Preparation method and application of CBD-AL fusion recombinant protein

Abstract

The invention provides a preparation method and application of a CBD-AL fusion recombinant protein, a variant Z region obtained by site-directed mutagenesis of a B region of SPA is connected in series with a B structural domain of a protein L, codon optimization is carried out, the front end of CBD is introduced into a BamHI restriction enzyme cutting site, a cysteine sequence, a termination codon TAA and an EcoRI restriction enzyme cutting site are added to the tail end of the B region of the recombinant protein L, and the CBD-AL fusion recombinant protein is obtained. BamHI and EcoRI are subjected to double enzyme digestion and then are connected to a PET28a vector subjected to double enzyme digestion, a recombinant protein CBD-AL gene expression vector of a structural domain of strongest binding IgG of protein A and protein L connected with CBD is constructed, the recombinant protein CBD-AL gene expression vector is transferred into an expression strain for amplification culture, an inducer is added for induction, bacterial sludge is collected, purification is performed by cellulose affinity chromatography, and the recombinant protein CBD-AL gene expression vector is obtained. A binding protein which has a cellulose binding function and can bind all types of immunoglobulins and subtypes thereof is prepared.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and particularly to a preparation method and application of a CBD-AL fusion recombinant protein. Background Art

[0002] Staphylococcal protein A (referred to as SPA) is a natural single-chain polypeptide present in the cell walls of most Staphylococcus aureus and is a major component of bacterial antigens. SPA consists of domains S, E, D, A, B, C, and X. Its five domains E, D, A, B, and C belong to the antibody-binding regions, and each domain has approximately 59 amino acids and can act on Fc alone. SPA does not contain cysteine, has no disulfide bonds or sulfhydryl groups, and thus has stable physicochemical properties. SPA has been discovered and widely used due to its immunological activity. Its most important immunological property is its ability to bind to immunoglobulins of humans and various mammalian cells, forming an immune precipitate that does not affect the immunological activity of the antibody. Recent studies have found that the binding site of immunoglobulin G (referred to as IgG) to the SPA molecule is the functional region at the junction of the CH3 segment and CH2, and SPA does not have a strong binding force with all mammalian IgGs, and its binding to mammalian IgGs is species-specific. Research has shown that the order of affinity of SPA for binding to mammalian IgGs is pig, dog, rabbit, human, monkey, rat, mouse, and cow. In addition, SPA does not react with all immunoglobulins. Research has shown that SPA mainly reacts with IgG subclasses IgG1, IgG2, and IgG4 in immunoglobulins and does not react with IgG3. In addition, SPA can also bind to a small amount of immunoglobulin M (referred to as IgM) and immunoglobulin A (referred to as IgA) in serum. The adsorption rates of SPA bacteria for various immunoglobulins are: 90%-98% for IgG, 2%-30% for IgM, and 1.5%-20% for IgA.

[0003] Protein L from Peptostreptococcus magnus (abbreviated as Protein L) is a cell wall surface protein from Peptostreptococcus magnus, which has broad IgG binding activity and was first discovered in 1895. Protein L is an elongated fibrous protein, and its specific binding site to antibodies is located in the variable region of the Kappa light chain, which is different from Protein A and Protein G that act on the Fc region. Some studies have found that Protein L has a total of 719 amino acids, including 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%. 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, which 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 to the variable region of the light chain are all located in the repetitive conserved sequence FR framework region and do not exert 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.

[0004] Protein A has been widely used in immunology due to its broad spectrum of action types, including: Western blot analysis to detect various antigen-antibody complexes on nitrocellulose membranes. Moreover, Protein A is widely used as a ligand coupled to resins in affinity chromatography for antibody purification. The most important application is the Protein A immunoadsorption column, which uses Protein A as a ligand and a high molecular polymer such as agarose gel as a carrier to prepare a protein affinity chromatography medium for separating antigens or fragments. Due to its advantages such as safety, effectiveness, low side effects, and high specificity, Protein A adsorption has gradually replaced traditional hemodialysis and has been widely used in the medical industry. Protein L has a broader spectrum of action types compared to Protein A and can act on the IgG3 subtype and antibody fragments. Therefore, the protein expressed by recombining the functions of Protein A and Protein L can basically cover almost all antibodies and fragments. Currently, the recombinant human IgG-binding proteins on the market contain tandem or pairwise combinations of the domains of Protein A, Protein G, and Protein L, and use traditional tags such as His tags 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. With the in-depth study of the functions of immunoglobulin-binding proteins, the market demand for Protein A and various recombinant ligands and their affinity packing materials will increase rapidly. Currently, worldwide, there are relatively few two-combined recombinant protein ligands, 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.

[0005] In view of this, it is necessary to design a preparation method for an improved CBD-AL fusion recombinant protein to solve the above problems. Summary of the Invention

[0006] Aiming at the defects of the above-mentioned existing technologies, the purpose of the present invention is to provide a binding protein that has cellulose-binding function and can bind all types of immunoglobulins and their subtypes.

[0007] To achieve the above purpose, the present invention provides a preparation method for a CBD-AL fusion recombinant protein, including the following steps:

[0008] S1. Construct a recombinant protein CBD-AL gene expression vector;

[0009] S2. Transform, induce expression, and purify the vector obtained in step S1 to obtain the CBD-AL fusion recombinant protein.

[0010] As a further improvement of the present invention, in step S1, the method for constructing the recombinant protein CBD-AL gene expression vector includes the following steps:

[0011] S11. Tandemly link the variant Z region with site-directed mutagenesis in the B region of SPA to the B domain of protein L and perform codon optimization;

[0012] S12. Introduce a BamHΙ restriction site at the front end of CBD, add a cysteine sequence, a stop codon TAA, and an EcoRΙ restriction site consistent with the expression vector to the end of the B region of recombinant protein L;

[0013] S13. After double digestion with BamHΙ and EcoRΙ, ligate it to the PET28a vector digested with the same enzymes to construct a recombinant protein CBD-AL gene expression vector with the domain that binds IgG most strongly between protein A and protein L connected to CBD.

[0014] Furthermore, in step S2, the method of transformation, induction expression and purification includes the following steps:

[0015] S21. Transfer the recombinant protein CBD-AL gene expression vector into an expression strain for expanded culture, add an inducer for induction expression, continue culturing, and centrifuge to collect the bacterial cells;

[0016] S22. Lyse the collected bacterial cells, purify the protein supernatant collected by centrifugation using cellulose affinity chromatography, collect the elution peak, then dialyze and concentrate, and perform concentration replacement to obtain the CBD-AL fusion recombinant protein;

[0017] S23. Verify the expression by SDS-PAGE.

[0018] In step S21, the expression strain is Escherichia coli or yeast; the culture temperature is 30 - 37 °C, the induction temperature is 28 - 37 °C, and the inducer is IPTG (isopropyl β-D-thiogalactoside) or methanol.

[0019] In step S22, the lysis of bacterial cells is achieved by the following method: Add PBS solution to the bacterial cells at a volume ratio of 1:10, the ultrasonic power for lysis is 300 - 800 W, and the lysis time is 5 - 10 min.

[0020] The present invention also provides an application of the CBD-AL fusion recombinant protein, including the following steps:

[0021] S1. Couple the CBD-AL fusion recombinant protein prepared by the preparation method of the above-mentioned CBD-AL fusion recombinant protein with microspheres to obtain a coupling product;

[0022] S2. Perform IgG adsorption performance testing on the coupling product obtained in step S1.

[0023] Furthermore, in step S1, the coupling method includes the following steps:

[0024] S11. Rinse the microspheres thoroughly, add NaOH and epibromohydrin, and react after stirring.

[0025] S12. After the reaction is completed, rinse thoroughly, add buffer solution, adjust the pH value, add CBD-AL fusion recombinant protein, and react in a constant temperature system.

[0026] S13. Rinse the reaction product obtained in step S12, and add 20% ethanolamine solution to block the unreacted epoxy groups.

[0027] S14. After the reaction is completed, rinse to obtain the coupling product, and store it in a solution containing a preservative.

[0028] In step S11, the temperature of the reaction is 30-37 °C, and the reaction time is 1-2 h.

[0029] In step S12, the pH value is 7-9, the temperature of the constant temperature system is 30-37 °C, and the reaction time is 20-22 h.

[0030] In step S13, the temperature of the reaction is 20-22 °C, and the reaction time is 8-10 h.

[0031] Further, in step S2, the method for testing the IgG adsorption performance includes the following steps:

[0032] S21. Take the coupling product, add human plasma, and slowly shake well in a room temperature shaker to obtain a reaction solution.

[0033] S22. Add the reaction solution to an affinity chromatography column, first rinse with the equilibration solution, then elute with the eluent, and collect the elution peak.

[0034] 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.

[0035] The beneficial effects of the present invention are:

[0036] (1) The preparation method of a CBD-AL fusion recombinant protein provided by the present invention constructs an AL tandem structure by combining the variant Z region with site-directed mutation in the B region of SPA and the B fragment with the strongest antibody-binding ability in the protein L sequence, and successfully expresses it in an expression strain. The protein product has a relatively broad affinity, a relatively high loading capacity, a relatively simple recombinant protein purification process, a high yield, and can be mass-produced. At the same time, the present invention combines the cellulose-binding region with the AL tandem to prepare a recombinant protein that has both cellulose-binding ability and the ability to bind all antibodies and subtype antibody fragments with a high loading capacity.

[0037] (2) The gene sequence of the present invention is codon-optimized to highly express recombinant proteins in the prokaryotic system of the expression strain. The operation is simple, the culture period is short, and the production cost is greatly reduced.

[0038] (3) The CBD-AL fusion recombinant protein provided by the present invention is composed of the variant Z region with site-directed mutation in the B region of SPA and the B region of protein L in series, which can efficiently and specifically bind IgG antibodies and various subtypes, thus ensuring the removal effect on IgG antibodies.

[0039] (4) The CBD-AL fusion recombinant protein provided by the present invention has a cellulose-binding region and can be purified with a cellulose filler with a lower price. The target protein can specifically bind to cellulose, avoiding the influence of miscellaneous proteins, making protein purification simpler, with lower cost and higher purity of the purified protein.

[0040] (5) The CBD-AL fusion recombinant protein provided by the present invention can specifically bind to cellulose-containing microspheres, which is beneficial to increasing the loading capacity of the microspheres for the recombinant protein and achieving a higher coupling rate.

[0041] (6) Compared with the protein A pre-packed column on the market (20 - 50 mg / g human IgG), the CBD-AL fusion recombinant protein provided by the present invention has higher dynamic binding activity to human IgG and has high application potential in immunological fields such as antibody purification.

[0042] The amino acid sequence of the said SPA is shown as SEQ ID NO:1:

[0043] SEQ ID No:1

[0044] AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKEEDNKKPGKEDGNKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDNNKPGKEDGNKPGKEDNNKPGKEDGNKPGKEDGNKPGKEDGNGVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELVVD

[0045] The amino acid sequence of the protein L is shown in SEQ ID NO:2:

[0046] SEQ ID No:2

[0047] KEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKENGKYTADLEDGGYTINIRFAGKKVDEKPEEKEQVTIKENIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTINIRFAG

[0048] The amino acid sequence of the cellulose-binding region is shown in SEQ ID No:3:

[0049] SEQ ID No:3

[0050] TMATGPEDLGTGLLEALLRGDLAGAEALFRRGLRFWGPEGILEHLLLPVLREVGEAWHRGEIGVAEEHLASTFLRARLQELLDLAGFPPGPPVLVTTPPGERHEIGAMLAAYHLRRKGVPALYLGPDTPLPDLRALARRLGAGAVVLSALLSEPLRALPDGALKDLAPRVFLGGQGAGPEEARRLGAEYMEDLKGLAEALWLPRGPEKEAIPGEFSGSGSPG Description of the Drawings

[0051] Figure 1 SDS-PAGE diagram of the CBD-AL fusion recombinant protein provided for Example 1.

[0052] Figure 2 Standard curve graph for measuring IgG adsorption amount by the BCA method in Example 2. Detailed Implementation Modes

[0053] 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 embodiments.

[0054] Here, it also needs to be noted that in order to avoid obscuring the present invention due to 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.

[0055] In addition, it also needs to be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0056] The present invention provides a method for preparing a CBD-AL fusion recombinant protein, comprising the following steps:

[0057] S1. Construct a recombinant protein CBD-AL gene expression vector, and the specific steps are as follows:

[0058] S11. Connect the variant Z region obtained by site-directed mutagenesis of the B region of SPA in series with the B domain of protein L, and perform codon optimization.

[0059] S12. Introduce the CBD front end into the BamHΙ restriction site, add a cysteine sequence, the stop codon TAA, and the EcoRΙ restriction site consistent with the expression vector to the end of region B of recombinant protein L.

[0060] S13. After double digestion with BamHΙ and EcoRΙ, ligate it to the PET28a vector digested with the same double enzymes to construct a recombinant protein CBD-AL gene expression vector for the domain with the strongest binding IgG of protein A and protein L connected to CBD.

[0061] S2. Transform, induce expression, and purify the vector obtained in step S1 to obtain the CBD-AL fusion recombinant protein. The specific steps are as follows:

[0062] S21. Transfer the recombinant protein CBD-AL gene expression vector into an expression strain for expanded culture, add an inducer for induced expression, continue culturing, and centrifuge to collect the bacterial cells.

[0063] Specifically, transfer the recombinant protein CBD-AL gene expression vector into Escherichia coli or yeast for expanded culture. The culture temperature is 30-37°C, add the inducer IPTG (isopropyl β-D-thiogalactoside) or methanol for induced expression, and the induction temperature is 28-37°C.

[0064] S22. Lyse the collected bacterial cells, purify the protein supernatant collected by centrifugation using cellulose affinity chromatography, collect the elution peak, then dialyze and concentrate, and perform concentration replacement to obtain the CBD-AL fusion recombinant protein.

[0065] Specifically, the lysis of bacterial cells is achieved by the following method: Add PBS solution to the bacterial cells at a volume ratio of 1:10, the ultrasonic power for lysis is 300-800W, and the lysis time is 5-10 minutes.

[0066] S23. Verify the expression by SDS-PAGE.

[0067] The present invention also provides an application of the CBD-AL fusion recombinant protein, including the following steps:

[0068] S1. Couple the CBD-AL fusion recombinant protein prepared by the preparation method of the CBD-AL fusion recombinant protein according to any one of claims 1-5 with microspheres to obtain a coupling product.

[0069] Specifically, the coupling method includes the following steps:

[0070] S11. Rinse the microspheres thoroughly, add NaOH and epibromohydrin, and react after stirring.

[0071] Specifically, rinse the microspheres thoroughly, add NaOH and epibromohydrin, and react at 30 - 37 °C for 1 - 2 h after stirring.

[0072] S12. After the reaction is completed, rinse thoroughly, add buffer solution, adjust the pH value, add CBD-AL fusion recombinant protein, and react in a constant temperature system.

[0073] Specifically, after the reaction is completed, rinse thoroughly, add buffer solution, adjust the pH value to 7 - 9, add CBD-AL fusion recombinant protein, and react in a constant temperature system at 30 - 37 °C for 20 - 22 h.

[0074] S13. Rinse the reaction product obtained in step S12, and add 20% ethanolamine solution to block the unreacted epoxy groups.

[0075] Specifically, rinse the reaction product obtained in step S12, add 20% ethanolamine solution to block the unreacted epoxy groups, and react at 20 - 22 °C for 8 - 10 h.

[0076] S14. After the reaction is completed, rinse to obtain the coupling product, and store it in a solution containing preservatives.

[0077] S2. Perform IgG adsorption performance test on the coupling product obtained in step S1.

[0078] Specifically, the method for the IgG adsorption performance test includes the following steps:

[0079] S21. Take the coupling product, add human plasma, and shake gently in a room temperature shaker to obtain a reaction solution.

[0080] S22. Add the reaction solution into an affinity chromatography column, first rinse with the equilibration solution, then elute with the eluent, and collect the elution peak.

[0081] S23. Use an IgG antibody kit to detect the IgG content in the eluent, and calculate the IgG adsorption amount of the coupling product.

[0082] The following specifically describes the synthesis method of the recombinant protein AL gene expression vector provided by the present invention in conjunction with specific embodiments.

[0083] Example 1

[0084] This example provides a preparation method of CBD-AL fusion recombinant protein, including the following steps:

[0085] S1. Construct a recombinant protein CBD-AL gene expression vector, specifically including the following steps:

[0086] S11. The variant Z region with site-directed mutagenesis in the B region of SPA is concatenated with the B domain of protein L, and the gene sequence is codon-optimized according to the codon preference of E. coli expression.

[0087] S12. Introduce a BamHΙ restriction site at the front end of CBD, add a cysteine sequence, a stop codon TAA and an EcoRΙ restriction site consistent with the expression vector at the end of the monomer of the B domain of protein L.

[0088] S13. After double digestion with BamHΙ and EcoRΙ, it is ligated to the PET28a vector digested with the same enzymes to construct a recombinant protein CBD-AL gene expression vector of the domain with the strongest binding IgG of protein A and protein L connected to CBD.

[0089] S2. Transform, induce expression and purify the vector obtained in step S1 to obtain the CBD-AL fusion recombinant protein, which specifically includes the following steps:

[0090] S21. Transfer the recombinant protein CBD-AL gene expression vector into an E. coli expression strain, expand the culture at 37°C, and when the OD600 value of the bacterial cells reaches 0.8, add an inducer IPTG (isopropylthiogalactoside) for induced expression, continue to culture overnight, and centrifuge to collect the bacterial cells.

[0091] S22. Add the collected bacterial cells to PBS solution at a volume ratio of 1:10, ultrasonically disrupt at a power of 500 W for 8 min, purify the protein supernatant collected by centrifugation using cellulose affinity chromatography, collect the elution peak, dialyze and concentrate, and perform concentration replacement to obtain the CBD-AL fusion recombinant protein.

[0092] S23. Verify the expression by SDS-PAGE. The SDS-PAGE pattern of the CBD-AL fusion recombinant protein is as Figure 1 shown.

[0093] Example 2

[0094] This example provides an application of the CBD-AL fusion recombinant protein, which includes the following steps:

[0095] S1. Couple the CBD-AL fusion recombinant protein prepared in Example 1 with microspheres to obtain a coupling product. Specifically includes the following steps:

[0096] S11. Wash SP Focurose HPR with 10 times the volume of injection water, add NaOH and epibromohydrin, stir, and react at 37°C for 1.5 h.

[0097] S12. After the reaction is completed, rinse it thoroughly with water for injection, then add buffer solution, adjust the pH value to 7.5, add the CBD-AL fusion recombinant protein prepared in Example 1, and react in a constant temperature system at 37 °C for 20 h.

[0098] S13. Rinse the packing material with 10 times the volume of water for injection. After rinsing thoroughly, add 20% ethanolamine solution to block the unreacted epoxy groups, and react at 20 °C for 10 h.

[0099] S14. After the reaction is completed, rinse with a large amount of water for injection to obtain the coupling product, and store it in a solution containing preservatives.

[0100] S2. Perform IgG adsorption performance test on the coupling product obtained in step S1. Specifically, it includes the following steps:

[0101] S21. Take 500 μL of the coupling product obtained in step S1, add 5 mL of human plasma, and slowly shake it evenly in a room temperature shaker for 1 h to obtain a reaction solution.

[0102] S22. Add the reaction solution in step S21 to an affinity chromatography column, first rinse it with the equilibration solution, then elute it with the elution solution, and collect the elution peak.

[0103] S23. Use an IgG antibody kit to detect the content of IgG in the eluate, and calculate the adsorption amount of the CBD-AL fusion recombinant protein to IgG. The standard curve for measuring the IgG adsorption amount by the BCA method is as Figure 2 shown.

[0104] The CBD-AL fusion recombinant protein provided by the present invention has the advantages of strong IgG binding activity, high purity and high yield. The yield of the target protein is 22.6 mg / L. The coupling rate of the CBD-AL fusion recombinant protein of the present invention with the microspheres is 92%, and the dynamic binding activity with human IgG is: 80 mg IgG / mg CBD-AL. Compared with the recombinant protein 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.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention rather than 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 CBD-AL fusion recombinant protein, characterized in that, The following steps are involved: S1. Construction of recombinant protein CBD-AL gene expression vector; S2. Transform, induce expression and purify the vector obtained in step S1 to obtain CBD-AL fusion recombinant protein.

2. The preparation method of the CBD-AL fusion recombinant protein according to claim 1, characterized in that, In step S1, the method for constructing a recombinant protein CBD-AL gene expression vector comprises the following steps: S11. The variant Z region obtained by site-directed mutation of the B region of SPA is connected in series with the B domain of protein L and codon optimization is performed; S12. The front end of CBD was introduced into the BamHΙ restriction site, and the end of the B region of the recombinant protein L was added with a cysteine ​​sequence, a stop codon TAA and an EcoRΙ restriction site consistent with the expression vector; S13. After double digestion with BamHΙ and EcoRΙ, it was connected to the PET28a vector that was also double digested with enzymes to construct a recombinant protein CBD-AL gene expression vector with the strongest IgG binding domain of protein A and protein L connected to CBD.

3. The preparation method of the CBD-AL fusion recombinant protein according to claim 1, characterized in that In step S2, the method of transformation, induction expression and purification comprises the following steps: S21. The recombinant protein CBD-AL gene expression vector is transferred into the expression strain for expansion and culture, an inducer is added to induce expression, the culture is continued, and the cells are collected by centrifugation; S22. The collected cells were crushed, the protein supernatant collected by centrifugation was purified by cellulose affinity chromatography, the elution peak was collected and then dialyzed and concentrated, and concentrated and replaced to obtain CBD-AL fusion recombinant protein; S23. SDS-PAGE was used to verify the expression.

4. The preparation method of the CBD-AL fusion recombinant protein according to claim 3, wherein In step S21, the expression strain is Escherichia coli or yeast; the culture temperature is 30-37°C, the induction temperature is 28-37°C, and the inducer is IPTG (isopropylthiogalactoside) or methanol.

5. The preparation method of the CBD-AL fusion recombinant protein according to claim 3, characterized in that, In step S22, the bacterial cells are crushed by the following method: the bacterial cells are added with PBS solution at a volume ratio of 1:10, the ultrasonic crushing power is 300-800W, and the crushing time is 5-10min.

6. Use of a CBD-AL fusion recombinant protein, characterized in that, The following steps are involved: S1. coupling the CBD-AL fusion recombinant protein obtained by the method for preparing the CBD-AL fusion recombinant protein according to any one of claims 1 to 5 with microspheres to obtain a coupling product; S2. The coupling product obtained in step S1 is subjected to an IgG adsorption performance test.

7. Use of the CBD-AL fusion recombinant protein according to claim 6, characterized in that, In step S1, the coupling method comprises the following steps: S11. Rinse the microspheres, add NaOH and epibromopropane, and stir to react; S12. After the reaction is completed, rinse thoroughly, add buffer, adjust the pH value, add CBD-AL fusion recombinant protein, and react in a constant temperature system; S13. Rinse the reaction product obtained in step S12, and add 20% ethanolamine solution to block the unreacted epoxy groups; S14. After the reaction is completed, the product is washed to obtain a coupling product, which is then stored in a solution containing a preservative.

8. Use of the CBD-AL fusion recombinant protein according to claim 7, characterized in that, In step S11, the reaction temperature is 30-37°C, and the reaction time is 1-2h; in step S12, the pH value is 7-9, the temperature of the constant temperature system is 30-37°C, and the reaction time is 20-22h.

9. Use of the CBD-AL 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 CBD-AL 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 well in a room temperature shaker to obtain a reaction solution; S22. Add the reaction solution into an affinity chromatography column, first rinse with a balance solution, then elute with an eluent, and collect the elution peak; S23. Use an IgG antibody kit to detect the IgG content in the eluent and calculate the IgG adsorption amount of the coupling product.

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