PTP sigmaIg1-3 fusion protein as well as preparation method and application thereof

Abstract

The invention discloses PTP (protein tyrosine phosphatase) sigma Ig1-3 fusion protein as well as a preparation method and application thereof, and belongs to the field of genetic engineering. The recombinant fusion protein is obtained by adding an induction peptide to the N end of a PTP sigma Ig1-3 structural domain and sequentially adding a TEV restriction enzyme cutting site, a GFP fluorescent protein and a His tag to the C end of the PTP sigma Ig1-3 structural domain, the amino acid sequence of the PTP sigma Ig1-3 structural domain is as shown in SEQ ID NO.1, the amino acid sequence of the induction peptide is as shown in SEQ ID NO.2, and the amino acid sequences of the TEV restriction enzyme cutting site, the GFP fluorescent protein and the His tag are as shown in SEQ ID NO.3. Experiments prove that the recombinant fusion protein has obvious binding activity with glycosaminoglycans such as Haitong heparin sodium, chondroitin sulfate and the like, and has a good application prospect in mechanism research of axonal injury and regeneration and drug delivery therapy.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to a PTPσ_Ig1-3 fusion protein and a preparation method and application thereof. Background Art

[0002] Protein Tyrosine Phosphatase Receptor Type Sigma (PTPσ) is a transmembrane receptor phosphatase belonging to the type IIB receptor tyrosine phosphatase (RPTP) family. It plays a key role in central nervous system development, axon regeneration, glial scar regulation, and tissue repair. Its extracellular domain, composed of multiple immunoglobulin-like (Ig-like) domains and fibronectin type III (FNIII) repeats, specifically recognizes extracellular matrix (such as chondroitin sulfate proteoglycans) or cell surface ligands, thereby regulating intracellular signaling pathways (such as RhoA / ROCK and PI3K / Akt), affecting cell adhesion, migration, and regeneration. In recent years, research on the PTPσ extracellular domain has become a hot topic in the treatment of neurodegenerative diseases (such as spinal cord injury and stroke) and autoimmune diseases (such as multiple sclerosis). Studies have shown that by blocking the interaction between the extracellular domain of PTPσ and its ligand, glial scar formation can be effectively inhibited and axon regeneration can be promoted, which has significant clinical translation potential.

[0003] Existing expression systems include prokaryotic systems (such as Escherichia coli) with low cost and simple operation, but they cannot achieve glycosylation of eukaryotic proteins and are prone to low renaturation efficiency due to inclusion body formation. Mammalian cell systems (such as CHO and HEK293) can achieve complex modifications, but they face problems such as low expression yield, high cost, and insufficient secretion efficiency. Yeast or insect cell systems offer a compromise between cost and modification capabilities, but their glycosylation patterns differ significantly from those of mammals, affecting function. Therefore, the exploration of new protein expression methods is urgently needed, which is of great significance for the in-depth study and application of PTPσ in clinical practice. Summary of the Invention

[0004] The purpose of the present invention is to provide a PTPσ_Ig1-3 fusion protein and its preparation method and application to solve the problems existing in the above-mentioned prior art. The PTPσ_Ig1-3 fusion protein has obvious binding activity with glycosaminoglycans such as Haitongheparin sodium and chondroitin sulfate, and has good application prospects in the study of the mechanism of axonal injury and regeneration and drug delivery therapy.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a recombinant fusion protein. The recombinant fusion protein is obtained by adding an inducing peptide to the N-terminus of the PTPσ_Ig1-3 domain, and sequentially adding a TEV cleavage site, a GFP fluorescent protein, and a His tag to the C-terminus. The amino acid sequence of the PTPσ_Ig1-3 domain is shown in SEQ ID NO.1, the amino acid sequence of the inducing peptide is shown in SEQ ID NO.2, and the amino acid sequences of the TEV cleavage site, the GFP fluorescent protein, and the His tag are shown in SEQ ID NO.3.

[0007] The His tag mentioned above is 6×HisTag, but is not limited thereto and may also be 3×FlagTag.

[0008] The present invention also provides a gene encoding the recombinant fusion protein, and the nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0009] The invention also provides a recombinant vector containing the gene.

[0010] The present invention also provides a recombinant bacterium containing the recombinant vector.

[0011] Preferably, the host of the recombinant bacteria is a mammalian cell.

[0012] The present invention also provides the use of the recombinant fusion protein, the gene, the recombinant vector or the recombinant bacteria in studying the binding properties of the PTPσ_Ig1-3 fusion protein and glycosaminoglycans.

[0013] Preferably, the method for studying the binding properties of PTPσ_Ig1-3 protein and glycosaminoglycans is fluorescence spectroscopy or isothermal titration calorimetry.

[0014] The present invention also provides a method for constructing the recombinant fusion protein, comprising the following steps:

[0015] Connecting the gene sequence encoding the recombinant fusion protein to the plasmid vector to obtain a recombinant plasmid;

[0016] transfecting the recombinant plasmid into mammalian eukaryotic cells, culturing, and collecting the culture fluid containing the target recombinant fusion protein;

[0017] The culture fluid containing the target recombinant fusion protein is purified by nickel ion affinity chromatography, and the purified eluate is then subjected to ultrafiltration, concentration, and desalination to obtain the recombinant fusion protein.

[0018] Preferably, the plasmid vector is pcDNA3.1, and / or the mass volume ratio of the recombinant plasmid to the transfection reagent is 1 μg:1.25 μL, and the transfection reagent includes a polyethyleneimine transfection reagent.

[0019] The present invention also provides a method for studying the binding properties of PTPσ_Ig1-3 protein and glycosaminoglycans in vitro for non-diagnostic and non-therapeutic purposes, comprising the method shown in (1) or (2):

[0020] (1) The recombinant fusion protein of claim 1 and glycosaminoglycan are dissolved in water, mixed, and the fluorescence spectrum is measured to determine the binding performance of the PTPσ_Ig1-3 protein and glycosaminoglycan by the fluorescence spectrum;

[0021] (2) The PTPσ_Ig1-3 protein was isothermally titrated with glycosaminoglycan solution, and the binding properties of the PTPσ_Ig1-3 protein and glycosaminoglycan were determined by measuring the binding thermodynamic parameters.

[0022] The binding of the above recombinant fusion protein to glycosaminoglycans (Haitongheparin sodium and chondroitin sulfate A) can quench the fluorescence of the GFP fluorescent protein. The thermodynamic parameters of the binding can be studied by ITC.

[0023] The present invention discloses the following technical effects:

[0024] The recombinant fusion protein constructed by the present invention has a GFP protein sequence inserted during the vector design process, which can be simply expressed and identified by fluorescence without the need for identification through complex protein characterization methods; a TEV enzyme cleavage site is inserted, and in order not to affect the protein activity research, the GFP protein sequence and His tag can be removed by TEV enzyme to obtain a tag-free pure protein.

[0025] The purification method of the recombinant fusion protein disclosed in the present invention is simple, has mild conditions, does not affect the activity of the protein, and the obtained protein can be used for studying the activity of the protein and the interaction between the protein and glycosaminoglycan substances.

[0026] The present invention verifies that the recombinant fusion protein has obvious binding activity with glycosaminoglycans such as Haitongheparin sodium and chondroitin sulfate, and has good application prospects in the study of the mechanism of axonal injury and regeneration and drug delivery therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the pcDNA3.1(+)-PTPσ_Ig1-3 plasmid map;

[0029] Figure 2Figure 2 is the SDS-PAGE result of PTPσ_Ig1-3 protein purification; M: protein molecular weight standard (10-150kD), 1: untransfected cell culture medium, 2: culture medium before purification, 3: culture medium after purification, 4: liquid eluted with loading buffer, 5: liquid eluted with equilibration buffer, 6: liquid eluted with the first column volume of elution buffer, 7: liquid eluted with the second column volume of elution buffer, 8: liquid eluted with the third column volume of elution buffer;

[0030] Figure 3 Figure 2 is the Western Blot test result after protein concentration; A: protein solution before concentration, B: protein solution after concentration;

[0031] Figure 4 is the fluorescence spectrum of PTPσ_Ig1-3 fusion protein;

[0032] Figure 5 Fluorescence spectra of PTPσ_Ig1-3 protein binding to glycosaminoglycans; A: Hep, B: CSA;

[0033] Figure 6 Figure 2 shows the ITC results of PTPσ_Ig1-3 binding to Hep; A: power difference DP, B: enthalpy change △H;

[0034] Figure 7 Figure 2 shows the ITC results of the binding of PTPσ_Ig1-3 to CSA; A: power difference DP, B: enthalpy change △H. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0038] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0039] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0040] Example 1

[0041] 1. Design, construction and expression of fusion protein expression vector

[0042] S1, by searching the NCBI database, the gene sequence encoding PTPσ_Ig1-3 is obtained, the amino acid sequence is shown as SEQ ID NO. 1, the leading peptide sequence SEQ ID NO. 2 is added at the N terminal, the TEV enzyme cutting site, the GFP fluorescent protein sequence and 6xHisTag are inserted at the C terminal, the amino acid sequence is shown as SEQ ID NO. 3, and the complete target gene sequence (SEQ ID NO. 4) is obtained;

[0043] S2, the target gene fragment is synthesized by a biological company, and then cloned into a pcDNA3.1(+) expression vector containing ampicillin resistance through KpnI and XhoI enzyme cutting sites, the ligation product is introduced into JM109 E. coli competent cells, and after coating in LB solid medium containing ampicillin, it is cultured for 12h, and a single colony is picked and added into 30mL LB liquid medium containing ampicillin and cultured at 37℃, 220rpm for 16h;

[0044] S3, the plasmid of the strain after expansion expression is extracted by a plasmid small amount extraction kit, and the plasmid concentration is determined by a Nanodrop spectrophotometer, and the target plasmid pcDNA3.1(+)-PTPσ_Ig1-3 (see Figure 1 ) is obtained.

[0045] 2. Plasmid transfection and fusion protein expression

[0046] S1. Transfection was performed using polyethyleneimine (PEI) transient transfection method: 8 μg of target plasmid was mixed with 10 μL of PEI transfection reagent, gently pipetted, incubated for 3 min, 2 mL of DMEM high-glucose medium was added, and gently pipetted evenly. After incubation for 30 min, 3 mL of DMEM high-glucose medium was added and pipetted evenly to obtain a transfection complex;

[0047] S2 and HEK 293T cells were cultured in a 37°C, 5% CO2 incubator in a medium containing 1% penicillin-streptomycin (P / S), 10% FBS, and 89% DMEM. Well-grown 1% P / S 293T cells in the logarithmic growth phase were cultured in 10 mm culture dishes and transfected at a cell density of 70%.

[0048] S3. Discard the original culture medium in the culture dish, add 3 mL of PBS buffer and gently rinse, add the transfection complex obtained in step S1 above, discard the transfection complex after 8 hours, add 10 mL of growth medium and continue to culture for 48-72 hours, change the medium every 24 hours, collect the expressed culture medium, filter through a 0.22 μm filter membrane, and obtain the culture medium containing the target protein to be purified.

[0049] 3. Protein purification

[0050] S1. The culture medium containing the target protein to be purified prepared above was concentrated and enriched by centrifugation through a 10 kDa ultrafiltration tube at 5000 rpm and 4°C for 30 min, and then 2 volumes of protein purification loading buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole) were added to obtain a sample to be purified;

[0051] S2. Fill the purification system pump pipeline with deionized water, connect the nickel ion chelate column to the system, and tighten it; rinse out the storage buffer (20% ethanol) with 4 column volumes of deionized water, and equilibrate the chromatographic column with 5 column volumes of loading buffer; load 4-5 column volumes of the sample to be purified using a constant flow pump, equilibrate the chromatographic column with 5 column volumes of equilibration buffer (50mM NaH2PO4, 300mM NaCl, 25mM imidazole), and perform one-step elution with elution buffer (50mM NaH2PO4, 300mM NaCl, 100mM imidazole); and ultrafiltration and concentration of the eluted product using a 10,000Da ultrafiltration tube;

[0052] S3, using Sephadex G-25 desalting column and PBS buffer (pH = 7.4) to desalt and replace the buffer of the protein solution obtained in step S2, freeze-dry the desalted protein solution to obtain the target protein, and store it at -80 ° C. The results of SDS-PAGE detection of the purified protein are as follows Figure 2As shown, the results showed that the target protein was obtained after purification.

[0053] After the target protein was concentrated, Western Blot detection was performed, and the results were as follows Figure 3 shown.

[0054] Detect the target protein by fluorescence spectrum detection, the results are as follows Figure 4 As shown, under an excitation wavelength of 488 nm, strong fluorescence was detected at 520 nm, corresponding to the GFP fluorescent protein in the target protein, indicating that the fusion protein was successfully expressed.

[0055] SEQ ID NO.1:

[0056] EEPPRFIKEPKDQIGVSGGVASFVCQATGDPKPRVTWNKKGKKVNSQRFETIEFDESAGAVLRIQPLRTPRDENVYECVAQNSVGEITVHAKLTVLREDQLPSGFPNIDMGPQLKVVERTRTATMLCAASGNPDPEITWFKDFLPV DPSASNGRIKQLRSGALQIESSEETDQGKYECVATNSAGVRYSSPANLYVRVRRVAPRFSILPMSHEIMPGGNVNITCVAVGSPMPYVKWMQGAEDLTPEDDMPVGRNVLELTDVKDSANYTCVAMSSLGVIEAVAQITVKSLPKA.

[0057] SEQ ID NO.2:

[0058] MAPEPAPGRTMVPLVPALVMLGLVAG.

[0059] SEQ ID NO.3:

[0060] FENLYFQGMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKHHHHHH.

[0061] SEQ ID NO.4:

[0062] ATGGCC CCTGAG CCTGCT CCTGGA AGAACC ATGGTG CCACTG GTGCCCGCCCTG GTGATGCTGGGC CTGGTC GCCGGC GAAGAG CCCCCC AGGTTT ATCAAAGAACCC AAGGAC CAGATC GGCGTGTCGGGG GGTGTG GCCTCT TTCGTG TGTCAGGCCACG GGTGAC CCCAAG CCACGA GTGACC TGGAACAAGAAG GGCAAG AAGGTCAACTCT CAGCGC TTTGAG ACGATT GAGTTT GATGAG AGTGCA GGGGCAGTGCTGAGGATC CAGCCG CTGAGG ACACCG CGGGAT GAAAAC GTGTAC GAGTGT GTGGCCCAGAACTCGGTT GGGGAG ATCACA GTCCAT GCCAAG CTTACT GTCCTC CGAGAGGACCAG CTGCCC TCTGGCTTCCC AACATC GACATG GGCCCA CAGTTG AAGGTGGTGGAG CGGACA CGGACA GCCACC ATGCTCTGTGCA GCCAGC GGCAAC CCTGACCCTGAG ATCACC TGGTTC AAGGAC TTCCTG CCTGTG GATCCTAGTGCC AGCAATGGACGC ATCAAA CAGCTG CGATCA GGAGCC CTGCAG ATTGAA AGCAGTGAGGAAACCGAC CAGGGC AAATAT GAGTGT GTGGCC ACCAAC AGCGCC GGCGTG CGCTACTCCTCACCTGCC AACCTC TACGTG CGAGTC CGCCGC GTGGCC CCGCGC TTCTCCATCCTG CCCATG AGCCACGAGATC ATGCCA GGGGGC AACGTG AACATC ACCTGCGTGGGCC GTGGGC TCGCCC ATGCCA TACGTGAAGTGG ATGCAG GGGGCC GAGGACCTGACC CCGAG GATGAC ATGCCC GTGGGT CGGAAC GTGCTGGAACTC ACAGATGTCAAG GACTCG GCCAAC TACACCTGCGTG GCCATG TCCAGC CTGGGCGTCATTGAGGCG GTTGCT CAGATC ACGGTG AAATCT CTCCCC AAAGCT TTTGAG AACCTGTACTTCCAGGGC ATGAGT AAAGGA GAAGAA CTTTTC ACTGGA GTTGTT CCAATTCTTGTT GAATTA GATGGTGATGTT AATGGG CACAAA TTTTCT GTCAGT GGAGAGGGTGAA GGTGAT GCAACA TACGGA AAACTTACCCTT AAATTT ATTTGC ACTACTGGAAAA CTACCT GTTCCA TGGCCA ACACTT GTCACT ACTTTCGCCTAT GGTGTTCAATGC TTTTCA AGATAC CCAGAT CATATG AAACGG CATGAC TTTTTCAAGAGTGCCATG CCCGAA GGTTAT GTACAG GAAAGA ACTATA TTTTTC AAAGAT GACGGGAACTACAAGACA CGTGCT GAAGTC AAGTTT GAAGGT GATACC CTTGTT AATAGAATCGAG TTAAAA GGTATTGATTTT AAAGAA GATGGA AACATT CTTGGA CACAAATTGGAA TACAAC TATAAC TCACAC AATGTATACATC ATGGCA GACAAA CAAAAGAATGGA ATCAAA GTTAAC TTCAAA ATTAGA CACAAC ATTGAAGATGGA AGCGTTCAACTA GCAGAC CATTAT CAACAA AATACT CCAATT GGCGAT GGCCCTGTCCTTCTACCA GACAAC CATTAC CTGTCC ACACAA TCTGTC CTTTCG AAAGAC CCCAACGAAAAGAGAGAC CACATG GTCCTT CTTGAG TTTGTA ACAGCT GCTGGG ATTACACATGGCATGGATGAACTATACAAACATCAC CACCAC CACCAC。

[0063] 实施例2

[0064] 1. Screening of transfection conditions:

[0065] S1, 30×10 4 293T cells were seeded into six-well plates at a density of cells / well and transfected when the cell density reached 70%;

[0066] S2, according to the ratio of 2 μg plasmid per well, plasmid and transfection reagent were mixed at the ratio of none, 1:1, 1:1.25, 1:1.5, 1:1.75, and 1:2 (μg:μL), incubated for 3 minutes, added 400 μL DMEM high glucose medium to each well, pipetted evenly, incubated for 30 minutes, and added 600 μL DMEM high glucose medium to mix well to obtain transfection complexes with different ratios;

[0067] S3. Discard the original culture medium in the six-well plate and gently rinse by adding 1 mL of PBS buffer (pH 7.4) along the wall. Discard the PBS and add transfection complexes of various ratios. After 8 hours, discard the transfection complexes and replace with growth medium. After 24 hours, collect the medium and filter through a 0.22 μm filter.

[0068] S4. Semi-quantitative analysis of the fusion protein in the culture supernatant of different transfection ratios obtained in step S3 above was performed using immunoblotting to determine that the optimal ratio of plasmid to transfection reagent was 1:1.25.

[0069] Example 3 Fluorescence spectroscopy (FL) and isothermal titration calorimetry (ITC) verification of the binding affinity between PTPσ_Ig1-3 fusion protein and Haitong heparin sodium

[0070] 1. FL method detection

[0071] S1. The purified and desalted PTPσ_Ig1-3 protein (1 μM) was mixed with different concentrations of heparin sodium (Hep) and chondroitin sulfate (CSA) solutions to make their concentrations 0 μM, 0.5 μM, 1 μM, 2 μM, and 5 μM, respectively.

[0072] S2. Vortex and ultrasonicate to mix thoroughly to obtain PTPσ_Ig1-3-Hep and PTPσ_Ig1-3-CSA complex solutions of different concentrations. The fluorescence spectra were measured. After adding Hep or CSA, the fluorescence intensity of PTPσ_Ig1-3 was significantly reduced and saturated. Figure 5 .

[0073] 2. ITC method detection

[0074] The purified and desalted PTPσ_Ig1-3 protein (10 μM) was isothermally titrated using Hep and CSA solutions with a concentration of 20 μM to obtain its various binding thermodynamic parameters.

[0075] like Figure 6 As shown, the KD value of 10 μM PTP_Ig1-3 fusion protein binding to 20 μM Haitong heparin sodium was 6.75e-9±14.3e-9, the ΔH value was -335±61.2 kJ / mol, and the ΔG value was -46.7 kJ / mol.

[0076] like Figure 7 As shown, the KD value of 10 μM PTP_Ig1-3 fusion protein binding to 20 μM Haitong heparin sodium was 36.4e-9±37.4e-9, the ΔH value was -335±56.3 kJ / mol, and the ΔG value was -42.5 kJ / mol.

[0077] The above results indicate that the recombinant fusion protein has significant binding activity with glycosaminoglycans such as Haitongheparin sodium and chondroitin sulfate. Therefore, the recombinant fusion protein has good application prospects in the study of the mechanism of axonal injury and regeneration and drug delivery therapy.

[0078] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A recombinant fusion protein, characterized in that The recombinant fusion protein is obtained by adding an inducing peptide to the N-terminus of the PTPσ_Ig1-3 domain, and sequentially adding a TEV cleavage site, a GFP fluorescent protein, and a His tag to the C-terminus, wherein the amino acid sequence of the PTPσ_Ig1-3 domain is shown in SEQ ID NO.1, the amino acid sequence of the inducing peptide is shown in SEQ ID NO.2, and the amino acid sequences of the TEV cleavage site, the GFP fluorescent protein, and the His tag are shown in SEQ ID NO.

3.

2. The gene encoding the recombinant fusion protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. A recombinant vector, characterized in that It contains the gene according to claim 2.

4. A recombinant bacterium, characterized in that It contains the recombinant vector according to claim 3.

5. The recombinant bacterium according to claim 4, characterized in that The host of the recombinant bacteria is a mammalian cell.

6. Use of the recombinant fusion protein according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant bacteria according to any one of claims 4 to 5 in studying the binding properties of PTPσ_Ig1-3 protein to glycosaminoglycans.

7. The use according to claim 6, characterized in that The method for studying the binding performance of PTPσ_Ig1-3 protein and glycosaminoglycan is fluorescence spectroscopy or isothermal titration calorimetry.

8. A method for constructing the recombinant fusion protein according to claim 1, characterized in that: The following steps are involved: Connecting the gene sequence encoding the recombinant fusion protein to the plasmid vector to obtain a recombinant plasmid; transfecting the recombinant plasmid into mammalian eukaryotic cells, culturing, and collecting the culture fluid containing the target recombinant fusion protein; The culture fluid containing the target recombinant fusion protein is purified by nickel ion affinity chromatography, and the purified eluate is then subjected to ultrafiltration, concentration, and desalination to obtain the recombinant fusion protein.

9. The construction method according to claim 8, wherein: The plasmid vector is pcDNA3.1, and / or the mass volume ratio of the recombinant plasmid and the transfection reagent is 1 μg:1.25 μL, and the transfection reagent includes a polyethyleneimine transfection reagent.

10. A method for studying the binding properties of PTPσ_Ig1-3 protein to glycosaminoglycans in vitro for non-diagnostic and non-therapeutic purposes, characterized in that: The method includes the following (1) or (2): (1) The recombinant fusion protein of claim 1 and glycosaminoglycan are dissolved in water, mixed, and the fluorescence spectrum is measured to determine the binding performance of the PTPσ_Ig1-3 protein and glycosaminoglycan by the fluorescence spectrum; (2) The PTPσ_Ig1-3 protein was isothermally titrated with glycosaminoglycan solution, and the binding properties of the PTPσ_Ig1-3 protein and glycosaminoglycan were determined by measuring the binding thermodynamic parameters.

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