Preparation method and application of MBP-GL fusion recombinant protein

Abstract

The invention provides a preparation method and application of MBP-GL fusion recombinant protein. A C3 region of SPG and a B structural domain of protein L are connected in series, codon optimization is performed on a gene sequence, the front end of MBP is introduced into a BamHI restriction enzyme cutting site, a cysteine sequence, a termination codon TAA and an EcoRI restriction enzyme cutting site consistent with an expression vector are added to the tail end of the B region of the protein L, and the MBP-GL fusion recombinant protein is obtained. BamHI and EcoRI are subjected to double enzyme digestion and then are connected to a carrier subjected to the same double enzyme digestion, a recombinant protein MBP-GL gene expression carrier of a structural domain of strongest binding IgG of protein G and protein L connected with MBP is constructed, the carrier is subjected to transformation, induced expression and purification, and the recombinant protein MBP-GL gene expression carrier which not only has glucan ligand specific binding capacity, but also can be used for preparing the recombinant protein MBP-GL gene expression carrier. And the recombinant protein can be combined with all antibodies, subtypes and antibody fragments with high load.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant proteins, and particularly to a preparation method and application of an MBP-GL fusion recombinant protein. Background Art

[0002] Streptococcal protein G (SPG) is a protein composed of nearly 600 amino acids present in the cell wall of streptococcus. The protein properties of streptococcal protein G are quite different from those of Staphylococcus aureus. SPG can bind to IgG of various mammals (humans, horses, sheep, cattle, rats, guinea pigs, cats, mice, birds, rabbits, etc.), acting not only on the Fc segment but also on the Fab segment, while protein A has a very low or no binding ability to IgG of some mammals and cannot bind to IgG3, which accounts for 8% of the total human IgG. On the other hand, SPG cannot act on other types of IgG antibodies such as IgG1, IgG2, IgG4, IgM, and IgA compared with SPA. Compared with protein A, SPG has a stronger binding force and a wider binding spectrum; at the same time, SPG has a lower binding level with serum proteins, with high product purity and low ligand detachment. The gene of SPG consists of a serum albumin binding region, a Fab binding region, an Fc binding region, a cell wall and cell membrane binding region, and the realization of their functions does not affect each other. The SPG structure starts from the hydrophobic end with a homologous structure composed of 4 amino acids, including A1, A, and A3. At the same time, the A1, A2, and A3 homologous structures are separated by the homologous regions B1 and B. The homologous structure consists of 51 amino acids, followed by a spacer region S, and then 3 homologous structures C1, C, and C3 separated by D1 and D2. This homologous structure consists of 55 amino acids. Behind the C3 region is the W region, which is also a hydrophilic region, and finally the M region. Research shows that the C region of SPG is related to the binding of the Fc end of IgG, and the C3 structure has a stronger binding ability to IgG than C1 and C2.

[0003] The SPG protein was initially obtained by papain digestion of Streptococcus, followed by purification through column chromatography and affinity chromatography. Only 3 mg of pure SPG could be obtained from every 50 g of bacteria, and the pathogenicity of Streptococcus was not conducive to the safe acquisition of SPG. Additionally, while SPG binds a large amount of IgG, its leukocyte binding site can bind some serum albumin (serum albumin is a major contaminant in the source of antibodies). In recent years, there have been many successful cases of the application of recombinant expression of SPG through genetic engineering. For example, expressing SPG using Escherichia coli does not require protease digestion of the bacterial cell wall, effectively preventing product degradation, and can design the gene of recombinant SPG to remove non-specific binding sites such as albumin and cell surface binding sites, reducing non-specific binding and cross-reaction, and expressing recombinant SPG with stronger specific binding more efficiently and safely. Thus, recombinant SPG has obtained a higher affinity than natural protein and can replace the secondary antibody, being widely used in fields such as immunochemistry.

[0004] Protein L from Peptostreptococcus magnus (abbreviated as Protein L) is a cell wall surface protein from Peptostreptococcus magnus and has broad IgG binding activity. It was first discovered in 1895. Some studies have found that Protein L has a total of 719 amino acids, containing 4 functional regions: A, B, C, W, and two signal peptide sequences SS, which are excised after being transported to the target. Among them, region B includes 5 highly homologous domains: B1, B2, B3, B4, B5, where the homology of B1 - B4 is 72% - 94%, while the homology of B5 with other B domains is only 57%. Protein L is an elongated fibrous protein, and its specific binding site with the antibody is located in the variable region of the Kappa light chain, different from Protein A and Protein G which act on the Fc region. Compared with Protein A / G, the advantage of Protein L is that its specific binding site is located in the variable region of the antibody Kappa light chain. It can not only specifically adsorb full-length antibodies (IgG1, IgG2, IgG3, IgG4, IgM, IgA), but also specifically adsorb antibody fragments containing the variable region of the light chain, such as Fab, sc Fvsd Ab, etc. At the same time, its binding sites with the variable region of the light chain are all located within the repetitive conserved sequence FR framework region and do not exhibit any biological activity. Therefore, even if Protein L is bound, it will not affect the binding of its CDR region to the antigen. In addition, Protein L has two interfaces in its binding to Fab, which also gives it an advantage in the binding force to polyclonal antibodies or multiple antibody fragments.

[0005] Both Protein G and Protein L are powerful reagents for detecting IgG and are thus antigens against which these antibodies are directed. They are used in Western blot analysis to detect various antigen-antibody complexes on nitrocellulose membranes. Moreover, Protein G and Protein L are widely used as ligands coupled to resins in affinity chromatography for antibody purification. Protein L has a broader spectrum of action types compared to Protein G and can act on IgG3 subtypes and antibody fragments. Therefore, the protein expressed by recombining the functions of Protein G and Protein L can basically cover almost all antibodies and fragments. The recombinant human IgG-binding proteins on the market currently contain tandem or pairwise combinations of the domains of Protein A, Protein G, and Protein L, and use traditional tags such as His-tag for affinity chromatography purification, with a static adsorption capacity for human IgG of 20 - 50 mg human IgG / mg protein. With the development of the biological and medical industries, the market has an increasing demand for recombinant proteins that are more convenient to purify and have a higher adsorption capacity. On the other hand, with the in-depth study of the functions of immunoglobulin-binding proteins, the market demand for various recombinant ligands and their affinity packing materials will increase rapidly. Currently, worldwide, the two-combined recombinant protein ligands are relatively few, and domestic related products have deficiencies such as low yield, poor activity, and low loading capacity. Therefore, there is an urgent need for an improved recombinant protein with high loading capacity, high specificity, and high yield.

[0006] In view of this, it is necessary to design an improved MBP-GL fusion recombinant protein to solve the above problems. Summary of the Invention

[0007] Aiming at the defects of the above-mentioned existing technologies, the purpose of the present invention is to provide a preparation method and application of an MBP-GL fusion recombinant protein. By combining maltose-binding protein (MBP) with a GL tandem, a recombinant protein is prepared that has both the ability to bind to polysaccharide agarose and a high loading capacity to bind all antibodies and subtypes and antibody fragments.

[0008] To achieve the above purpose, the present invention provides a preparation method of an MBP-GL fusion recombinant protein, including the following steps:

[0009] S1. Construct a recombinant protein MBP-GL gene expression vector;

[0010] S2. Transform, induce expression, and purify the vector obtained in step S1 to obtain the MBP-GL fusion recombinant protein.

[0011] As a further improvement of the present invention, in step S1, the method for constructing the recombinant protein MBP-GL gene expression vector includes the following steps:

[0012] S11. Tandem the C3 region of SPG with the B domain of Protein L and optimize the gene sequence for codons;

[0013] S12. Introduce the MBP front end into the BamHΙ cleavage site, add a cysteine sequence, a stop codon TAA, and an EcoRΙ cleavage site consistent with the expression vector at the end of the B region of protein L;

[0014] S13. After double digestion with BamHΙ and EcoRΙ, ligate it to the vector digested with the same two enzymes to construct a recombinant protein MBP-GL gene expression vector for the domain with the strongest binding IgG of protein G and protein L connected to MBP.

[0015] In step S13, the vector digested with the same two enzymes is PET28a or pPICZaA.

[0016] Furthermore, in step S2, the methods of transformation, induction expression and purification include the following steps:

[0017] S21. Transfer the recombinant protein MBP-GL gene expression vector into an expression strain for expanded culture, add an inducer for induction expression, continue culturing, and perform centrifugation after culturing is completed;

[0018] S22. Purify the protein supernatant collected by centrifugation with a polysaccharide resin and collect the elution peak;

[0019] S23. Further optimize the purity of the elution liquid collected in step S22 by hydrophobic chromatography to obtain the MBP-GL fusion recombinant protein;

[0020] S24. Verify the expression by SDS-PAGE.

[0021] In step S21, the expression strain is Escherichia coli or Pichia pastoris; the culture temperature is 30-37 °C, and the inducer is isopropyl thiogalactoside or methanol.

[0022] As a further improvement of the present invention, the present invention also provides an application of the MBP-GL fusion recombinant protein, including the following steps:

[0023] S1. Couple the MBP-GL fusion recombinant protein prepared by the above preparation method of the MBP-GL fusion recombinant protein with microspheres to obtain a coupling product;

[0024] S2. Test the IgG adsorption performance of the coupling product obtained in step S1.

[0025] Furthermore, in step S1, the coupling method includes the following steps:

[0026] S11. Rinse the microspheres thoroughly, add NaOH and epibromohydrin, and react after stirring;

[0027] After the reaction is completed, rinse thoroughly, add buffer solution, adjust the pH value, add the MBP-GL fusion recombinant protein, and carry out the reaction in a constant temperature system;

[0028] Rinse the reaction product obtained in step S12, add 20% ethanolamine solution to block the unreacted epoxy groups, and carry out the reaction at a predetermined temperature;

[0029] After the reaction is completed, rinse to obtain the coupling product, and store it in a solution containing preservatives.

[0030] In step S11, the temperature of the reaction is 35 - 38°C, and the reaction time is 1 - 2 h; in step S12, the pH value is 7 - 8, the temperature of the constant temperature system is 36.5 - 37.5°C, and the reaction time is 20 - 22 h.

[0031] In step S13, the temperature of the reaction is 20 - 22°C, and the reaction time is 8 - 10 h.

[0032] Furthermore, in step S2, the method for testing the IgG adsorption performance includes the following steps:

[0033] Take the coupling product, add human plasma, and shake gently in a room temperature shaker to obtain a reaction solution;

[0034] Add the reaction solution obtained in S21 to an affinity chromatography column, first rinse with the equilibration buffer, then elute with the elution buffer, and collect the elution peak;

[0035] Use an IgG antibody kit to detect the content of IgG in the eluate, and calculate the adsorption amount of the coupling product to IgG.

[0036] The beneficial effects of the present invention are:

[0037] (1) The present invention provides a preparation method and application of an MBP-GL fusion recombinant protein, which combines maltose-binding protein (MBP) with a GL tandem, to prepare a recombinant protein that has both the ability to bind to polysaccharide agar and a high loading capacity to bind all antibodies and subtypes and antibody fragments; constructs a GL tandem structure by combining C3 of SPG with the B fragment in the protein L sequence that has the strongest antibody-binding ability, and successfully expresses it in an expression strain. The affinity of its protein product is relatively broad, the loading capacity is relatively high, and the recombinant protein purification process is relatively simple, with high yield. It not only avoids the pathogenic bacteria risk of natural protein extraction but also enables large-scale production.

[0038] (2) The gene sequence of the present invention is codon-optimized to efficiently express recombinant proteins in the prokaryotic system of the expression strain. Compared with using papain to digest streptococcus, this method not only effectively avoids the pathogenicity of streptococcus, but also has simpler operation, shorter culture period, and greatly reduces the production cost.

[0039] (3) The MBP-GL fusion recombinant protein of the present invention is composed of the C3 region of SPG and the B region of protein L in series, and can efficiently and specifically bind IgG antibodies and various subtypes, thereby ensuring the removal effect of IgG antibodies.

[0040] (4) The MBP-GL fusion recombinant protein of the present invention has a maltose-binding protein, which can increase the solubility of the recombinant protein overexpressed in bacteria. At the same time, it can be purified with a polysaccharide agar filler with a lower price, saving the production cost.

[0041] (5) The MBP-GL fusion recombinant protein of the present invention can specifically bind to microspheres containing polysaccharides, which is beneficial to increasing the loading capacity of the microspheres for the recombinant protein and has a high coupling rate.

[0042] (6) After induction and purification of Escherichia coli in the present invention, the total amount of exogenous protein expression accounts for more than 60% of the total protein, with high expression level and short expression time, which is beneficial to the industrial production of recombinant protein G in genetic engineering.

[0043] (7) The MBP-GL fusion recombinant protein of the present invention replaces the traditional His tag, avoiding problems such as safety, detection, time and economic costs caused by subsequent Ni shedding; at the same time, the MBP tag can reduce the degradation of the recombinant protein and improve the solubility and expression level.

[0044] (8) The MBP-GL fusion recombinant protein of the present invention has high dynamic binding activity to human IgG. Compared with the protein A pre-packed column on the market (20 - 50 mg / g human IgG), the recombinant protein product provided by the present invention has high application potential in immunological fields such as antibody purification.

[0045] The amino acid sequence of the said SPG is as shown in SEQ ID NO:1;

[0046] SEQ ID NO:1

[0047] KAKQLLKQTQKLQKKPSNNTLTTTVLMVFGLMMMRLRPLRLNERRNNYSSRRRNCRKSLQTIRRQRCWCLDLCDDLYGNMKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEKAKQLLKQTQKLQKKPSNNTLTTTVLMVFGLMMMRLRPLRLMKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEMKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE

[0048] The amino acid sequence of the said protein L is shown as SEQ ID NO:2;

[0049] SEQ ID NO:2

[0050] KEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKENGKYTADLEDGGYTINIRFAGKKVDEKPEEKEQVTIKENIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTINIRFAG

[0051] The base sequence of the said maltose-binding protein (MBP) is shown as SEQ ID No:3;

[0052] SEQ ID No:3

[0053]

[0054] The amino acid sequence of the maltose-binding protein (MBP) is shown in SEQ ID No: 4;

[0055] SEQ ID No:4

[0056] KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT Description of the Drawings

[0057] Figure 1 SDS-PAGE diagram of the MBP-GL fusion recombinant protein obtained in Examples 1-2.

[0058] Figure 2 Standard curve diagram for measuring IgG adsorption amount by the BCA method in Example 3.

[0059] Reference Signs

[0060] 1 - Pichia pastoris cell precipitate; 2 - Pichia pastoris supernatant; 3, 4 - Pichia pastoris supernatant without inducer; 5 - Starch-agarose column purification balance sample (E. coli); 6 - Starch-agarose column purification elution sample (E. coli); 7 - Standard molecular weight size. Detailed Description of the Invention

[0061] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail below with reference to the drawings and specific examples.

[0062] Here, it should also be noted that in order to avoid obscuring the present invention with unnecessary details, only the structures and / or processing steps closely related to the solution of the present invention are shown in the drawings, while other details less related to the present invention are omitted.

[0063] In addition, it should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0064] The present invention provides a method for preparing an MBP-GL fusion recombinant protein, comprising the following steps:

[0065] S1. Construct a recombinant protein MBP-GL gene expression vector.

[0066] Specifically, the method for constructing the recombinant protein MBP-GL gene expression vector comprises the following steps:

[0067] S11. Tandemly connect the C3 region of SPG and the B domain of protein L, and optimize the gene sequence for codons.

[0068] S12. Introduce a BamHΙ restriction enzyme site at the front end of MBP, and add a cysteine sequence, a stop codon TAA and an EcoRΙ restriction enzyme site consistent with the expression vector at the end of the B region of protein L.

[0069] S13. After double digestion with BamHΙ and EcoRΙ, ligate it to the vector digested with the same enzymes to construct a recombinant protein MBP-GL gene expression vector of the domain with the strongest binding IgG of protein G and protein L connected to MBP.

[0070] Wherein, in step S13, the vector digested with the same enzymes is preferably PET28a or pPICZaA.

[0071] S2. Transform, induce expression and purify the vector obtained in step S1 to obtain an MBP-GL fusion recombinant protein.

[0072] Specifically, the method for transformation, induction expression and purification comprises the following steps:

[0073] S21. Transfer the recombinant protein MBP-GL gene expression vector into an expression strain for enlarged culture, add an inducer for induction expression, continue culturing, and perform centrifugation after culturing is completed.

[0074] Specifically, the expression strain is preferably Escherichia coli or Pichia pastoris.

[0075] The temperature of the culture is preferably 30-37 °C.

[0076] The inducer is preferably isopropylthiogalactoside or methanol.

[0077] S22. Purify the centrifugally collected protein supernatant with polysaccharide resin and collect the elution peak.

[0078] S23. Further optimize the purity of the elution liquid collected in step S22 by hydrophobic chromatography to obtain the MBP-GL fusion recombinant protein.

[0079] S24. Verify the expression by SDS-PAGE.

[0080] The present invention also provides an application of the MBP-GL fusion recombinant protein, including the following steps:

[0081] S1. Couple the MBP-GL fusion recombinant protein prepared by the preparation method of the above MBP-GL fusion recombinant protein with microspheres to obtain a coupling product.

[0082] Specifically, the coupling method includes the following steps:

[0083] S11. Rinse the microspheres thoroughly, add NaOH and epibromohydrin, and react after stirring.

[0084] Specifically, the reaction temperature is preferably 35-38 °C, and the reaction time is preferably 1-2 h.

[0085] S12. After the reaction is completed, rinse thoroughly, add buffer solution, adjust the pH value, add the MBP-GL fusion recombinant protein, and react in a constant temperature system.

[0086] Specifically, the pH value is preferably 7-8, the temperature of the constant temperature system is preferably 36.5-37.5 °C, and the reaction time is preferably 20-22 h.

[0087] S13. Rinse the reaction product obtained in step S12, add a 20% ethanolamine solution to block the unreacted epoxy groups, and react at a predetermined temperature.

[0088] Specifically, the reaction temperature is preferably 20-22 °C, and the reaction time is preferably 8-10 h.

[0089] S14. After the reaction is completed, rinse to obtain the coupling product and store it in a solution containing a preservative.

[0090] S2. Perform IgG adsorption performance test on the coupling product obtained in step S1.

[0091] Specifically, the method for the IgG adsorption performance test includes the following steps:

[0092] S21. Take the coupling product, add human plasma, and slowly shake well in a room temperature shaker to obtain a reaction solution;

[0093] S22. Add the reaction solution obtained in S21 into an affinity chromatography column, first rinse it with the equilibration solution, then elute it with the elution solution, and collect the elution peak.

[0094] S23. Use an IgG antibody kit to detect the content of IgG in the eluate, and calculate the adsorption amount of the conjugate to IgG.

[0095] The following specifically describes in detail the preparation method and application of the MBP-GL fusion recombinant protein provided by the present invention in combination with specific examples.

[0096] Example 1

[0097] This example provides a preparation method of an MBP-GL fusion recombinant protein, and the expression strain is Escherichia coli, including the following steps:

[0098] S1. Construct a recombinant protein MBP-GL gene expression vector:

[0099] S11. Tandemly connect the C3 region of SPG and the B domain of protein L, and optimize the gene sequence according to the codon preference of Escherichia coli expression.

[0100] S12. Introduce a BamHΙ restriction site at the front end of MBP, and add a cysteine sequence, a stop codon TAA and an EcoRΙ restriction site consistent with the expression vector at the end of the B domain monomer of protein L.

[0101] S13. After double digestion with BamHΙ and EcoRΙ, ligate it to the PET28a vector digested with the same enzymes, and construct a recombinant protein MBP-GL gene expression vector of the domain with the strongest binding IgG of protein G and protein L connected to MBP.

[0102] S2. Transform, induce expression and purify the vector obtained in step S1 to obtain an MBP-GL fusion recombinant protein:

[0103] S21. Transfer the successfully constructed recombinant protein MBP-GL gene expression vector into Escherichia coli BL21(DE3) for expansion culture. The culture temperature is 37°C. When the OD600 value of the bacteria reaches 0.8, add IPTG (isopropyl β-D-thiogalactoside) for induction expression, continue to culture overnight, and centrifuge to collect the bacteria.

[0104] S22. Add the collected bacteria to a PBS solution at a volume ratio of 1:10, ultrasonically disrupt them at a power of 500 W for 8 min, and affinity purify the protein supernatant collected by centrifugation with a polysaccharide resin, and collect the elution peak.

[0105] S23. Further subject the collected eluate to hydrophobic purification with MMC to obtain an MBP-GL fusion recombinant protein.

[0106] The expression was verified by S24.SDS-PAGE, and the results are as Figure 1 shown.

[0107] Example 2

[0108] This example provides a method for preparing the MBP-GL fusion recombinant protein. The expression strain is Pichia pastoris, and the method includes the following steps:

[0109] S1. Construct a recombinant protein MBP-GL gene expression vector:

[0110] S11. Tandemly connect the C3 region of SPG and the B domain of protein L, and optimize the codons of the gene sequence according to the codon preference of yeast expression.

[0111] S12. Introduce a BamHΙ restriction site at the front end of MBP, and add a cysteine sequence, a stop codon TAA and an EcoRΙ restriction site consistent with the expression vector at the end of the B domain monomer of protein L.

[0112] S13. After double digestion with BamHΙ and EcoRΙ, ligate it to the pPICZaA vector digested with the same enzymes to construct a recombinant protein MBP-GL gene expression vector with the domain that binds IgG strongest between protein G and protein L connected to MBP.

[0113] S2. Transform, induce expression and purify the vector obtained in step S1 to obtain the MBP-GL fusion recombinant protein:

[0114] S21. Insert the successfully constructed recombinant protein MBP-GL gene expression vector into the Pichia pastoris S44 cells to express the recombinant plasmid pPICZaA-GL. The recombinant plasmid is linearized with Sal I and then electrotransformed into Pichia pastoris S44; expand the culture of the expressed strain at a culture temperature of 30 °C. After culturing the bacteria for 18 h, centrifuge at 2000 rpm for 5 min at room temperature to collect the bacteria; resuspend the collected bacteria with YPD medium to make the OD600 about 1.0; place the bacterial solution in a shake flask and culture at 30 °C and 200 rpm, and add methanol with a concentration of 1% every 24 h; after culturing for 72 h, perform centrifugation.

[0115] S22. Affinity chromatography purify the supernatant collected by centrifugation with polysaccharide resin, and collect the elution peak.

[0116] S23. Further perform hydrophobic purification on the collected elution liquid with MMC to obtain the MBP-GL fusion recombinant protein.

[0117] S24.SDS-PAGE verifies the expression situation, and the results are as Figure 1 shown.

[0118] Example 3

[0119] This example provides an application of MBP-GL fusion recombinant protein, including the following steps:

[0120] S1. Couple the MBP-GL fusion recombinant protein obtained in Examples 1 and 2 with microspheres of SP Focurose HPR (purchased from Wuhan Huiyan Biotechnology) to obtain a coupling product;

[0121] S11. Wash SP Focurose HPR with 10 volumes of injection water, add NaOH and epibromohydrin, stir, and react at 37 °C for 1.5 h.

[0122] S12. After the reaction is completed, wash it with injection water, then add buffer solution, adjust the pH value to 7.5, add MBP-GL fusion recombinant protein, and react in a constant temperature system at 37 °C for 20 h.

[0123] S13. After the reaction stops, wash the reaction product obtained in step S12 with about 10 volumes of injection water. After washing, add 20% ethanolamine solution to block the unreacted epoxy groups and react at 20 °C for 10 h.

[0124] S14. After the reaction is completed, wash it with a large amount of injection water to obtain a coupling product, and store it in a solution containing a preservative.

[0125] S2. Test the IgG adsorption performance of the coupling product obtained in step S1.

[0126] S21. Take 500 μL of the coupling product, add 5 mL of human plasma, and slowly shake it evenly in a room temperature shaker for 1 h to obtain a reaction solution.

[0127] S22. Add the reaction solution obtained in S21 to an affinity chromatography column, first wash it with a balance solution, then elute it with an eluent, and collect the elution peak.

[0128] S23. Use an IgG antibody kit to detect the content of IgG in the eluent, calculate the adsorption amount of IgG by the coupling product, and the standard curve for measuring the IgG adsorption amount by the BCA method is as Figure 2 shown.

[0129] The expression product of the recombinant GL gene produced by the present invention, the MBP-GL fusion recombinant protein, has the advantages of strong IgG binding activity, binding to a broad spectrum of antibodies and high yield. The yield of the target protein is 20 mg / l. The coupling rate of the MBP-GL fusion recombinant protein of the present invention with the microspheres is 88%. The dynamic binding activity with human IgG is: 60 mg IgG / mg MBP-GL. Compared with the recombinant protein pre-packed column on the market (20-50 mg / g human IgG), the recombinant protein product MBP-GL fusion recombinant protein provided by the present invention has high application potential in immunological fields such as antibody purification.

[0130] In summary, the MBP-GL fusion recombinant protein provided by the present invention has the ability to specifically bind to dextran ligands, can also bind to all types of immunoglobulins and their subtypes, and has a relatively simple purification process, high protein purity, high yield, and can be mass-produced.

[0131] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a MBP-GL fusion recombinant protein, characterized in that, It includes the following steps: S1. Construct a recombinant protein MBP-GL gene expression vector; S2. Transform, induce expression and purify the vector obtained in step S1 to obtain the MBP-GL fusion recombinant protein.

2. The preparation method of the MBP-GL fusion recombinant protein according to claim 1, characterized in that, In step S1, the method for constructing the recombinant protein MBP-GL gene expression vector includes the following steps: S11. Tandemly connect the C3 region of SPG and the B domain of protein L, and optimize the gene sequence for codons; S12. Introduce a BamHΙ restriction enzyme site at the front end of MBP, add a cysteine sequence, a stop codon TAA and an EcoRΙ restriction enzyme site consistent with the expression vector at the end of the B region of protein L; S13. After double digestion with BamHΙ and EcoRΙ, ligate it to the vector digested with the same enzymes to construct a recombinant protein MBP-GL gene expression vector of the domain with the strongest binding IgG of protein G and protein L connected to MBP.

3. The preparation method of the MBP-GL fusion recombinant protein according to claim 2, characterized in that, In step S13, the vector digested with the same enzymes is PET28a or pPICZaA.

4. The preparation method of the MBP-GL fusion recombinant protein according to claim 1, wherein In step S2, the method for transformation, induction expression and purification includes the following steps: S21. Transfer the recombinant protein MBP-GL gene expression vector into an expression strain for expanded culture, add an inducer for induction expression, continue culturing, and perform centrifugation after culturing ends; S22. Purify the protein supernatant collected by centrifugation with a polysaccharide resin and collect the elution peak; S23. Further optimize the purity of the elution liquid collected in step S22 by hydrophobic chromatography to obtain the MBP-GL fusion recombinant protein; S24. Verify the expression by SDS-PAGE.

5. The preparation method of the MBP-GL fusion recombinant protein according to claim 4, characterized in that, In step S21, the expression strain is Escherichia coli or Pichia pastoris; the culture temperature is 30-37 °C, and the inducer is isopropyl thiogalactoside or methanol.

6. Use of an MBP-GL fusion recombinant protein, characterized in that, It includes the following steps: S1. Couple the MBP-GL fusion recombinant protein prepared by the preparation method of the MBP-GL fusion recombinant protein according to any one of claims 1-5 with microspheres to obtain a coupling product; S2. Perform an IgG adsorption performance test on the coupling product obtained in step S1.

7. Use of the MBP-GL fusion recombinant protein according to claim 6, characterized in that, In step S1, the coupling method includes the following steps: S11. Rinse the microspheres clean, add NaOH and epibromohydrin, and react after stirring; S12. After the reaction ends, rinse clean, add a buffer solution, adjust the pH value, add the MBP-GL fusion recombinant protein, and react in a constant temperature system; S13. Rinse the reaction product obtained in step S12, add a 20% ethanolamine solution to block the unreacted epoxy groups, and react at a predetermined temperature; S14. After the reaction is completed, rinse to obtain a coupling product, and store it in a solution containing a preservative.

8. Use of the MBP-GL fusion recombinant protein according to claim 7, characterized in that, In step S11, the reaction temperature is 35-38 °C, and the reaction time is 1-2 h; in step S12, the pH value is 7-8, the temperature of the constant temperature system is 36.5-37.5 °C, and the reaction time is 20-22 h.

9. Use of the MBP-GL fusion recombinant protein according to claim 7, characterized in that, In step S13, the reaction temperature is 20-22 °C, and the reaction time is 8-10 h.

10. Use of the MBP-GL fusion recombinant protein according to claim 6, characterized in that, In step S2, the method for testing the IgG adsorption performance includes the following steps: S21. Take the coupling product, add human plasma, and slowly shake it evenly in a room temperature shaker to obtain a reaction solution; S22. Add the reaction solution obtained in S21 into an affinity chromatography column, first rinse it with a balance solution, and then elute it with an eluent, and collect the elution peak; S23. Use an IgG antibody kit to detect the content of IgG in the eluent, and calculate the adsorption amount of the coupling product to IgG.

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