A PTPσ_Ig1-3 fusion protein, its preparation method and application

Abstract

This invention discloses a PTPσ_Ig1-3 fusion protein, its preparation method, and its applications, belonging to the field of genetic engineering. The recombinant fusion protein is obtained by adding an inducible peptide to the N-terminus of the PTPσ_Ig1-3 domain, and sequentially adding a TEV restriction site, 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 inducible peptide is shown in SEQ ID NO.2, and the amino acid sequences of the TEV restriction site, GFP fluorescent protein, and His tag are shown in SEQ ID NO.3. Experimental verification showed that this recombinant fusion protein has significant binding activity with glycosaminoglycans such as heparin sodium and chondroitin sulfate, showing promising applications in the study of axonal injury and regeneration mechanisms and in drug delivery therapy.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a PTPσ_Ig1-3 fusion protein, its preparation method, and its applications. Background Technology

[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 crucial role in central nervous system development, axonal regeneration, glial scar regulation, and tissue repair. Its extracellular domain consists of multiple immunoglobulin-like domains and fibronectin type III (FNIII) repeat sequences, enabling it to specifically recognize extracellular matrix (such as chondroitin sulfate proteoglycan) or cell surface ligands, thereby regulating intracellular signaling pathways (such as RhoA / ROCK, PI3K / Akt, etc.) and influencing cell adhesion, migration, and regeneration. In recent years, research targeting the extracellular domain of PTPσ 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 blocking the interaction between the extracellular domain of PTPσ and its ligand can effectively inhibit glial scar formation and promote axonal regeneration, demonstrating significant clinical translational potential.

[0003] Among existing expression systems, prokaryotic systems (such as E. coli) are low-cost and simple to operate, but they cannot achieve glycosylation of eukaryotic proteins and are prone to low refolding efficiency due to inclusion body formation. Mammalian cell systems (such as CHO and HEK293) can perform complex modifications, but they face problems such as low expression levels, high costs, 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, there is an urgent need to explore new protein expression methods, which is of great significance for the in-depth research and application of PTPσ in clinical practice. Summary of the Invention

[0004] The purpose of this invention is to provide a PTPσ_Ig1-3 fusion protein, its preparation method, and its applications, in order to solve the problems existing in the prior art. This PTPσ_Ig1-3 fusion protein has significant binding activity with glycosaminoglycans such as heparin sodium and chondroitin sulfate, and has good application prospects in the study of axonal injury and regeneration mechanisms and in drug delivery therapy.

[0005] To achieve the above objectives, the present invention provides the following solution:

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

[0007] The His tag mentioned above is 6×HisTag, but it is not limited to this and can also be 3×FlagTag.

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

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

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

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

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

[0013] Preferably, the method for studying the binding performance of PTPσ_Ig1-3 protein to 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] The gene sequence encoding the recombinant fusion protein is ligated to a plasmid vector to obtain a recombinant plasmid;

[0016] The recombinant plasmid was transfected into mammalian eukaryotic cells, cultured, and the culture medium containing the target recombinant fusion protein was collected.

[0017] The culture medium containing the target recombinant fusion protein was purified by nickel ion affinity chromatography. The purified eluent was then concentrated by ultrafiltration and desalted to obtain the recombinant fusion protein.

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

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

[0020] (1) Dissolve the recombinant fusion protein and glycosaminoglycan according to claim 1 in water respectively, mix them, and measure the fluorescence spectrum. The binding performance of PTPσ_Ig1-3 protein to glycosaminoglycan is determined by the presence or absence of fluorescence spectrum.

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

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

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

[0024] The recombinant fusion protein constructed in this invention incorporates a GFP protein sequence during vector design, allowing for simple expression identification and characterization via fluorescence, eliminating the need for complex protein characterization methods. Furthermore, the insertion of a TEV restriction site allows for the removal of the GFP protein sequence and His tag using TEV enzymes without affecting protein activity studies, resulting in a tag-free pure protein.

[0025] The purification method for the recombinant fusion protein disclosed in this invention is simple, mild, and does not affect protein activity. The obtained protein can be used for the study of protein activity and the interaction between the protein and glycosaminoglycans.

[0026] This invention verifies that the recombinant fusion protein has significant binding activity with glycosaminoglycans such as heparin sodium and chondroitin sulfate, and has good application prospects in the study of axonal injury and regeneration mechanisms and drug delivery therapy. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Map of pcDNA3.1(+)-PTPσ_Ig1-3 plasmid;

[0029] Figure 2SDS-PAGE results for 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: elution buffer, 5: elution buffer, 6: elution buffer of the first column volume, 7: elution buffer of the second column volume, 8: elution buffer of the third column volume.

[0030] Figure 3 The image shows the Western Blot results after protein concentration; A: protein solution before concentration, B: protein solution after concentration;

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

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

[0033] Figure 6 The diagram shows the ITC results of PTPσ_Ig1-3 combined with Hep; A: power difference DP, B: enthalpy change ΔH;

[0034] Figure 7 The diagram shows the ITC results of combining PTPσ_Ig1-3 with CSA; A: power difference DP, B: enthalpy change ΔH. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example 1

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

[0042] S1. By searching the NCBI database, the gene sequence encoding PTPσ_Ig1-3 was obtained, and its amino acid sequence is shown in SEQ ID NO.1. The induction membrane penetration leader peptide sequence SEQ ID NO.2 was added to its N-terminus, and the TEV restriction site, GFP fluorescent protein sequence and 6×HisTag were inserted to its C-terminus, and the amino acid sequence is shown in SEQ ID NO.3, to obtain the complete target gene sequence (SEQ ID NO.4).

[0043] S2. The biotechnology company synthesized the target gene fragment and then cloned it into the ampicillin-resistant pcDNA3.1(+) expression vector through KpnI and XhoI restriction sites. The ligation product was introduced into JM109 Escherichia coli competent cells, plated in LB solid medium containing ampicillin and cultured for 12 h. Single colonies were picked and added to 30 mL of LB liquid medium containing ampicillin and cultured at 37 °C and 220 rpm for 16 h.

[0044] S3. Plasmids from the amplified bacterial strain were extracted using a plasmid mini-extraction kit, and the plasmid concentration was measured using a Nanodrop spectrophotometer to obtain the target plasmid pcDNA3.1(+)-PTPσ_Ig1-3 (see...). Figure 1 ).

[0045] 2. Plasmid transfection and fusion protein expression

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

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

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

[0049] 3. Protein purification

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

[0051] S2. Fill the purification system pump tubing with deionized water, connect the nickel ion chelating column to the system, and tighten. Rinse the column with 4 column volumes of deionized water to remove the storage buffer (20% ethanol), and equilibrate the 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 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). Use a 10000Da ultrafiltration tube to concentrate the elution product through ultrafiltration.

[0052] S3. The protein solution obtained in step S2 was desalted and buffer-replaced using a Sephadex G-25 desalting column and PBS buffer (pH = 7.4). The desalted protein solution was then freeze-dried to obtain the target protein and stored at -80°C. The SDS-PAGE results of the purified protein are shown below. Figure 2As shown in the figure, the results indicate that the target protein was obtained after purification.

[0053] After concentrating the target protein, Western blotting was performed, and the results are as follows: Figure 3 As shown.

[0054] The target protein was detected by fluorescence spectroscopy, and the results are as follows: Figure 4 As shown, strong fluorescence was detected at 520 nm under an excitation wavelength of 488 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] Example 2

[0064] 1. Screening of transfection conditions:

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

[0066] S2. Mix 2 μg of plasmid per well with the plasmid and transfection reagent at ratios of none, 1:1, 1:1.25, 1:1.5, 1:1.75, and 1:2 (μg:μL), respectively. After incubation for 3 min, add 400 μL of DMEM high-glucose medium to each well, mix well by pipetting, and incubate for 30 min. Then add 600 μL of DMEM high-glucose medium and mix well to obtain transfection complexes with different ratios.

[0067] S3. Discard the original culture medium in the six-well plate, add 1 mL of PBS buffer (pH=7.4) along the wall and gently rinse. After discarding the PBS, add the transfection complex in each proportion. After 8 hours, discard the transfection complex and replace it with growth medium. After 24 hours, collect the culture medium and filter it through a 0.22 μm filter membrane.

[0068] S4. Use immunoblotting to perform semi-quantitative analysis on the fusion protein in the culture medium supernatant with different transfection ratios obtained in step S3 above, and determine that the optimal ratio of plasmid to transfection reagent is 1:1.25.

[0069] Example 3: Verification of the binding affinity between PTPσ_Ig1-3 fusion protein and heparin sodium using fluorescence spectroscopy (FL) and isothermal titration calorimetry (ITC).

[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. The mixture was thoroughly homogenized by vortex sonication to obtain PTPσ_Ig1-3-Hep and PTPσ_Ig1-3-CSA complex solutions of different concentrations. Their fluorescence spectra were measured. The addition of Hep or CSA significantly reduced the fluorescence intensity of PTPσ_Ig1-3, and saturation was observed. (See...) Figure 5 .

[0073] 2. ITC method for detection

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

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

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

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

[0078] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope 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 inducible peptide to the N-terminus of the PTPσ_Ig1-3 domain and sequentially adding a TEV restriction site, GFP fluorescent protein, and 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 inducible peptide is shown in SEQ ID NO.2, and the amino acid sequences of the TEV restriction site, GFP fluorescent protein, and His tag are shown in SEQ ID NO.

3.

2. A gene encoding the recombinant fusion protein of 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 described in claim 2.

4. A recombinant bacterium, characterized in that, It contains the recombinant vector as described in claim 3.

5. The recombinant bacteria of claim 4, wherein The host of the recombinant bacteria is a mammalian cell.

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

7. Use according to claim 6, wherein The methods used to study the binding performance of PTPσ_Ig1-3 protein to glycosaminoglycans were fluorescence spectroscopy or isothermal titration calorimetry.

8. A method of constructing a recombinant fusion protein according to claim 1, characterized by, Includes the following steps: The gene sequence encoding the recombinant fusion protein is ligated to a plasmid vector to obtain a recombinant plasmid; The recombinant plasmid was transfected into mammalian eukaryotic cells, cultured, and the culture medium containing the target recombinant fusion protein was collected. The culture medium containing the target recombinant fusion protein was purified by nickel ion affinity chromatography. The purified eluent was then concentrated by ultrafiltration and desalted to obtain the recombinant fusion protein.

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

10. A method for in vitro study of the binding properties of PTPσ_Ig1-3 protein to glycosaminoglycans for non-diagnostic and therapeutic purposes, characterized by, This includes the methods shown in (1) or (2) below: (1) Dissolve the recombinant fusion protein and glycosaminoglycan according to claim 1 in water respectively, mix them, and measure the fluorescence spectrum. The binding performance of PTPσ_Ig1-3 protein to glycosaminoglycan is determined by the presence or absence of fluorescence spectrum. (2) PTPσ_Ig1-3 protein was isothermally titrated with glycosaminoglycan solution, and the binding performance of PTPσ_Ig1-3 protein to glycosaminoglycan was determined by measuring the binding thermodynamic parameters.

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