Preparation method and application of serum antibody affinity separation material

Abstract

The present invention provides a method for preparing a serum antibody affinity separation material, which comprises washing an agarose matrix, epoxy activation reaction, and coupling with a recombinant Streptococcus Protein G (SPG) with agarose as a matrix to prepare the serum antibody affinity separation material; or washing an agarose matrix, epoxy activation reaction, amination reaction of the activated agarose, and coupling to prepare the serum antibody affinity separation material. Also provided is an application of the serum antibody affinity separation material prepared by the present invention, which is used for adsorbing immunoglobulin G (IgG). The serum antibody affinity separation material prepared by the present invention does not need to use the classic but highly toxic cyanogen bromide as a coupling reagent, and has good biosafety.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation and purification, and specifically relates to a preparation method and application of a serum antibody affinity separation material. Background Art

[0002] The existing biomimetic affinity / ligand-ligand separation and purification methods have the limitation of slightly low specificity. The existing SPG affinity separation and purification methods also have limitations, including the following two points: (1) the activation reagent cyanogen bromide is highly toxic and has low biosafety; (2) the ligand is easily detached. During the purification of clinical pharmaceutical antibodies, the detached SPG affinity ligand is likely to cause inflammation or hypersensitivity immune response in patients. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for preparing a serum antibody affinity separation material and its application in view of the above-mentioned deficiencies in the prior art. The serum antibody affinity separation material prepared by the present invention has adsorption properties for antibodies.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing serum antibody affinity separation material, the method comprising:

[0005] S1. Washing of agarose matrix: Place agarose in a sand core funnel and wash with ultrapure water 10 to 20 times, and obtain dried agarose by suction filtration;

[0006] S2, epoxy activation reaction: add epichlorohydrin solution to the dried agarose obtained in S1, preheat for 30 min in a shaker at 25°C to 60°C and 170 rpm / min to 220 rpm / min, then continue to react for 4 h in a shaker at 25°C to 60°C and 170 rpm / min to 220 rpm / min, detect pH once every 1 h, and adjust pH to alkaline condition with NaOH dry powder or NaOH solution with a concentration of 1 mol / L to 8 mol / L. After the reaction, wash 10 times with ultrapure water to obtain activated agarose gel;

[0007] S3. Preparation of serum antibody affinity separation materials:

[0008] The activated agarose gel obtained in S2 is coupled with the recombinant SPG protein to obtain the serum antibody affinity separation material. The specific method is as follows:

[0009] Coupling of recombinant SPG protein: the recombinant SPG protein purified by Ni metal chelate chromatography column is replaced by Superdex G75 gel filtration column: the protein solution is collected by PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0, and after vacuum freeze drying, a frozen SPG protein powder is obtained, and then a PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0 is added to dissolve to obtain a recombinant SPG protein solution with a concentration of 4 to 16 mg / mL, and the recombinant SPG protein solution is added to the activated agarose gel obtained in S2, and coupled for 24 hours at a temperature of 16°C to 37°C and a rotation speed of 70 rpm / min to 180 rpm / min, and then a NaCl solution with a concentration of 0.1 mol / L to 2.0 mol / L is added, and then washed with double distilled water to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material;

[0010] Alternatively, the activated agarose gel obtained in S2 is subjected to an amination reaction, coupled with cyanuric chloride, and then subjected to a substitution reaction with the amino residues on the recombinant SPG protein to finally obtain a serum antibody affinity separation material. The specific method is as follows:

[0011] Amination reaction: adding an amination reagent to the activated agarose gel obtained in S2, adjusting the pH to 12.0-13.0, reacting for 12-16 hours at a temperature of 50° C.-60° C. and a rotation speed of 170 rpm / min-200 rpm / min, and then washing with ultrapure water for 10-20 times to obtain ammoniated agarose;

[0012] Indirect arm connection: suspend the amino-modified agarose in ultrapure water at 0°C to 4°C, add a cyanuric chloride acetone solution precooled at -20°C for 3h, react in an ice bath at a speed of 170rpm / min for 5.5h, detect the pH every 0.5h during the reaction, and adjust the pH to 7.0-8.0 with a saturated Na2CO3 solution. After the reaction, wash with ultrapure water 10 times, and then filter to obtain the agarose after indirect arm connection; the ratio of cyanuric chloride to acetone solution in the cyanuric chloride acetone solution is 0.03g:1mL;

[0013] The agarose to which the indirect arm is connected is coupled with a purified recombinant SPG protein solution with a concentration of 4 mg / mL to 16 mg / mL at a temperature of 25° C. and a rotation speed of 200 rpm / min for 24 hours to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material.

[0014] Preferably, the mass volume ratio of the drained agarose and the epichlorohydrin solution in S2 is 1 g:2.1 mL.

[0015] Preferably, when the activated agarose gel obtained in S2 is subjected to an amination reaction and indirect arm connection to obtain a serum antibody affinity separation material, the amination reagent in S3 includes ammonia water, hydrazine hydrate or 1,6-hexanediamine.

[0016] Preferably, when the activated agarose gel obtained in S2 is subjected to an amination reaction and indirect arm connection to obtain a serum antibody affinity separation material, the amount ratio of the amination agarose and the cyanuric chloride acetone solution in S3 is 1 g:2.1 mL.

[0017] Preferably, when the activated agarose gel obtained in S2 is subjected to amino reaction and indirect arm connection to obtain serum antibody affinity separation material, the dosage ratio of the agarose after indirect arm connection to the recombinant SPG protein solution in S3 is 1g:0.7mL.

[0018] Preferably, the preparation method of the recombinant SPG protein in the recombinant SPG protein solution in S3 is:

[0019] S301, preparing a gene encoding a recombinant SPG protein by chemical synthesis (the nucleotide sequence is shown in SEQ ID NO: 1); then performing codon optimization to obtain a nucleotide sequence of the recombinant SPG protein (as shown in SEQ ID NO: 4), and then using the gene sequence as a template to design primer sequences for PCR amplification to obtain a PCR product;

[0020] The reaction system of the PCR amplification is: 10 μL of 5×PS buffer, 4 μL of dNTP with a concentration of 2.5 mmol / L, 1 μL of upstream primer with a concentration of 10 μmol / L, 1 μL of downstream primer with a concentration of 10 μmol / L, 1.0 μL of DNA polymerase with a concentration of 2.5 U / μL, 1 μL of genomic DNA to be tested, and double distilled water to 50 μL; the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3;

[0021] The PCR amplification reaction procedure is: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 80 s, a total of 32 cycles; 72°C extension for 10 min, 4°C storage;

[0022] S302, after electrophoresis of the PCR product in S301, the gel was cut to recover the band at about 500 bp to obtain the purified target gene, and the purified target gene and the expression vector pET-28a were double-digested with NdeⅠ / XhoⅠ restriction endonucleases to obtain the target gene double-digestion product and the expression vector pET-28a double-digestion product respectively;

[0023] The enzyme digestion reaction system is: 6 μL of purified target gene or expression vector pET-28a, 16 μL of 10×FastDigestBuffer, 1 μL of FastDigest NdeⅠ, 1 μL of FastDigest XhoⅠ, and 36 μL of ddH2O;

[0024] S303, respectively recovering the target gene double-enzyme digestion product and the expression vector pET-28a double-enzyme digestion product obtained in S302 using an AxyPrepTM DNA GelExtraction Kit gel recovery kit to obtain a double-enzyme digestion gel-recovered SPG gene and a double-enzyme digestion pET-28a gel recovery product, respectively, and subjecting the double-enzyme digestion gel-recovered SPG gene and the double-enzyme digestion pET-28a gel recovery product to a ligation reaction at 22° C. for 1.5 h to obtain a ligation product;

[0025] The ligation product was transformed into competent cells E. coli DH5α by heat shock method, and positive clones were screened by LB agar plate pre-added with 1 mM kanamycin, and then cultured in LB liquid medium, and positive clones were verified by colony PCR. The recombinant clone strain with correct sequencing results was named pET-28a-SPG-DH5a strain;

[0026] The ligation reaction system is: 2 μL of 5×Ligase Reaction Buffer, 4 μL of SPG gene recovered from gel after double enzyme digestion, 8 μL of pET-28a gel recovery product from double enzyme digestion, 2 μL of T4 DNA ligase with a concentration of 5 U / μL, and 4 μL of double distilled water;

[0027] S304, expression of recombinant SPG protein: The plasmid of the logarithmic growth phase of the pET28a-SPG-DH5a strain obtained in S303 was extracted using a plasmid extraction kit and transformed into the expression host bacteria E. coli by heat shock. BL21 (DE3) competent cells, inoculate the positive monoclonal colonies screened on the solid LB plate into LB liquid medium pre-added with 1 mM kanamycin, culture overnight at 37°C and 180 rpm / min, then transfer to a new 500 mL LB liquid medium pre-added with 1 mM kanamycin, continue to culture at 37°C and 180 rpm / min until the absorbance of the bacterial solution at 600 nm reaches 0.4-0.6, add isopropyl-β-D-thiogalactoside solution to make the final concentration of the expression host bacteria 1.0 mmol / L, take 1 mL after induction for 8 hours, centrifuge at 10000 rpm / min, collect the bacterial sludge, dilute with 10 mmol / L phosphate buffer, add 5× Loading Buffer, heat in a 100°C water bath for 10 min, and detect by SDS-PAGE to obtain the expression host bacteria containing the target protein;

[0028] S305. Purification of recombinant SPG protein: After the expression host bacteria containing the target protein obtained in S304 are subjected to three stages of activation, transfer and bacterial enrichment culture, the expression host bacteria containing the target protein is resuspended in a phosphate buffer with a concentration of 10 mmol / L at a mass volume ratio of 1 g:20 mL, and the bacterial cell wall is ultrasonically broken under ice bath conditions at 0°C to 4°C, and then centrifuged at 8000 rpm / min for 30 min, the supernatant is collected, and purified by Ni metal chelate column chromatography to obtain recombinant SPG protein; the ultrasonic conditions are: first ultrasonically break the bacteria for 2 seconds, then rest for 3 seconds, and repeat the ultrasonic breaking and intermittent operation for a total of 40 minutes, with a power of 55 W; the nucleotide sequence of the recombinant SPG protein is shown in SEQ ID NO:4.

[0029] The recombinant SPG protein involved in the present invention is recombinant Streptococcus Protein G (SPG for short).

[0030] The present invention also provides the application of the serum antibody affinity separation material prepared above, wherein the serum antibody affinity separation material is used for adsorbing antibody IgG.

[0031] IgG stands for immunoglobulin G (IgG).

[0032] In the present invention, the matrix activated by epichlorohydrin can directly use the amino group of the recombinant SPG protein to complete the epoxy ring-opening reaction; or agarose is activated by epichlorohydrin under strong alkaline conditions; the activated medium is washed, then reacted with ammonia water, and the medium is washed with dimethyl sulfoxide and water. Then cyanuric chloride is connected under alkaline conditions. Finally, the purified recombinant SPG protein amino group is directly used to undergo a substitution reaction with the chlorine atom of cyanuric chloride to form a covalent bond. The amination reagents for reacting with epoxy groups described in this scheme include but are not limited to ammonia water, and such amino-containing compounds include a series of diamino compounds such as hydrazine hydrate and 1,6-hexanediamine.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] The present invention can directly select epichlorohydrin-activated agarose, and use the amino group of the recombinant SPG protein to complete the ring-opening reaction; at the same time, the agarose is activated by epichlorohydrin under strong alkaline conditions, reacted with an amination reagent after washing, and washed with dimethyl sulfoxide and water, and then connected with cyanuric chloride under alkaline conditions, and finally the amino group of the purified recombinant SPG protein is directly used to undergo a substitution reaction with the chlorine atom of cyanuric chloride to form a covalent bond. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the SDS-PAGE electrophoresis diagram of the recombinant SPG protein induced to express PET28a-SPG-BL21 (DE3) in Example 2 of the present invention.

[0036] Figure 2 It is the SDS-PAGE electrophoresis diagram of affinity chromatography of serum antibody by recombinant SPG protein affinity medium at different time points in Example 4 of the present invention.

[0037] Figure 3 This is the SDS-PAGE electrophoresis diagram of the affinity chromatography sample of serum antibody using the affinity medium of the activation medium with different spacer arms coupled to the recombinant SPG protein in Example 5 of the present invention. DETAILED DESCRIPTION

[0038] Example 1

[0039] Codon optimization of the recombinant Streptococcus G protein (SPG) encoding gene.

[0040] Codon optimization for protein recombinant expression refers to the different codon preferences used by different species to encode a certain amino acid; in essence, the tRNA abundance corresponding to the amino acid in different species is different. Therefore, this study optimized the codons of Streptococcus G protein. The rare codons of E. coli tRNA in the SPG gene were eliminated, and codon optimization was completed in the following ways: (1) replacing the original sequence codons with synonymous codons of similar frequency in the E. coli host; (2) randomly replacing the original gene codons with synonyms according to the host codon frequency. The expression rate of the protein in the body determines its activity. (3) GC content adjustment; (4) avoidance of base duplication, etc. The nucleotide sequence of the recombinant SPG protein obtained after codon optimization is shown in SEQ ID NO:4, and the nucleotide sequence of the original codon is shown in SEQ ID NO:1.

[0041] Example 2

[0042] This embodiment is a method for preparing a recombinant SPG protein, which is:

[0043] S301, preparing a gene encoding a recombinant SPG protein by chemical synthesis, wherein the nucleotide sequence of the gene encoding the recombinant SPG protein is shown in SEQ ID NO: 1; then codon optimization is performed to obtain a nucleotide sequence of the recombinant SPG protein (as shown in SEQ ID NO: 4), and then using the gene sequence as a template, a primer sequence is designed to perform PCR amplification to obtain a PCR product;

[0044] The reaction system of the PCR amplification is: 10 μL of 5×PS buffer, 4 μL of dNTP with a concentration of 2.5 mmol / L, 1 μL of upstream primer with a concentration of 10 μmol / L, 1 μL of downstream primer with a concentration of 10 μmol / L, 1.0 μL of DNA polymerase with a concentration of 2.5 U / μL, 1 μL of genomic DNA to be tested, and double distilled water to 50 μL; the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3;

[0045] The PCR amplification reaction procedure is: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 80 s, a total of 32 cycles; 72°C extension for 10 min, 4°C storage;

[0046] S302, subjecting the PCR product in S301 to 1.5% agarose gel electrophoresis and then cutting the gel to recover the band at about 500 bp to obtain the purified target gene, and double-digesting the purified target gene and the expression vector pET-28a with NdeⅠ / XhoⅠ restriction endonucleases to obtain the target gene double-digestion product and the expression vector pET-28a double-digestion product, respectively;

[0047] The enzyme digestion reaction system is: 6 μL of purified target gene or expression vector pET-28a, 16 μL of 10×FastDigestBuffer, 1 μL of FastDigest NdeⅠ, 1 μL of FastDigest XhoⅠ, and 36 μL of ddH2O;

[0048] S303, respectively recovering the target gene double-enzyme digestion product and the expression vector pET-28a double-enzyme digestion product obtained in S302 using an AxyPrepTM DNA Gel Extraction Kit to obtain a double-enzyme digestion gel-recovered SPG gene and a double-enzyme digestion pET-28a gel-recovered product, respectively, and subjecting the double-enzyme digestion gel-recovered SPG gene and the double-enzyme digestion pET-28a gel-recovered product to a ligation reaction at 22° C. for 1.5 h to obtain a ligation product;

[0049] The ligation product was transformed into competent cells E. coli DH5α (cloning host bacteria) by heat shock method, and positive clones were screened by agar plate LB (1% Tryptone, 0.5% Yeast Extract and 1% NaCl, 2% agarose, dissolved in water, sterilized at 121°C for 20 min) pre-added with 1 mM kanamycin, and then cultured in LB liquid medium (1% Tryptone, 0.5% Yeast Extract and 1% NaCl, dissolved in water, sterilized at 121°C for 20 min), colony PCR was used to verify the positive clones, and the recombinant clone strain with correct sequencing results was named pET-28a-SPG-DH5a strain;

[0050] The ligation reaction system is: 5×Ligase Reaction Buffer 2 μL, SPG gene recovered from gel after double enzyme digestion 4 μL, pET-28a gel recovered product from double enzyme digestion 8 μL, T4 DNA ligase with a concentration of 5 U / μL 2 μL, double distilled water 4 μL;

[0051] S304, expression of recombinant SPG protein: The plasmid of the logarithmic growth phase of the pET28a-SPG-DH5a strain obtained in S303 was extracted using a plasmid extraction kit and transformed into the expression host bacteria E. coli by heat shock. BL21 (DE3) competent cells, the positive monoclonal colonies screened by solid LB plate were inoculated into LB liquid medium pre-added with 1mM kanamycin, cultured overnight at 37°C and 180rpm / min, and then transferred to a new volume of 500mL LB liquid medium pre-added with 1mM kanamycin, and continued to be cultured at 37°C and 180rpm / min until the absorbance of the bacterial solution at a wavelength of 600nm reached 0.4-0.6, and isopropyl-β-D-thiogalactoside (IPTG) solution was added to make the final concentration of the expression host bacteria 1.0mmol / L. After induction for 8h, 1mL was taken and centrifuged at 10000rpm / min to collect the bacterial sludge, which was diluted with 10mmol / L phosphate buffer (PBS) and added with 5× Loading Buffer, heated in a 100℃ water bath for 10 min, and detected by SDS-PAGE to obtain the expression host bacteria containing the target protein; the expression amount of the recombinant SPG protein in the genetically modified Escherichia coli BL21 (DE3) host bacteria increased by about 24.3%;

[0052] S305. Purification of recombinant SPG protein: After the expression host bacteria containing the target protein obtained in S304 are subjected to three stages of activation, transfer and bacterial enrichment culture, the expression host bacteria containing the target protein is resuspended in a phosphate buffer with a concentration of 10 mmol / L at a mass volume ratio of 1 g:20 mL, and the bacterial cell wall is ultrasonically broken under ice bath conditions at 0°C to 4°C, and then centrifuged at 8000 rpm / min for 30 min, the supernatant is collected, and purified by Ni metal chelate column chromatography to obtain recombinant SPG protein; the ultrasonic conditions are: first ultrasonically break the bacteria for 2 seconds, then rest for 3 seconds, and repeat the ultrasonic breaking and intermittent operation for a total of 40 minutes, with a power of 55 W; the nucleotide sequence of the recombinant SPG protein is shown in SEQ ID NO:4.

[0053] The method of Ni metal chelate column chromatography is:

[0054] (1) Ni packing balance: Use Buffer A (500mmol / L NaCl, 20mmol / L Tris0, finally adjust to pH 8.0 with HCl) and 4% (v / v) Buffer B (500mmol / L NaCl, 20mmol / L Tris, 500mmol / L midazole), finally adjust to pH 8.0 with HCl; after mixing, wash the Ni column (His-tag bind resin Ni+ ), flow rate 3 mL / min, flush more than 20 column volumes until the baseline is flat, and the baseline is zeroed;

[0055] (2) Loading: The filtered supernatant was loaded at a flow rate of 2 mL / min. After loading, the Ni+ column was rinsed with the equilibration buffer described in step (1), and the flow-through was collected and named flow-through 1, flow-through 2, ..., flow-through N.

[0056] (3) Washing: Wash the Ni column with Buffer A + 10% (v / v) Buffer B at a flow rate of 2 mL / min. Collect the washing solutions and name them as Wash 1, Wash 2, ..., Wash N. Wash until the baseline of the entire UV absorption peak is flat.

[0057] (4) Elution: Wash the Ni column with Buffer B at a flow rate of 3 mL / min. When the OD280 rises to 0.02 Au, start collecting the elution peak. When the OD280 returns to near the baseline, stop collecting. The eluent components collected during this period are named elution 1, elution 2, ... elution N. Finally, the eluent peak is temporarily stored at 4 °C. If necessary, it can be stored in a -20 °C refrigerator.

[0058] (5) Ni filler regeneration: After flushing the AKATA preparative liquid chromatography system and the Ni column with ultrapure water and 20% ethanol at a flow rate of 1 mL / min for 20 column volumes, the Ni+ affinity column was removed and stored in a refrigerator at 4°C.

[0059] Take 100 μL of each of the solutions in different components (1: bacterial sludge before induction, 2: bacterial sludge after induction, 3: supernatant after lysis, 4: precipitate after lysis, 5: flow-through of purified recombinant SPG protein, 6: first wash of purified recombinant SPG protein, 7: final wash of purified recombinant SPG protein, 8: first elution of purified recombinant SPG protein and 9: second elution of purified SPG protein) and add them to 25 μL of 5× Loading buffer. Heat in a 100°C water bath for 10 min and then perform SDS-PAGE electrophoresis. The results are shown in the attached figure. Figure 1 As shown; 20 μL of each component was taken to determine the protein content using the BCA method, and the remaining samples were stored at -20°C for later use.

[0060] All the eluates were collected and then ultrafiltrated and centrifuged to concentrate the target protein. After SDS-PAGE electrophoresis, Coomassie Brilliant Blue staining was performed. The final electrophoresis results were scanned in grayscale, and the purity of the purified recombinant SPG was 91.2±1.72%. Protein BCA quantification determined that the concentration of the recombinant SPG protein was 3.18±0.34μg / μL; the above recombinant SPG protein concentration value was obtained after 3 biological replicates and BCA protein quantification.

[0061] The recombinant SPG protein involved in this embodiment is the recombinant Streptococcus Protein G (SPG for short).

[0062] The recombinant SPG protein with a concentration of 3.18±0.34 μg / μL prepared in this example was lyophilized, and the lyophilized SPG protein powder was dissolved in a PBS buffer with a pH of 8.0 and a final concentration of 0.2 moL / L Na2SO4 to obtain a recombinant SPG protein solution with a final concentration of 4 mg / mL to 16 mg / mL for the preparation of serum antibody affinity separation materials in Examples 3-6.

[0063] Example 3

[0064] The preparation method of the serum antibody affinity separation material of this embodiment is as follows:

[0065] S1. Washing of agarose matrix: Place agarose (Sepharose 6FF) in a sand core funnel and wash 20 times with ultrapure water, and obtain dried agarose by suction filtration;

[0066] Sepharose 6FF was purchased from Xi'an Lanxiao Biotechnology Co., Ltd.;

[0067] S2, epoxy activation reaction: add epichlorohydrin solution to the dried agarose obtained in S1, preheat for 30 min in a shaker at 25°C and 220 rpm / min, then continue to react for 4 h in a shaker at 25°C and 220 rpm / min, detect pH once every 1 h, and adjust pH to alkaline conditions with NaOH powder. After the reaction, wash with ultrapure water 10 times to obtain activated agarose gel; the mass volume ratio of the dried agarose and epichlorohydrin solution is 1 g:2.1 mL; the pH value of the activated agarose gel is 7.4;

[0068] Weigh 1g of the activated agarose gel as the experimental group, and weigh 1g of untreated Sepharose 6FF as the control group; add 20mL of 1.3mol / L sodium thiosulfate solution and 10μL of neutral red indicator respectively, place in a 37℃ incubator for 30min, shake once every 10min, and after the reaction is completed, titrate with 1mol / L HCl until a deep pink color appears, and record the amount of HCl used; calculate the epoxy group density;

[0069] The density of matrix-coupled epoxy groups is positively correlated with the amount of small molecule ligands bound thereto. With the goal of obtaining a high epoxy activation density, the present invention uses an orthogonal experiment to screen out the reaction conditions with the highest epoxy density. That is, after washing Sepharose 6FF with ddH2O, soak it in a 100% DMSO solution for 15 minutes, add a reaction solution with a volume ratio of 65% to 35% of DMSO:ECH to the agarose matrix according to a matrix-to-reaction solution mass volume ratio of 1:2 (g / mL), use NaOH powder as a catalyst to maintain the alkaline state of the reaction solution (pH>8.0), and finally obtain an activated matrix with an epoxy density of 111.42±7.72μmol / g; the above experimental results are the best experimental results determined by multiple experiments.

[0070] S3. Preparation of serum antibody affinity separation materials:

[0071] The activated agarose gel obtained in S2 is coupled with the recombinant SPG protein to obtain the serum antibody affinity separation material. The specific method is as follows:

[0072] Coupling of recombinant SPG protein: The recombinant SPG protein purified by Ni metal chelate chromatography column was replaced by Superdex G75 gel filtration column: the protein solution was collected with PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0, and after vacuum freeze drying, a frozen SPG protein powder was obtained, and then a PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0 was added to dissolve it to obtain a recombinant SPG protein solution with a concentration of 4 mg / mL, and the recombinant SPG protein solution was added to the activated agarose gel obtained in S2, coupled at a temperature of 16°C and a rotation speed of 70 rpm / min for 24 hours, and then a NaCl solution with a concentration of 0.1 mol / L was added, and then washed with double distilled water to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material;

[0073] The preparation method of the recombinant SPG protein in this example is the same as that in Example 2.

[0074] In this embodiment, the matrix activated by epichlorohydrin can directly use the amino group of the recombinant SPG protein to complete the epoxy ring-opening reaction.

[0075] The serum antibody affinity separation material prepared in this example is used to adsorb antibody IgG (IgG is immunoglobulin G), and is purified in one step by the SPG affinity matrix prepared in this example. After three repeated experiments, the adsorption amount, purity and protein recovery rate of serum antibody IgG obtained are 14.78±1.34 mg / mL, 83.9±1.75%, and 88.35±5.23%, respectively.

[0076] Example 4

[0077] The preparation method of the serum antibody affinity separation material of this embodiment is as follows:

[0078] S1. Washing of agarose matrix: Place agarose (Sepharose 6FF) in a sand core funnel and wash 10 times with ultrapure water, and obtain dried agarose by suction filtration;

[0079] S2, epoxy activation reaction: add epichlorohydrin solution to the dried agarose obtained in S1, preheat for 30 min in a shaker at 60°C and 170 rpm / min, then continue to react for 4 h in a shaker at 60°C and 170 rpm / min, detect pH once every 1 h, and adjust pH to alkaline conditions with 1 mol / L NaOH solution. After the reaction, wash with ultrapure water 10 times to obtain activated agarose gel; the mass volume ratio of the dried agarose and epichlorohydrin solution is 1 g:2.1 mL; the pH value of the activated agarose gel is 7.4;

[0080] S3. Preparation of serum antibody affinity separation materials:

[0081] The activated agarose gel obtained in S2 is coupled with the recombinant SPG protein to obtain the serum antibody affinity separation material. The specific method is as follows:

[0082] Coupling of recombinant SPG protein: The recombinant SPG protein purified by Ni metal chelate chromatography column was replaced by Superdex G75 gel filtration column: the protein solution was collected with PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0, and after vacuum freeze drying, a frozen SPG protein powder was obtained, and then a PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0 was added to dissolve it to obtain a recombinant SPG protein solution with a concentration of 16 mg / mL, and the recombinant SPG protein solution was added to the activated agarose gel obtained in S2, coupled at a temperature of 37°C and a rotation speed of 180 rpm / min for 24 hours, and then a NaCl solution with a concentration of 2.0 mol / L was added, and then washed with double distilled water to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material;

[0083] This embodiment also conducted an experiment with different coupling times of the recombinant SPG protein at a concentration of 10.2 mg / mL in step S3, that is, 5 mL of the prepared serum antibody affinity separation material was taken at 2h, 8h, 24h, 48h, and 72h of the coupling of the recombinant SPG protein to evaluate the separation effect of the affinity matrix on serum samples at different time points.

[0084] Take serum antibody affinity separation materials prepared with recombinant SPG protein at different time periods to purify bovine serum antibodies. In brief, take 1 mL of affinity matrix coupled with SPG protein at 2h, 8h, 24h, 48h, and 72h, respectively, and immediately load it into a 10 mL chromatography column to determine the sedimentation volume required for the experiment, and use 10 column volumes of ultrapure water and PBS to balance the affinity matrix with 10 column volumes of ddH2O and PBS respectively; take 5 mL of 10 mg / mL calf serum and balance it with 1 mL of PBS, then repeatedly blow the gel with a Pasteur pipette, incubate for 15 minutes, and collect all the flow through. The medium was washed with 10 column volumes of PBS 10 times, and the first (first tube) washing solution and the last (last tube) washing solution of the purified recombinant SPG protein were collected; the medium was repeatedly washed twice with 1 mL of 0.05 mol / L, pH 2.5 Gly-HCl solution, and all eluted samples were collected to obtain the eluate of purified bovine serum antibody IgG; the medium was washed with 10 column volumes of ultrapure water 10 times, dried, and stored in 20% ethanol by volume at room temperature.

[0085] The recombinant SPG protein was quantitatively detected by BCA method and SDS-PAGE electrophoresis was performed to analyze the effect of recombinant SPG protein affinity medium on antibody IgG specific adsorption at different times. The results of SDS-PAGE electrophoresis are shown in Figure 2 As shown (1: calf serum diluted 30 times, 2: medium flow-through after coupling for 24 hours, 3: first wash solution after coupling for 24 hours, 4: last wash solution after coupling for 24 hours, 5: elution solution after coupling for 2 hours, 6: elution solution after coupling for 8 hours, 7: elution solution after coupling for 24 hours, 8: elution solution after coupling for 48 hours, and 9: elution solution after coupling for 72 hours.

[0086] It can be clearly seen from the SDS-PAGE electrophoresis that the 24h-coupled affinity medium has the best separation effect on calf serum samples and the antibody adsorption amount (BCA protein quantification) is also the highest.

[0087] The serum antibody affinity separation material prepared in this example is used to adsorb antibody IgG. The recombinant SPG protein is coupled to the epoxy-activated matrix for 24 hours in this example, and the prepared SPG affinity matrix is ​​purified in one step. After three repeated experiments, the adsorption amount, purity and protein recovery rate obtained are: 15.47±1.63 mg / mL, 88.92±2.17%, and 87.95±4.68% of serum antibody IgG, respectively.

[0088] Example 5

[0089] The preparation method of the serum antibody affinity separation material of this embodiment is as follows:

[0090] S1. Washing of agarose matrix: Place agarose (Sepharose 6FF) in a sand core funnel and wash 10 times with ultrapure water, and obtain dried agarose by suction filtration;

[0091] S2, epoxy activation reaction: add epichlorohydrin solution to the dried agarose obtained in S1, preheat for 30 min in a shaker at 40°C and 200 rpm / min, then continue to react for 4 h in a shaker at 40°C and 200 rpm / min, detect pH once every 1 h, and adjust pH to 12.0 with 8 mol / L NaOH solution. After the reaction, wash with ultrapure water 10 times to obtain activated agarose gel; the mass volume ratio of the dried agarose and epichlorohydrin solution is 1 g:2.1 mL; the pH value of the activated agarose gel is 7.4;

[0092] S3. Preparation of serum antibody affinity separation materials:

[0093] The activated agarose gel obtained in S2 is subjected to amino reaction and indirect arm connection to obtain a serum antibody affinity separation material. The specific method is as follows:

[0094] Amination reaction: adding an amination reagent to the activated agarose gel obtained in S2, adjusting the pH to 12.0, reacting for 12 hours at a temperature of 50° C. and a rotation speed of 200 rpm / min, and then washing with ultrapure water 10 times to obtain an amination-treated agarose; the amination reagent is 1,6-hexanediamine or hydrazine hydrate;

[0095] Indirect arm connection: suspend the amino-treated agarose in ultrapure water at 4°C, add a cyanuric chloride acetone solution precooled at -20°C for 3h, and then react in an ice bath at a speed of 170rpm / min for 5.5h. During the reaction, the pH is detected every 0.5h, and the pH is adjusted to 8.0 with a saturated Na2CO3 solution. After the reaction, the agarose is washed with ultrapure water 10 times, and then filtered to obtain an indirect arm-connected agarose; the ratio of the amino-treated agarose to the cyanuric chloride acetone solution is 1g:2.1mL; the ratio of cyanuric chloride to the acetone solution in the cyanuric chloride acetone solution is 0.03g:1mL;

[0096] The agarose after the indirect arm connection is coupled with the purified recombinant SPG protein solution with a concentration of 10.2 mg / mL at a temperature of 25°C and a rotation speed of 200 rpm / min for 24 hours to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material; the usage ratio of the agarose after the indirect arm connection to the recombinant SPG protein solution is 1 g:2.0 mL.

[0097] In this embodiment, calf serum antibodies were purified by coupling 24 the epoxy-activated Sepharose 6FF matrix with 1,6-hexamethylenediamine-cyanuric chloride and recombinant SPG protein to obtain an affinity matrix; and by coupling 24 the epoxy-activated Sepharose 6FF matrix with hydrazine hydrate-cyanuric chloride and recombinant SPG protein to obtain an affinity matrix. That is, 1 mL of the recombinant SPG protein affinity matrix coupled for 24 hours was taken respectively, and immediately loaded into a 10 mL chromatography column after taking out to determine the sedimentation volume required for the experiment, and the affinity matrix was equilibrated with 10 column volumes of ddH2O and PBS respectively with 10 column volumes of ultrapure water and PBS; 5 mL of 10 mg / mL calf serum was taken and equilibrated with 1 mL of PBS, and the gel was repeatedly blown with a Pasteur pipette, incubated for 15 minutes, and all the flow-through liquid was collected; the medium was washed 10 times with 10 column volumes of PBS, and the first (first tube) washing liquid and the last (last tube) washing liquid of the purified recombinant SPG protein were collected; the medium was repeatedly washed twice with 1 mL of Gly-HCl solution with a concentration of 0.05 mol / L and a pH of 2.5, and all eluted samples were collected to obtain the eluate of the purified bovine serum antibody IgG; the medium was washed 10 times with 10 column volumes of ultrapure water, dried, and stored in 20% ethanol by volume at room temperature.

[0098] The recombinant SPG protein was quantitatively detected by BCA method and SDS-PAGE electrophoresis was performed to analyze the effect of recombinant SPG protein affinity medium on antibody IgG specific adsorption at different times. The results of SDS-PAGE electrophoresis are shown in Figure 3 As shown:

[0099] 1: Calf serum diluted 35 times;

[0100] 2: The method described in this example couples the affinity matrix of the recombinant SPG protein, and the affinity matrix obtained by coupling the recombinant SPG protein with hydrazine hydrate-cyanuric chloride for 24 hours is used to purify the bovine serum antibody;

[0101] 3: The final washing solution when purifying bovine serum antibody with affinity matrix obtained by coupling hydrazine hydrate-cyanuric chloride with recombinant SPG protein for 24 hours; M: protein marker;

[0102] 4 and 5 are respectively: eluent 1 and eluent 2 obtained by eluting the conjugate with Gly-HCl at pH 2.5 when purifying bovine serum antibody by affinity matrix coupled with recombinant SPG protein for 24 hours with hydrazine hydrate-cyanuric chloride;

[0103] 6: The method described in this example couples the affinity matrix of the recombinant SPG protein, and the matrix is ​​coupled with hydrazine hydrate-cyanuric chloride for 24 hours to separate the flow-through obtained from calf serum;

[0104] 7: The method described in Example 5 is to couple the affinity matrix of the recombinant SPG protein, and the matrix is ​​coupled with 1,6-hexanediamine-cyanuric chloride for 24 hours to separate the impurities obtained from the calf serum;

[0105] 8 and 9 are respectively: Gly-HCl eluent 1 and eluent 2 at pH 2.5 used for affinity separation of calf serum by coupling recombinant SPG protein with 1,6-hexanediamine-cyanuric chloride for 24 hours.

[0106] The preparation method of the recombinant SPG protein in this example is the same as that in Example 2.

[0107] In this embodiment, Sepharose 6FF is activated by epichlorohydrin under strong alkaline conditions; the activated medium is washed, then reacted with ammonia water, and the medium is washed with dimethyl sulfoxide and water, and then connected with cyanuric chloride under alkaline conditions, and finally the amino group of the purified recombinant SPG protein is directly substituted with the chlorine atom of cyanuric chloride to form a covalent bond. In this embodiment, the amino-containing compounds that react with the epoxy group include but are not limited to ammonia water, and such amino-containing compounds include hydrazine hydrate, 1,10-diaminodecane, 1,4-butanediamine, 1,6-hexanediamine and a series of diamino-containing compounds.

[0108] The serum antibody affinity separation material prepared in this embodiment is used to adsorb antibody IgG. After the recombinant SPG protein is activated with epoxy and coupled with hydrazine-cyanuric chloride matrix for 24 hours as described in this embodiment, the prepared SPG affinity matrix is ​​purified in one step, and the adsorption amount, purity and protein recovery rate are respectively: 15.28±2.12mg / mL, 87.78±4.32%, and 84.36±6.23% of serum antibody IgG. The above experimental data are obtained by analyzing the data of three separation and purification of calf serum by recombinant SPG protein affinity matrix; and after the recombinant SPG protein is activated with epoxy and coupled with 1,6-hexanediamine-cyanuric chloride matrix for 24 hours as described in this embodiment, the prepared SPG affinity matrix is ​​purified in one step, and after three repeated experiments, the adsorption amount, purity and protein recovery rate are respectively: 13.14±1.69mg / mL, 84.25±5.16%, and 83.27±5.47% of serum antibody IgG.

[0109] Example 6

[0110] The preparation method of the serum antibody affinity separation material of this embodiment is as follows:

[0111] S1. Washing of agarose matrix: Place agarose in a sand core funnel and wash with ultrapure water 20 times, and obtain dried agarose by suction filtration;

[0112] S2, epoxy activation reaction: add epichlorohydrin solution to the dried agarose obtained in S1, preheat for 30 min in a shaker at 60°C and 220 rpm / min, then continue to react for 4 h in a shaker at 60°C and 170 rpm / min, detect pH once every 1 h, and adjust pH to 13.0 with 7 mol / L NaOH solution. After the reaction, wash with ultrapure water 10 times to obtain activated agarose gel; the mass volume ratio of the dried agarose and epichlorohydrin solution is 1 g:2.1 mL; the pH value of the activated agarose gel is about 7.4;

[0113] S3. Preparation of serum antibody affinity separation materials:

[0114] The activated agarose gel obtained in S2 is subjected to amino reaction and indirect arm connection to obtain a serum antibody affinity separation material. The specific method is as follows:

[0115] Amination reaction: adding an amination reagent to the activated agarose gel obtained in S2, adjusting the pH to 13.0, reacting for 12 to 16 hours at a temperature of 60° C. and a rotation speed of 170 rpm / min, and then washing with ultrapure water 20 times to obtain an amination-treated agarose; the amination reagent is ammonia water;

[0116] Indirect arm connection: suspend the amino-treated agarose in ultrapure water at 4°C, add a cyanuric chloride acetone solution precooled at -20°C for 3h, and then react in an ice bath at a speed of 170rpm / min for 5.5h. During the reaction, the pH is detected every 0.5h, and the pH is adjusted to 8.0 with a saturated Na2CO3 solution. After the reaction, the agarose is washed with ultrapure water 10 times, and then filtered to obtain an indirect arm-connected agarose; the ratio of the amino-treated agarose to the cyanuric chloride acetone solution is 1g:2.1mL; the ratio of cyanuric chloride to the acetone solution in the cyanuric chloride acetone solution is 0.03g:1mL;

[0117] The agarose after the indirect arm connection is coupled with the purified recombinant SPG protein solution with a concentration of 10 mg / mL at a temperature of 25°C and a rotation speed of 200 rpm / min for 24 hours to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material; the usage ratio of the agarose after the indirect arm connection to the recombinant SPG protein solution is 1g:2.0mL.

[0118] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a serum antibody affinity separation material, characterized in that: The method is: S1. Washing of agarose matrix: Place agarose in a sand core funnel and wash with ultrapure water 10 to 20 times, and obtain dried agarose by filtration; S2, epoxy activation reaction: add epichlorohydrin solution to the dried agarose obtained in S1, preheat for 30 min in a shaker at 25°C to 60°C and 170 rpm / min to 220 rpm / min, then continue to react for 4 h in a shaker at 25°C to 60°C and 170 rpm / min to 220 rpm / min, detect pH once every 1 h, and adjust pH to alkaline condition with NaOH dry powder or NaOH solution with a concentration of 1 mol / L to 8 mol / L. After the reaction, wash 10 times with ultrapure water to obtain activated agarose gel; S3. Preparation of serum antibody affinity separation materials: The activated agarose gel obtained in S2 is coupled with the recombinant SPG protein to obtain the serum antibody affinity separation material. The specific method is as follows: Coupling of recombinant SPG protein: the recombinant SPG protein purified by Ni metal chelate chromatography column was replaced by Superdex G75 gel filtration column: the protein solution was collected by PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0, and the frozen SPG protein powder was obtained after vacuum freeze drying, and then dissolved by adding PBS buffer containing a final concentration of 0.2 mol / L Na2SO4 and a pH of 8.0 to obtain a recombinant SPG protein solution with a concentration of 4 to 16 mg / mL, and the recombinant SPG protein solution was added to the activated agarose gel obtained in S2, and coupled for 24 hours at a temperature of 16°C to 37°C and a rotation speed of 70 rpm / min to 180 rpm / min, and then a NaCl solution with a concentration of 0.1 mol / L to 2.0 mol / L was added, and then washed with double distilled water to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material; Alternatively, the activated agarose gel obtained in S2 is subjected to an amination reaction, coupled with cyanuric chloride, and then subjected to a substitution reaction with the amino residues on the recombinant SPG protein to finally obtain a serum antibody affinity separation material. The specific method is as follows: Amination reaction: adding an amination reagent to the activated agarose gel obtained in S2, adjusting the pH to 12.0-13.0, reacting for 12-16 hours at a temperature of 50° C.-60° C. and a rotation speed of 170 rpm / min-200 rpm / min, and then washing with ultrapure water for 10-20 times to obtain ammoniated agarose; Indirect arm connection: suspend the amino-modified agarose in ultrapure water at 0°C to 4°C, add a cyanuric chloride acetone solution precooled at -20°C for 3h, react in an ice bath at a speed of 170rpm / min for 5.5h, detect the pH every 0.5h during the reaction, and adjust the pH to 7.0-8.0 with a saturated Na2CO3 solution. After the reaction, wash with ultrapure water 10 times, and then filter to obtain the agarose after indirect arm connection; the ratio of cyanuric chloride to acetone solution in the cyanuric chloride acetone solution is 0.03g:1mL; The agarose to which the indirect arm is connected is coupled with a purified recombinant SPG protein solution with a concentration of 4 mg / mL to 16 mg / mL at a temperature of 25° C. and a rotation speed of 200 rpm / min for 24 hours to obtain a recombinant SPG affinity medium, which is a serum antibody affinity separation material.

2. The method for preparing a serum antibody affinity separation material according to claim 1, characterized in that: The mass volume ratio of the dried agarose and epichlorohydrin solution described in S2 is 1 g:2.1 mL.

3. The method for preparing a serum antibody affinity separation material according to claim 1, characterized in that: When the activated agarose gel obtained in S2 is subjected to an amination reaction and indirect arm connection to obtain a serum antibody affinity separation material, the amination reagent in S3 includes ammonia water, hydrazine hydrate or 1,6-hexanediamine.

4. The method for preparing a serum antibody affinity separation material according to claim 1, characterized in that: When the activated agarose gel obtained in S2 is subjected to an amination reaction and indirect arm connection to obtain a serum antibody affinity separation material, the amount ratio of the amination agarose and the cyanuric chloride acetone solution in S3 is 1 g:2.1 mL.

5. The method for preparing a serum antibody affinity separation material according to claim 1, characterized in that: When the activated agarose gel obtained in S2 is subjected to amino reaction and indirect arm connection to obtain the serum antibody affinity separation material, the usage ratio of the agarose after indirect arm connection to the recombinant SPG protein solution in S3 is 1 g:0.7 mL.

6. The method for preparing a serum antibody affinity separation material according to claim 1, characterized in that: The preparation method of the recombinant SPG protein in the recombinant SPG protein solution described in S3 is: S301, preparing a gene encoding a recombinant SPG protein by chemical synthesis, performing codon optimization, obtaining a nucleotide sequence of the recombinant SPG protein, and then using the gene sequence as a template to design primer sequences for PCR amplification to obtain a PCR product; the nucleotide sequence of the gene encoding the recombinant SPG protein is shown in SEQ ID NO: 4; The reaction system of the PCR amplification is: 10 μL of 5×PS buffer, 4 μL of dNTP with a concentration of 2.5 mmol / L, 1 μL of upstream primer with a concentration of 10 μmol / L, 1 μL of downstream primer with a concentration of 10 μmol / L, 1.0 μL of DNA polymerase with a concentration of 2.5 U / μL, 1 μL of genomic DNA to be tested, and double distilled water to 50 μL; the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3; The PCR amplification reaction procedure is: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 80 s, a total of 32 cycles; 72°C extension for 10 min, 4°C storage; S302, after electrophoresis of the PCR product in S301, the gel was cut to recover the band at about 500 bp to obtain the purified target gene, and the purified target gene and the expression vector pET-28a were double-digested with NdeⅠ / XhoⅠ restriction endonucleases to obtain the target gene double-digestion product and the expression vector pET-28a double-digestion product respectively; The enzyme digestion reaction system is: 6µL of purified target gene or expression vector pET-28a, 16µL of 10×FastDigestBuffer, 1µL of FastDigest NdeⅠ, 1µL of FastDigest XhoⅠ, and 36µL of ddH2O; S303, respectively recovering the target gene double-enzyme digestion product and the expression vector pET-28a double-enzyme digestion product obtained in S302 using an AxyPrepTM DNA Gel Extraction Kit to obtain a double-enzyme digestion gel-recovered SPG gene and a double-enzyme digestion pET-28a gel-recovered product, respectively, and subjecting the double-enzyme digestion gel-recovered SPG gene and the double-enzyme digestion pET-28a gel-recovered product to a ligation reaction at 22° C. for 1.5 h to obtain a ligation product; The ligation product was transformed into competent cells E. coli DH5α by heat shock method, and positive clones were screened by LB agar plate pre-added with 1 mM kanamycin, and then cultured in LB liquid medium, and positive clones were verified by colony PCR. The recombinant clone strain with correct sequencing results was named pET-28a-SPG-DH5a strain; The system of the ligation reaction is: 2 μL of 5×Ligase Reaction Buffer, 4 μL of SPG gene recovered from gel after double enzyme digestion, 8 μL of pET-28a gel recovery product from double enzyme digestion, 2 μL of T4 DNA ligase with a concentration of 5 U / μL, and 4 μL of double distilled water; S304, expression of recombinant SPG protein: The plasmid of the logarithmic growth phase of the pET28a-SPG-DH5a strain obtained in S303 was extracted using a plasmid extraction kit and transformed into the expression host bacteria E. coli by heat shock. BL21 (DE3) competent cells, inoculate the positive monoclonal colonies screened on the solid LB plate into LB liquid medium pre-added with 1mM kanamycin, culture overnight at 37°C and 180rpm / min, then transfer to a new 500mL LB liquid medium pre-added with 1mM kanamycin, continue to culture at 37°C and 180rpm / min until the absorbance of the bacterial solution at 600nm reaches 0.4-0.6, add isopropyl-β-D-thiogalactoside solution to make the final concentration of the expression host bacteria 1.0mmol / L, take 1mL after induction for 8h, centrifuge at 10000rpm / min, collect the bacterial sludge, dilute with 10mmol / L phosphate buffer, add 5× Loading Buffer, heat in a 100°C water bath for 10min, and detect by SDS-PAGE to obtain the expression host bacteria containing the target protein; S305. Purification of recombinant SPG protein: After the expression host bacteria containing the target protein obtained in S304 are subjected to three stages of activation, transfer and bacterial enrichment culture, the expression host bacteria containing the target protein is resuspended in a phosphate buffer with a concentration of 10 mmol / L at a mass volume ratio of 1 g:20 mL, and the bacterial cell wall is ultrasonically broken in an ice bath at 0°C to 4°C, and then centrifuged at 8000 rpm / min for 30 min, the supernatant is collected, and purified by Ni metal chelate column chromatography to obtain recombinant SPG protein; the ultrasonic conditions are: first ultrasonically break the bacteria for 2 seconds, then rest for 3 seconds, and repeat the ultrasonic breaking and intermittent operation for a total of 40 minutes, with a power of 55 W; the nucleotide sequence of the recombinant SPG protein is shown in SEQ ID NO:

4.

7. Use of a serum antibody affinity separation material prepared by any one of claims 1 to 6, characterized in that: The serum antibody affinity separation material is used to adsorb antibody IgG.

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