure 2 c), D372A, D372N and E647A have certain binding abilities, and the binding abilities of D372A and D372N are stronger.

[0084] 6. Immunochemiluminescence analysis of SpNanA D372N Characterization of the binding specificity of mutants:

[0085] (1) First, fetuin (molar ratio 1:100) was desialylated using α2,3 / 6 / 8-sialyl hydrolase SpNanA and α2,3-linked sialyl hydrolase RgNanH and incubated at 37°C for 1 h.

[0086] (2) At the same time, take an equal amount of untreated fetuin, add an equal amount of 5× loading buffer to the three samples, and heat at 95°C for 10 min.

[0087] (3) Three fetuin samples (10 μg) were subjected to 10% SDS gel electrophoresis and transferred to a membrane, and then blocked with TBST containing 5% BSA for one hour.

[0088] (4) Preparation of SpNanA D372N (20 μg / mL) in TBST and incubated with the proteins on the membrane at 4°C overnight; washed three times with TBST, each wash for 5 min.

[0089] (5) Incubate the membrane with HRP-conjugated Anti-His antibody at a final concentration of 0.2 μg / mL at room temperature for 1 h; wash three times with TBST, each wash for 5 min.

[0090] (6) The membrane was scanned and imaged using Tanon. The results showed that ( Figure 2 d), SpNanA D372N Can bind fetuin, and this binding is completely dependent on the sialic acid content.

[0091] 7. Use ITC to detect SpNanA D372N Characterization of the binding ability of mutants:

[0092] (1) The protein was dissolved in PBS at a concentration of 100 μM, and Neu5Ac(α2,3)-Galactose(β1,4)-GlcNAc(3'SLN) and Neu5Ac(α2,6)-Galactose(β1,4)-GlcNAc(6'SLN) were dissolved in PBS at a concentration of 1 mM.

[0093] (2) Perform ITC isothermal titration, and the parameter settings are shown in Table 2:

[0094] Table 2 ITC program parameter settings

[0095]

[0096] (3) The original data was fitted using Origin7.0 to obtain K d The values ​​were 36 μM (3'SLN) and 18 μM (6'SLN), respectively.

[0097] (4) The results show that SpNanA D372N It binds to both α2,3- and α2,6-linked sialylated glycans without significant preference.

[0098] 8. Characterization of the binding specificity of mutants using flow cytometry:

[0099] (1) Construction of CMAS knockout Hela cell line using CRISPER-Cas9 technology

[0100] CMAS is the only CMP-Neu5Ac synthase in mammalian cells, and the cell line with this gene knockout theoretically has no sialylation modification. Here, we used CRISPERPick software to design gRNA targeting a specific gene and constructed a plasmid px458-sgRNA that can express gRNA, Cas9 and GFP.

[0101] We propagated Hela cells to six-well plates and grew them to 70% confluence, and transfected px458-gRNA into the cells using the highly efficient eukaryotic transfection reagent VigoFect. 48 hours after transfection, we used the AriaIII high-performance flow cytometer to sort GFP-positive single cells into 96-well plates and continued to culture for 2 weeks. We extracted the genome of the obtained monoclonal cell line and sequenced the target gene. The cells with successful knockout of the CMAS gene were CMAS - / - Hela.

[0102] The gRNA sequences are shown in the table below.

[0103]

[0104] (2) Obtaining Hela cells pretreated with α2,3 / 6 / 8-sialyl hydrolase SpNanA: After normal Hela cells were digested, they were washed twice with PBS buffer, and wild-type sialyl hydrolase SpNanA (purified in the above Example 1.2) was taken and incubated with Hela cell suspension in PBS buffer (final concentration of 20 μg / mL) at room temperature for 45 minutes; after washing twice with PBS buffer, Hela cells pretreated with sialyl hydrolase SpNanA were obtained.

[0105] (3)SpNanA D372N (20 μg / mL) was used in combination with three cell lines: wild-type HeLa (WT HeLa), HeLa pretreated with α2,3 / 6 / 8-sialyl hydrolase SpNanA, and CMAS knockout cells. - / - Hela was incubated for 1 h at room temperature and washed twice with PBS.

[0106] (4) Incubate with anti-His fluorescent antibody conjugated to APC (20 μg / mL) at room temperature for 30 min, wash twice with PBS, and then detect using flow cytometry.

[0107] (5) Sialidase pretreatment can cut off most of the sialic acid on the surface of Hela cells, CMAS - / - Hela cells have no sialic acid on their surface, but both cells contain all monosaccharides and polysaccharides except sialic acid. Figure 2 g), WT Hela can be induced by SpNanA D372N Efficient labeling of Hela cells and CMAS pretreated with sialidase - / - Hela cells cannot be recognized and bound by the mutant protein, indicating that the binding of the mutant to the cells depends entirely on the content of sialic acid on the cell surface.

[0108] At this point, due to SpNanA D372N The substrate selectivity of the mutants was defined as RGB-SiaTotal.

[0109] Example 2 Development and Characterization of RGB-Sia3:

[0110] The α2,3-sialyl hydrolase RgNanH from Ruminococcus gnavus was used as a substrate. The α2,3-sialyl hydrolase RgNanH can hydrolyze α2,3-linked sialic acid but not α2,6 / 8-linked sialic acid. Its nucleotide sequence is sequence 5. The key catalytic site D282 was mutated, and RgNanH was expressed and purified. D282A and RgNanH D282N , and characterize the binding ability and specificity of the mutants. The specific methods are as follows:

[0111] 1. Expression and purification of RgNanH mutant protein

[0112] The RgNanH gene sequence was synthesized de novo by the company after codon optimization. Its amino acid sequence was obtained by changing the 282nd position of sequence 3 from A to D, and was constructed into the pET-15b vector. All RgNanH mutants were constructed with reference to the operation steps of the Rapid Site-Directed Mutagenesis Kit (Transgen). PCR amplification and point-selected sequencing were as described above. D282A and RgNanH D282N The two mutants were subsequently characterized. D282A and RgNanH D282N The amino acid sequences are shown in Sequence 6 and Sequence 7 in the sequence listing.

[0113] Table 3 Primers used to construct RgNanH mutants

[0114]

[0115] 2. Identification of RgNanH mutant protein activity:

[0116] (1) First, the wild-type RgNanH and the four mutants were incubated with fetuin (molar ratio 1:100) at 37°C for 1 h.

[0117] (2) At the same time, an equal amount of fetuin sample was taken and an equal amount of 5× loading buffer was added, and heated at 95° C. for 10 min; the position of the fetuin band was observed by polyacrylamide gel electrophoresis (10% SDS-PAGE).

[0118] (3) The results show that Figure 3b), the positions of the serum protein bands after treatment with the two mutants were the same as those of the untreated serum proteins, and had obvious differences in band positions compared with the serum proteins treated with the wild type, indicating that the two mutants had lost their hydrolysis activity.

[0119] 3. Screening of RgNanH mutant binding ability using flow cytometry:

[0120] (1) First, the wild-type or mutant protein (20 μg / mL) with His-tagged protein was incubated with Hela cells in PBS buffer at room temperature for 1 h and then washed twice with PBS.

[0121] (2) Incubate with anti-His fluorescent antibody conjugated to APC (20 μg / mL) at room temperature for 30 min and wash twice with PBS.

[0122] (3) Flow cytometry was used for detection. The results showed that ( Figure 3 c), RgNanH D282A The mutant has a stronger ability to bind to sialylated glycans on the cell surface.

[0123] 4. Western blot analysis of RgNanH D282A Characterization of the binding specificity of mutants:

[0124] (1) First, fetuin (molar ratio 1:100) was desialylated using α2,3 / 6 / 8-sialyl hydrolase SpNanA and α2,3-linked sialyl hydrolase RgNanH and incubated at 37°C for 1 h.

[0125] (2) At the same time, take an equal amount of untreated fetuin, add an equal amount of 5× loading buffer to the three samples, and heat at 95°C for 10 min.

[0126] (3) Three BSA samples (10 μg) were subjected to 10% SDS gel electrophoresis and transferred to a membrane, and then blocked for one hour using TBST containing 5% BSA.

[0127] (4) Preparation of RgNanH D282A (20 μg / mL) in TBST and incubated with the proteins on the membrane at 4°C overnight; washed three times with TBST, each wash for 5 min.

[0128] (5) Incubate the membrane with HRP-conjugated Anti-His antibody at a final concentration of 0.2 μg / mL at room temperature for 1 h; wash three times with TBST, each wash for 5 min.

[0129] (6) Use Tanon to scan and image the membrane.

[0130] (7) The results show that Figure 3 e), RgNanH D282A Can bind fetuin and is completely dependent on the α2,3-sialic acid content.

[0131] 5. Characterization of the binding specificity of mutants using flow cytometry:

[0132] (1) Hela cells were pretreated with α2,3-sialyl hydrolase RgNanH. 20 μg / mL of the enzyme was incubated with the cell suspension in PBS buffer at room temperature for 45 min. The cells were washed twice with PBS buffer.

[0133] (2)RgNanH D282A (20 μg / mL) was used in combination with three cell lines: WT Hela, Hela pretreated with α2,3-sialyl hydrolase RgNanH, and CMAS knockout cells containing CMP-Neu5Ac synthase CMAS. - / - Hela was incubated for 1 h at room temperature and washed twice with PBS.

[0134] (3) Incubate with anti-His fluorescent antibody conjugated to APC (20 μg / mL) at room temperature for 30 min, wash twice with PBS, and then detect using flow cytometry.

[0135] (4) RgNanH sialidase pretreatment can cleave off most of the α2,3-sialic acid on the surface of Hela cells, CMAS - / - Hela cells have no sialic acid on their surface, but both cell types contain all monosaccharides and polysaccharides except sialic acid.

[0136] (5) The results show that Figure 3 d) WT Hela can be expressed by RgNanH D282A Efficient labeling of Hela cells and CMAS pretreated with sialidase RgNanH - / - Hela cells cannot be recognized and bound by the mutant protein, indicating that the binding of the mutant to the cells depends entirely on the content of α2,3-sialic acid on the cell surface.

[0137] At this point, due to RgNanH D282A Substrate selectivity of the mutant, which was defined as RGB-Sia3.

[0138] Example 3 Development and Characterization of RGB-Gal:

[0139] Using β-galactosidase LacZ from Escherichia coli (E. coli) as a substrate, its key catalytic site E537 was mutated, and LacZ was expressed and purified E537Aand LacZ E537Q , and characterized the binding ability and specificity of the mutant, whose nucleotide sequence is sequence 8. The specific method is as follows:

[0140] After design and mutation, the enzyme can hydrolyze terminal β-linked galactose but not α-linked galactose.

[0141] 1. Expression and purification of mutant proteins

[0142] The lacZ gene sequence was obtained from the Escherichia coli genome and constructed into the pET-28a vector. All LacZ mutants were constructed by referring to the operation steps of the Rapid Site-Directed Mutagenesis Kit (Transgen), and PCR amplification and point-selection sequencing were as described above. Since the mutation of the E537 site has been reported in the literature to reduce the enzymatic activity of LacZ by more than 160,000-300,000, it is equivalent to an inactivating mutation. Therefore, the hydrolysis activity after the mutation was not characterized in this example. LacZ was constructed E537A and LacZ E537Q The two mutants were subsequently characterized. The specific method was the same as in Example 1. LacZ E537A and LacZ E537Q The amino acid sequences are shown in Sequence 9 and Sequence 10 in the sequence listing.

[0143] Table 4 Primers used to construct LacZ mutants

[0144]

[0145] 2. Characterization of the binding specificity of mutants using flow cytometry:

[0146] (1)LacZ E537A With LacZ E537Q (40 μg / mL) and WT Hela and CMAS - / - Hela cells were incubated at room temperature for 1 h and washed twice with PBS.

[0147] (2) Incubate with anti-His fluorescent antibody conjugated to APC (20 μg / mL) at room temperature for 30 min, wash twice with PBS, and then detect using flow cytometry.

[0148] (3) The results show that Figure 4 b) Since WT Hela cells are highly sialylated and have little galactose exposed on their surface, the labeling signal of the mutant is very weak. - / -When Hela cells are used, since there is no sialic acid at the end, a large amount of terminal galactose is exposed on the cell surface, which can be recognized and bound by the two LacZ mutants. This also indirectly reflects the selectivity of LacZ mutants for terminal galactose.

[0149] 3. Western blot analysis of LacZ E537Q Characterization of the binding specificity of mutants:

[0150] (1) First, fetuin (molar ratio 1:100) was desialylated using α2,3 / 6 / 8-sialyl hydrolase SpNanA and α2,3-linked sialyl hydrolase RgNanH and incubated at 37°C for 1 h.

[0151] (2) Take an equal amount of untreated fetuin at the same time, add an equal amount of 5× loading buffer to the three samples, and heat at 95°C for 10 min.

[0152] (3) Three BSA samples (10 μg) were subjected to 10% SDS gel electrophoresis and transferred to a membrane, and then blocked for one hour using TBST containing 5% BSA.

[0153] (4) Preparation of LacZ E537Q (40 μg / mL) in TBST and incubated with the proteins on the membrane at 4°C overnight; washed three times with TBST, each wash for 5 min.

[0154] (5) Incubate the membrane with HRP-conjugated Anti-His antibody at a final concentration of 0.2 μg / mL at room temperature for 1 h; wash three times with TBST, each wash for 5 min.

[0155] (6) Use Tanon to scan and image the membrane.

[0156] (7) The results show that Figure 4 c) The results show that as the terminal galactose residues exposed by BSA increase after sialidase treatment, LacZ E537Q This also reflects the selectivity of the LacZ mutant for terminal galactose.

[0157] At this point, due to LacZ E537Q Substrate selectivity of the mutants, which were defined as RGB-Gal.

[0158] Example 4 Construction and binding properties of RGB polymers:

[0159] This paper proposes to construct RGB into a polymer to improve the labeling effect of RGB on cell surface sugars, shorten the labeling time, and achieve one-pot labeling of multiple sugar forms ( Figure 5a). The core of this method is to assemble RGB monomers and streptavidin into polymers through the interaction between the site-specific modified biotin on RGB and streptavidin, thereby improving its affinity for cell surface sugars. In order to obtain RGB with site-specific modified biotin, the target gene was recombined into the pET-Dual-His-Avi-MCS-BirA vector by homologous recombination. This vector carries dual tags His-tag and Avi-tag, and has two T7 promoters, which can simultaneously express the target protein and the biotin ligase BirA. BirA can transfer the additional biotin added in the culture medium to the Avi-tag of the target protein, thereby obtaining the site-specific biotin-modified target protein, and can be directly affinity purified through the His tag without additional steps.

[0160] 1. Obtain biotin-modified RGB monomer:

[0161] (1) Preparation of linearized vector: pET-Dual-His-Avi-MCS-BirA was digested with restriction endonucleases BamHI and SalI, incubated at 37°C for 2 h, and then heated at 75°C to inactivate the enzymes.

[0162] (2) Amplification of target gene: PCR primers are shown in Table 5: Add 5 ng of target gene, 1 μL of 10 μM forward and reverse primers, and 2× Phanta Max Master Mix, then mix well and perform PCR amplification. The PCR reaction is annealed at 55°C for 30 seconds, extended at 72°C for 2 minutes, and repeated for 30 cycles. The molecular weight of the target band amplified by PCR is detected by 1% agarose gel electrophoresis.

[0163] Table 5 Primers used to construct biotinylated RGB monomers

[0164]

[0165] (3) Homologous recombination: IIOne Step Cloning Kit instructions for homologous recombination, 37℃ for 30min, and then inactivate the enzyme at 70℃. After transformation and plating, single clones were picked, plasmids were extracted, and plasmid sequences were verified by sequencing.

[0166] (4) Protein purification: The correctly sequenced plasmid was transferred into BL21 (DE3) competent cells. After plating, picking single clones and expanding the culture, when the OD600 of the bacterial solution reached 0.6-0.8, IPTG with a final concentration of 500 μM was added to induce expression. At the same time, 50 μM Biotin was added and the cells were cultured at 25°C and 180 rpm / min for 20 hours.

[0167] (5) The subsequent protein purification, identification and quantification steps are the same as above.

[0168] 2. Optimization of RGB polymer assembly ratio

[0169] Dilute the biotin-modified RGB monomer obtained in the previous step to 0.2 mg / mL (3.6 μM) PBS solution in a total of 20 μL as the standard assembly system, and add different concentrations of streptavidin coupled to the AF647 fluorescent group to make the final concentration of the streptavidin polymer 4.5 μM, 1.8 μM, 0.9 μM, 0.45 μM, 0.18 μM, 0.09 μM and 0.045 μM, corresponding to the molar ratio of RGB monomer to streptavidin monomer of 1:5, 1:2, 1:1, 2:1, 5:1, 10:1 and 20:1. Premix at 4°C for 1 hour or overnight.

[0170] (1) The premixed RGB polymers with different assembly ratios were added to a Hela cell suspension (cell concentration was 1.5×10^6 cells / mL) at a dilution ratio of 1:100 (total RGB concentration was 2 μg / mL), and the cells were incubated at room temperature for 1 hour. The cells were washed once with PBS solution and characterized by flow cytometry.

[0171] (2) A two-step method with the same concentration was used as a control: RGB monomer (2 μg / mL) was added to a cell suspension of equal concentration (1.5×10^6 cells / mL) and incubated at room temperature for 1 h; after washing twice with PBS, 2 μg / mL of streptavidin conjugated to AF647 fluorescent group was used for incubation at room temperature for 30 min. After washing once with PBS, flow cytometry was used for characterization.

[0172] (1) The results show that Figure 5 bf), compared with the two-step labeling method, the method of premixing RGB monomers with streptavidin can greatly improve the labeling intensity and efficiency; among them, the premix with a molar ratio of RGB monomers to streptavidin monomers of 2:1 has the strongest labeling signal.

[0173] 3. Characterization of RGB polymers:

[0174] (1) Take 8 μL of the premix of the strongest labeling effect of RGB monomer and streptavidin monomer with a molar ratio of 2:1 and the second strongest premix of molar ratios of 1:1 and 5:1, and 8 μL of PBS solution containing only RGB monomer (3.6 μM) or only fluorescent-conjugated streptavidin (0.45 μM).

[0175] (2) Add 2 μL of non-denaturing and non-reducing loading buffer to the sample and mix well, then perform electrophoresis in a (4%-20% SDS-PAGE) polyacrylamide gradient gel.

[0176] (3) After electrophoresis, the fluorescent gel was photographed using Typhoon, followed by Coomassie Brilliant Blue staining to observe the band positions of RGB monomers, streptavidin, and polymers formed by RGB and streptavidin.

[0177] (4) The results show that Figure 5 g), monomers, dimers, trimers and polymers exist under the three mixing ratio conditions; under the 2:1 ratio condition with the strongest labeling signal in flow cytometry detection, the content of dimers, trimers and polymers is relatively higher, and the streptavidin reaction in the mixed system is complete. Therefore, unless otherwise specified, RGB polymers are prepared at this ratio.

[0178] 4. Characterization of the labeling effect of RGB polymers by flow cytometry:

[0179] (2) Biotin-modified RGB-SiaTotal and RGB-Sia3 (0.2 mg / mL) were premixed with streptavidin (25 μg / mL) carrying the AF647 fluorescent group at 4°C for 1 h. Under this condition, the molar ratio of RGB monomer to streptavidin was approximately 8:1.

[0180] (3) Hela cells were incubated with 2 μg / mL RGB polymer at room temperature for 1 h, and a two-step labeling method with the same protein concentration was used as a comparison, i.e., the cells were incubated with 2 μg / mL RGB monomer for 1 h; after incubation, the cells were washed twice with PBS.

[0181] (4) After incubation with 5 μg / mL fluorescent streptavidin for 30 min, the cells were washed twice with PBS; flow cytometry was used for characterization ( Figure 5 b).

[0182] (5) The results show that Figure 5 c, d), RGB polymers can greatly improve the labeling intensity, which is about 100-400 times that of the two-step labeling method.

[0183] 5. Characterization of Binding Specificity of RGB Multimers by Flow Cytometry

[0184] (1) To characterize the binding specificity of RGB polymers, CMAS - / - Hela cells and Hela cells pretreated with corresponding sialidase were used as negative controls, and wild-type Hela cells were used as positive controls. 2 μg / mL RGB-SiaToal polymers and RGB-Sia3 polymers were incubated with these three types of cells at room temperature for 1 hour respectively.

[0185] (2) After washing twice with PBS solution, the cells were detected using a flow cytometer.

[0186] (3) The results show that Figure 5 e, f), only WT Hela cells were labeled by RGB multimers, indicating that the multimers corresponding to RGB-SiaTotal and Sia3 retain good specificity for RGB itself to sugars while improving affinity and labeling intensity.

[0187] 6. Optimizing the labeling concentration and time of RGB polymers

[0188] (1) In the labeling concentration gradient experiment, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL of RGB-SiaToal multimers and RGB-Sia3 multimers were incubated with WT Hela cells at room temperature for 1 h;

[0189] (2) After washing twice with PBS solution, the cells were detected using a flow cytometer. The results are shown in ( Figure 6 a, b) as shown.

[0190] (3) In the labeling time gradient experiment, 1 μg / mL RGB-SiaToal multimer and RGB-Sia3 multimer were incubated with WT Hela cells at room temperature for 15 min, 30 min, and 60 min, respectively;

[0191] (4) After washing twice with PBS solution, the cells were detected using a flow cytometer. The results are shown in ( Figure 6 c, d), indicating that the labeling efficiency of RGB polymers is very high, and the labeling saturation state at this concentration can be reached in 15 minutes.

[0192] 7. Characterization of Cytotoxicity of RGB Polymers

[0193] (1) RGB-SiaTotal multimers (RGB protein concentration was 1 μg / mL) and RGB-Sia3 multimers were incubated with HEK293T, Hela, or mouse embryonic primary cells at room temperature for 1 h.

[0194] (2) Use trypan blue and a cell counter to detect cell viability.

[0195] (3) The results show that Figure 6 e, f), after RGB labeling, the cells still have good vitality.

[0196] 8. One-pot simultaneous labeling of multiple glycoforms using multiple RGB polymers

[0197] (1) RGB-SiaTotal (0.2 mg / mL) was premixed with streptavidin conjugated to AF488 (25 μg / mL) at 4°C for 1 h, and RGB-Sia3 (0.2 mg / mL) was premixed with streptavidin conjugated to AF647 (25 μg / mL) at 4°C for 1 h.

[0198] (2) WT Hela cells were labeled with RGB-SiaTotal polymer (1 μg / mL) and RGB-Sia3 polymer (1 μg / mL) separately or simultaneously at room temperature for 30 min.

[0199] (3) Wash twice with PBS solution and detect using flow cytometer.

[0200] (4) The results show that Figure 7 b), indicating that the labeling of these two RGB polymers does not interfere with each other, which also indicates the possibility of simultaneous labeling of multiple glycoforms with multiple RGB polymers in a one-pot method.

[0201] Example 5 Application of RGB in cell imaging:

[0202] (1) First, Hela cells were digested and seeded in an imaging dish at a cell density of 20%, and placed in a cell culture incubator (37°C, 5% CO2) overnight. When the cell density was about 60%-70%, the culture dish was taken out and washed twice with PBS buffer.

[0203] (2) Enzyme digestion (optional): Incubate the cells with α2,3 / 6 / 8-sialyl hydrolase SpNanA or α2,3-linked sialyl hydrolase RgNanH for 45 min (RT) for desialylation. After enzymatic digestion, wash twice with PBS buffer.

[0204] (3) Fixation: Fix with 4% PFA for 15 min and wash twice with PBS buffer.

[0205] (4) Sialic acid staining: The cells were incubated with RGB-SiaTota-AF647 polymer or RGB-Sia3-AF647 polymer at a final concentration of 2 μg / mL on a shaker at room temperature for 1 h; after the incubation, the cells were washed twice with PBS buffer.

[0206] (5) Nuclear staining: Use 0.2 mg / mL Hoechst 33342 to stain the nucleus for 20 min and wash twice with PBS.

[0207] (6) Imaging: The images were taken using a 20x fluorescence microscope.

[0208] (7) Imaging results are shown in Figure 8 ,from Figure 8 It can be seen that RGB-SiaTotal and RGB-Sia3 also have good specificity for sialylated glycans in cell imaging.

[0209] Example 6 Development and application of RGB-based single-cell polysaccharide profiling technology scRGB-seq

[0210] In this example, we developed scRGB-seq technology and applied it to the determination and characterization of total sialic acid content and α2,3-sialic acid content on the surface of mouse brain resident immune cells by premixing RGB-SiaTotal and RGB-Sia3 with DNA barcoded streptavidin.

[0211] 1. Acquisition of mouse brain-resident immune cells

[0212] After the mice were killed, the brains were removed, digested with papain, centrifuged by gradient centrifugation, filtered through a 45 μm filter, and then washed twice with PBS to obtain a single-cell suspension of the mouse brain.

[0213] 2. Premix RGB-SiaTotal and RGB-Sia3 with DNA barcoded streptavidin:

[0214] RGB-SiaTotal(0.2mg / mL) and TotalSeq TM -B0952 PE Streptavidin (5 μg / mL) was premixed at 4°C for 1 h; RGB-Sia3 (0.2 mg / mL) and TotalSeq TM -B0954 PE Streptavidin (5 μg / mL) was premixed at 4°C for 1 h. Under this condition, the ratio of RGB monomer to streptavidin monomer was about 10:1. The reason for using this ratio was to ensure that there was no free streptavidin in the system to avoid mutual interference, to save expensive reagents, and to ensure that the labeling intensity under this condition had reached the detection requirements (data not shown).

[0215] 3. Cell labeling, sorting, sequencing and analysis

[0216] (1) TotalSeq coupled with equal concentration (2 μg / mL) TM -B0952 barcoded RGB-SiaTotal multimer coupled with TotalSeq TM -B0954 PE RGB-Sia3 polymers and APC-conjugated CD45 fluorescent antibody (0.5 μg / mL) were incubated with mouse brain single cell suspension for 30 min ( Fig. 9 a), Wash twice with PBS.

[0217] (2) Flow cytometry sorting of all CD45-positive immune cells.

[0218] (3) The single-cell sequencing process is captured using the 10X Genomics v3 kit; the polyA-containing RNA and the labeled DNA barcode released after the cells are lysed in the droplets can complementarily pair with the poly(dT) and CaptureSeq 1 primers on the microspheres, respectively, and achieve reverse transcription, amplification, library construction, sequencing and analysis in the subsequent steps.

[0219] (4) The single-cell raw sequencing data were processed using Cell Ranger 4.0.0 software, and the results output by Cell Ranger were further processed and analyzed using Seurat v4.0 software. After data quality control filtering, we obtained a total of 3297 high-quality single cells. The analysis results of total sialylated glycans and α2,3-linked sialylated glycans on the surface of mouse brain resident immune cells are shown in the attached Fig. 9 b, c, d.

[0220] Example 7 Glycogen affinity enrichment based on RGB

[0221] (1) Take 40 μL of streptavidin resin and equilibrate it three times with 100 μL of PBS.

[0222] (2) Incubate 250 μL of biotin-modified RGB-SiaTotal (0.8 mg / mL) with the equilibrated streptavidin resin at 4°C for 2 h; wash three times with 250 μL of PBS to obtain the Streptavidin-RGB-SiaTotal resin for use.

[0223] (3) Mix 20 μL of Streptavidin-RGB-SiaTotal resin with 200 μL of BSA or Fetuin (50 μg / mL) and incubate at 4°C for 3 h or overnight; after incubation, wash five times with 100 μL of PBS.

[0224] (4) The resin-bound protein was eluted twice using 20 μL of 10 mM sodium sialate (pH = 6), each incubation at room temperature for 15 min, with a total elution volume of 40 μL;

[0225] (5) Take 10 μL of the sample for SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining.

[0226] (6) The results show that Fig.10 ), RGB-SiaTotal can better enrich sialylated protein Fetuin, but has no binding and enrichment ability for BSA without sialic acid modification.

[0227] Example 8 Application of RGB in Immunohistochemistry

[0228] (1) The mouse brain was removed and embedded in OCT, then quickly frozen and prepared into frozen sections.

[0229] (2) After drying at room temperature, fix with 4°C cold acetone for 10 min; wash with PBS three times, 5 min each time.

[0230] (3) Use an immunohistochemistry pen to draw a circle around the tissue, add 3% hydrogen peroxide solution into the circle, and incubate at room temperature in the dark for 25 minutes; wash with PBS three times, 5 minutes each time.

[0231] (4) After the slides were dried, PBS solution containing 3% BSA was added dropwise and blocked at room temperature for 45 minutes.

[0232] (5) Prepare RGB-SiaTotal 10 μg / mL in PBS solution containing 1% BSA; after drying the sealed tissue slides, add RGB-SiaTotal solution to the circle and incubate in a wet box at 4°C overnight; after incubation, wash three times with PBS, each time for 5 minutes.

[0233] (6) Add 2 μg / mL horseradish peroxidase-labeled streptavidin and incubate in a wet box at room temperature for 45 min. After incubation, wash three times with PBS, each time for 5 min.

[0234] (7) Add freshly prepared DAB solution, monitor the color development time under a microscope, and rinse with tap water to stop the color development.

[0235] (8) Add hematoxylin solution for 5 minutes; rinse with tap water to terminate.

[0236] (9) Use the bright field of a microscope to take photos.

[0237] Immunohistochemistry results are attached. Fig.11 , Fig.11 It can be seen that compared with the control group, the RGB-SiaTotal group has a stronger brown signal, indicating that RGB-SiaTotal can be successfully applied to immunohistochemistry; and there is a stronger sialylation modification in the mouse brain.

[0238] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A recombinantly expressible carbohydrate binding protein, characterized in that: The carbohydrate binding protein is a protein having the ability to bind carbohydrates, obtained by mutating the nucleophilic attacking group or the amino acid containing the acid-base group in the glycoside hydrolase.

2. The recombinantly expressible carbohydrate binding protein according to claim 1, characterized in that The glycoside hydrolase is one of α2,3 / 6 / 8-sialyl hydrolase, α2,3-sialyl hydrolase and β-galactosidase.

3. The recombinantly expressible carbohydrate binding protein according to claim 2, characterized in that: The carbohydrate binding protein is any one of the proteins described in A1)-A6) below: A1) The amino acid sequence is the protein shown in Sequence 3 in the sequence list; A2) The amino acid sequence is the protein shown in Sequence 4 in the sequence list; A3) The amino acid sequence is the protein shown in Sequence 6 in the sequence list; A4) The amino acid sequence is the protein shown in Sequence 7 in the sequence listing; A5) The amino acid sequence is the protein shown in Sequence 9 in the sequence listing; A6) The amino acid sequence is the protein shown in Sequence 10 in the sequence listing; A7) A protein whose amino acid sequence comprises at least two of the above sequences.

4. The method for preparing a recombinantly expressible carbohydrate binding protein according to any one of claims 1 to 3, characterized in that: The steps include: 1) Using glycosyl hydrolase, the key catalytic site in the enzyme activity pocket was selected for mutation to obtain mutant 1; 2) The substrate binding ability and specificity of the enzyme of mutant 1 are evaluated, and mutants with good binding ability and specificity are screened as carbohydrate binding proteins.

5. The method for preparing the recombinantly expressible carbohydrate binding protein according to claim 4, characterized in that: The key sites are two amino acid residue sites containing carboxyl groups.

6. A method for preparing a carbohydrate binding protein polymer, characterized in that: Using the carbohydrate binding protein according to any one of claims 1 to 3 comprises the following steps: 1) modifying the N-terminus of the carbohydrate binding protein according to any one of claims 1 to 3 with biotin to obtain a biotin-modified carbohydrate binding protein; 2) The biotin-modified carbohydrate-binding protein is co-incubated with streptavidin to obtain carbohydrate-binding protein polymers.

7. Use of the recombinantly expressible carbohydrate binding protein according to any one of claims 1 to 3, the preparation method according to any one of claims 4 to 5, or the carbohydrate binding protein multimer according to claim 6 in any of the following: B1) Application in single cell polysaccharide profiling; B2) Application in carbohydrate affinity enrichment based on recombinantly expressed carbohydrate binding proteins.

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