CN116037072B - Preparation method and application of serum antibody affinity separation material - Google Patents
Preparation method and application of serum antibody affinity separation material Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
本发明提供了一种血清抗体亲和分离材料的制备方法，以琼脂糖为基质的琼脂糖基质的洗涤、环氧活化反应和重组链球菌G蛋白(Streptococcus ProteinG简称SPG)的偶联制备血清抗体亲和分离材料；或者琼脂糖基质的洗涤、环氧活化反应、活化后琼脂糖的氨基化反应和连接后制备血清抗体亲和分离材料的方法。还提供了一种本发明制备的血清抗体亲和分离材料的应用，用于吸附免疫球蛋白G(ImmunoglobulinG,IgG)。本发明制备的血清抗体亲和分离材料无需使用经典但有剧毒的溴化氰作偶联试剂，生物安全性良好。
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
The invention belongs to the technical field of protein separation and purification, and particularly relates to a preparation method and application of a serum antibody affinity separation material.
Background
The existing affinity separation and purification method based on bionic affinity/ligand-ligand has the limitation of slightly lower specificity, and the existing SPG affinity separation and purification method has the limitations of (1) large cyanogen bromide toxicity of an activating reagent and low biological safety, and (2) easy falling of ligand, and easy inflammation or hypersensitive immune reaction of a patient caused by the falling SPG affinity ligand when the clinical medicinal antibody is purified.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art and provides a preparation method and application of a serum antibody affinity separation material.
In order to solve the technical problems, the invention adopts the technical scheme that the method for preparing the serum antibody affinity separation material comprises the following steps:
s1, washing an agarose matrix, namely placing agarose into a sand core funnel, pumping and washing the agarose matrix with ultrapure water for 10-20 times, and performing pumping filtration to obtain pumped agarose;
S2, epoxy activation reaction, namely adding an epoxy chloropropane solution into the pumped agarose obtained in the step S1, preheating for 30min in a shaking table with the temperature of 25-60 ℃ and the rotating speed of 170-220 rpm/min, then continuing to react for 4h on the shaking table with the temperature of 25-60 ℃ and the rotating speed of 170-220 rpm/min, detecting the pH value for 1 time every 1h, regulating the pH value to an alkaline condition by using NaOH dry powder or NaOH solution with the concentration of 1-8 mol/L, and pumping and washing for 10 times by using ultrapure water after the reaction is finished to obtain activated agarose gel;
s3, preparation of serum antibody affinity separation materials:
coupling the activated agarose gel obtained in the step S2 with recombinant SPG protein to obtain a serum antibody affinity separation material, wherein the specific method comprises the following steps:
Coupling recombinant SPG protein, namely, subjecting the recombinant SPG protein obtained by purification of a Ni metal chelating chromatographic column to SuperdexG gel filtration column to replace a system, collecting protein solution by using PBS buffer with the pH value of Na 2SO4 with the final concentration of 0.2 moL/L=8.0, performing vacuum freeze drying to obtain frozen SPG protein powder, adding PBS buffer with the pH value of Na 2SO4 with the final concentration of 0.2 moL/L=8.0 for dissolving to obtain recombinant SPG protein solution with the concentration of 4-16 mg/mL, adding the recombinant SPG protein solution into activated agarose gel obtained in S2, performing coupling for 24 hours under the conditions of the temperature of 16-37 ℃ and the rotating speed of 70-180 rpm/min, then adding NaCl solution with the concentration of 0.1-2.0 moL/L, and performing double-evaporation water washing to obtain a recombinant SPG affinity medium, namely a serum antibody affinity separation material;
Or coupling the activated agarose gel obtained in the step S2 with cyanuric chloride after amination reaction, and then carrying out substitution reaction with amino residues on recombinant SPG protein to finally obtain the serum antibody affinity separation material, wherein the specific method comprises the following steps of:
An amination reaction, namely adding an amination reagent into the activated agarose gel obtained in the step S2, adjusting the pH value to 12.0-13.0, reacting for 12-16 hours under the conditions of 50-60 ℃ and 170-200 rpm/min, and then pumping and washing with ultrapure water for 10-20 times to obtain the aminated agarose;
Suspending the aminated agarose in ultrapure water with the temperature of 0-4 ℃, adding an acetone solution of cyanuric chloride pre-cooled for 3 hours at the temperature of-20 ℃, carrying out ice bath reaction for 5.5 hours under the condition of the rotating speed of 170rpm/min, detecting the pH value every 0.5 hour in the reaction, regulating the pH value to 7.0-8.0 by using a saturated Na 2CO3 solution, carrying out suction washing for 10 times by using ultrapure water after the reaction is finished, and carrying out suction filtration to obtain the agarose after the connection of the indirect arm, wherein the dosage ratio of cyanuric chloride to the acetone solution of cyanuric chloride in the acetone solution of cyanuric chloride is 0.03 g/1 mL;
Coupling agarose connected with the indirect arm with purified recombinant SPG protein solution with concentration of 4 mg/mL-16 mg/mL for 24h at the temperature of 25 ℃ and the rotating speed of 200rpm/min to obtain recombinant SPG affinity medium, namely the serum antibody affinity separation material.
Preferably, the mass to volume ratio of the pumped agarose to epichlorohydrin solution in S2 is 1g:2.1mL.
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 comprises ammonia, hydrazine hydrate or 1, 6-hexamethylenediamine.
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 dosage ratio of the agarose after amination to the cyanuric chloride acetone solution in S3 is 1g to 2.1mL.
Preferably, when the activated agarose gel obtained in the step S2 is subjected to an amination reaction and indirect arm connection to obtain the serum antibody affinity separation material, the dosage ratio of the agarose with the indirect arm connection to the recombinant SPG protein solution in the step S3 is 1g:0.7mL.
Preferably, the preparation method of the recombinant SPG protein in the recombinant SPG protein solution in the S3 comprises the following steps:
S301, preparing a gene for encoding the recombinant SPG protein by using a chemical synthesis method (the nucleotide sequence is shown as SEQ ID NO: 1), then performing codon optimization to obtain a nucleotide sequence of the recombinant SPG protein (shown as SEQ ID NO: 4), and then performing PCR amplification by taking the gene sequence as a template and designing a primer sequence to obtain a PCR product;
The PCR amplification reaction system comprises 5X PSbuffer mu L, 2.5mmol/L dNTP4 mu L, 10 mu mol/L upstream primer 1 mu L, 10 mu mol/L downstream primer 1 mu L, 2.5U/mu L DNA polymerase 1.0 mu L, 1 mu L genome DNA to be detected, and double distilled water to 50 mu L, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
The PCR amplification reaction procedure comprises 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 30s,65 ℃ annealing for 30s and 72 ℃ extension for 80s, wherein the total time is 32 cycles, 72 ℃ continuous extension for 10min and 4 ℃ preservation;
s302, carrying out electrophoresis on the PCR product in S301, cutting gel, recovering a band at about 500bp to obtain a purified target gene, and carrying out double enzyme digestion on the purified target gene and an expression vector pET-28a by NdeI/XhoI restriction enzyme to obtain a target gene double enzyme digestion product and an expression vector pET-28a double enzyme digestion product respectively;
The enzyme digestion reaction system comprises 6 mu L of purified target gene or expression vector pET-28a, 10X FASTDIGEST BUFFER mu L, FASTDIGEST NDEI mu L, FASTDIGEST XHOI mu L and ddH 2 O36 mu L;
S303, respectively recovering the target gene double enzyme digestion product obtained in the S302 and the expression vector pET-28a double enzyme digestion product by using a AxyPrepTM DNA GelExtraction Kit glue recovery kit to respectively obtain a double enzyme digestion post-glue recovery SPG gene and a double enzyme digestion pET-28a glue recovery product, and carrying out a connection reaction on the double enzyme digestion post-glue recovery SPG gene and the double enzyme digestion pET-28a glue recovery product for 1.5 hours under the condition that the temperature is 22 ℃ to obtain a connection product;
Transforming the connection product into competent cells E.coli DH5 alpha by a heat shock method, screening positive clones by agar plate LB with kana antibiotics with the concentration of 1mM, culturing by LB liquid culture medium, verifying positive clones by colony PCR, and obtaining the recombinant clone strain with correct sequencing result as pET-28a-SPG-DH5a strain;
The system of the ligation reaction is 5X Ligase Reaction Buffer mu L, SPG gene is recovered by double enzyme digestion, product is recovered by double enzyme digestion pET-28a, 2 mu L of T4DNA ligase with concentration of 5U/mu L and double distilled water is recovered by double enzyme digestion;
S304, expressing recombinant SPG protein, namely extracting plasmid in logarithmic phase of pET28a-SPG-DH5a strain obtained in S303 by using a plasmid extraction kit, converting expression host bacteria E.coli BL21 (DE 3) competent cells by heat shock, inoculating positive monoclonal colony screened by a solid LB plate to LB liquid culture medium of kana antibiotics with the pre-concentration of 1mM, culturing overnight at 37 ℃ and the rotating speed of 180rpm/min, transferring to new LB liquid culture medium of kana antibiotics with the pre-concentration of 1mM with the volume of 500mL, culturing continuously at 37 ℃ and the rotating speed of 180rpm/min until the light absorption value of bacterial liquid reaches 0.4-0.6 at the wavelength of 600nm, adding isopropyl-beta-D-thiogalactoside solution, obtaining 1mL after induction for 8 hours, centrifuging at the rotating speed of 10000rpm/min, collecting bacterial mud, detecting that the bacterial mud contains SDS-containing target protein by 10 ℃ and diluting with the phosphate buffer solution of 10 ℃ and carrying out heat dilution on the bacterial mud to obtain the target protein by detecting the SDS-containing the target protein by using the liquid of Loading Buffer ℃ after the bacterial mud;
S305, purifying recombinant SPG protein, namely, after three stages of activation, transfer and enrichment culture of the expression host bacteria containing the target protein obtained in S304, re-suspending the expression host bacteria containing the target protein in phosphate buffer with the concentration of 10mmol/L according to the mass-volume ratio of 1g to 20mL, ultrasonically crushing bacterial cell walls under the ice bath condition of 0-4 ℃, centrifuging for 30min at the rotating speed of 8000rpm/min, collecting supernatant, purifying by Ni metal chelating column chromatography to obtain the recombinant SPG protein, wherein the ultrasonic condition is that firstly ultrasonic bacteria breaking is carried out for 2S, then intermittent for 3S, the ultrasonic bacteria breaking and intermittent operation are repeated for 40min, the power is 55W, and the nucleotide sequence of the recombinant SPG protein is shown as SEQ ID NO: 4.
The recombinant SPG protein is recombinant streptococcus G protein (Streptococcus Protein G, SPG for short).
The invention also provides application of the prepared serum antibody affinity separation material, and the serum antibody affinity separation material is used for adsorbing antibody IgG.
IgG is Immunoglobulin G (IgG).
The matrix after the epichlorohydrin activation 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 the strong alkaline condition, and the medium after the activation is washed and then reacts with ammonia water, and dimethyl sulfoxide and water are used for washing the medium. Then the cyanuric chloride is connected under alkaline condition. And finally, directly carrying out substitution reaction on the purified recombinant SPG protein amino and the chlorine atom of cyanuric chloride to form a covalent bond. The amination reagents described in this embodiment that react with epoxy groups include, but are not limited to, ammonia, and such amino-containing compounds include a range of bis-amino-containing compounds such as hydrazine hydrate, 1,6 hexamethylenediamine, and the like.
Compared with the prior art, the invention has the following advantages:
The invention can directly select agarose after the activation of epoxy chloropropane, and use amino groups of recombinant SPG protein to complete ring-opening reaction, and at the same time, the agarose is activated by epoxy chloropropane under the condition of strong alkalinity, and then reacts with an amination reagent, and is washed by dimethyl sulfoxide and water, then is connected with cyanuric chloride under the condition of alkalinity, and finally, the amino groups of the purified recombinant SPG protein and chlorine atoms of cyanuric chloride are directly substituted to form covalent bonds.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of recombinant SPG protein induced to express PET28a-SPG-BL21 (DE 3) in example 2 of the present invention.
FIG. 2 is a SDS-PAGE electrophoresis of the affinity medium of recombinant SPG protein for serum antibodies at various time points in example 4 of the present invention.
FIG. 3 is an SDS-PAGE electrophoresis of serum antibody affinity chromatography samples of the different spacer arm activation medium coupled recombinant SPG protein affinity medium of example 5 of the present invention.
Detailed Description
Example 1
Codon optimization of recombinant Streptococcal Protein G (SPG) encoding genes.
The codon optimization of protein recombinant expression is that codon preference used by encoding an amino acid of different species is different, and the fact that tRNA abundance corresponding to the amino acid in different species is different is essential, so the research optimizes the codon of streptococcus G protein. The rare codons of the E.coli tRNA in the SPG gene were deleted and codon optimization was accomplished by (1) replacing the original sequence codons with synonymous codons of similar frequency in the E.coli host and (2) randomly synonymously replacing the original gene codons according to the host codon frequency. The rate of protein expression in the body determines its activity. (3) GC content adjustment, and (4) avoidance of base recurrence, etc. The nucleotide sequence of the recombinant SPG protein obtained through codon optimization is shown as SEQ ID NO. 4, and the nucleotide sequence of the original codon is shown as SEQ ID NO. 1.
Example 2
The preparation method of the recombinant SPG protein comprises the following steps:
s301, preparing a gene for encoding the recombinant SPG protein by using a chemical synthesis method, wherein the nucleotide sequence of the gene for encoding the recombinant SPG protein is shown as SEQ ID NO. 1, then performing codon optimization to obtain the nucleotide sequence (shown as SEQ ID NO. 4) of the recombinant SPG protein, and then performing PCR amplification by taking the gene sequence as a template and designing a primer sequence to obtain a PCR product;
The PCR amplification reaction system comprises 5X PSbuffer mu L, 2.5mmol/L dNTP 4 mu L, 10 mu mol/L upstream primer 1 mu L, 10 mu mol/L downstream primer 1 mu L, 2.5U/mu L DNA polymerase 1.0 mu L, 1 mu L genome DNA to be detected, and double distilled water to 50 mu L, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
The PCR amplification reaction procedure comprises 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 30s,65 ℃ annealing for 30s and 72 ℃ extension for 80s, wherein the total time is 32 cycles, 72 ℃ continuous extension for 10min and 4 ℃ preservation;
S302, cutting a gel after electrophoresis of a PCR product of the S301 with 1.5% agarose gel, recovering a band at about 500bp to obtain a purified target gene, and carrying out double enzyme digestion on the purified target gene and an expression vector pET-28a by NdeI/XhoI restriction enzyme to obtain a target gene double enzyme digestion product and an expression vector pET-28a double enzyme digestion product respectively;
The enzyme digestion reaction system comprises 6 mu L of purified target gene or expression vector pET-28a, 10× FASTDIGEST BUFFER 16 mu L, FASTDIGEST NDEI mu L, FASTDIGEST XHOI mu L and ddH 2 O36 mu L;
s303, respectively recovering the target gene double enzyme digestion product obtained in the S302 and the expression vector pET-28a double enzyme digestion product by using a AxyPrepTM DNA Gel Extraction Kit glue recovery kit to respectively obtain a double enzyme digestion post-glue recovery SPG gene and a double enzyme digestion pET-28a glue recovery product, and carrying out a connection reaction on the double enzyme digestion post-glue recovery SPG gene and the double enzyme digestion pET-28a glue recovery product for 1.5 hours under the condition that the temperature is 22 ℃ to obtain a connection product;
Transforming the connection product into competent cells E.coli DH5 alpha (cloning host bacteria) by a heat shock method, screening positive clones by an agar plate LB (1% of Tryptone,0.5% of Yeast Extract and 1% of NaCl,2% of agarose, dissolved in water and sterilized at 121 ℃ for 20 min) pre-adding kanavid with 1mM concentration, then culturing by an LB liquid medium (1% of Tryptone,0.5% of Yeast Extract and 1% of NaCl, dissolved in water and sterilized at 121 ℃ for 20 min), verifying positive clones by colony PCR, and obtaining the recombinant clone strain named as pET-28a-SPG-DH5a strain with correct sequencing result;
The system of the ligation reaction is 5X Ligase Reaction Buffer mu L, SPG gene is recovered by double enzyme digestion, product is recovered by double enzyme digestion pET-28a, 2 mu L of T4DNA ligase with concentration of 5U/mu L and double distilled water is recovered by double enzyme digestion;
s304, expressing recombinant SPG protein, namely extracting plasmid in logarithmic phase of pET28a-SPG-DH5a strain obtained in S303 by using a plasmid extraction kit, converting expression host bacteria E.coli BL21 (DE 3) competent cells by heat shock, inoculating positive monoclonal colony screened by a solid LB plate to LB liquid culture medium of kana antibiotics with the pre-concentration of 1mM, culturing overnight at 37 ℃ and the rotation speed of 180rpm/min, transferring to new LB liquid culture medium of kana antibiotics with the pre-concentration of 1mM with the volume of 500mL, culturing continuously at the temperature of 37 ℃ and the rotation speed of 180rpm/min until the light absorption value of bacterial liquid reaches 0.4-0.6 at the wavelength of 600nm, adding isopropyl-beta-D-thiogalactoside (IPTG) solution, obtaining 1mL after induction for 8 hours, centrifuging at the rotation speed of 10000rpm/min, collecting the bacterial liquid with the pre-concentration of 1mM, detecting that the bacterial liquid contains the target protein by 10mM of PBS, and diluting the host bacteria with the concentration of 10% of PBS (25 ℃ after the bacterial liquid is subjected to heat shock absorption of 10 ℃ to 20 ℃ after the bacterial liquid is subjected to heat shock absorption of water bath to the test to obtain the recombinant protein;
S305, purifying recombinant SPG protein, namely, after three stages of activation, transfer and enrichment culture of the expression host bacteria containing the target protein obtained in S304, re-suspending the expression host bacteria containing the target protein in phosphate buffer with the concentration of 10mmol/L according to the mass-volume ratio of 1g to 20mL, ultrasonically crushing bacterial cell walls under the ice bath condition of 0-4 ℃, centrifuging for 30min at the rotating speed of 8000rpm/min, collecting supernatant, purifying by Ni metal chelating column chromatography to obtain the recombinant SPG protein, wherein the ultrasonic condition is that firstly ultrasonic bacteria breaking is carried out for 2S, then intermittent for 3S, the ultrasonic bacteria breaking and intermittent operation are repeated for 40min, the power is 55W, and the nucleotide sequence of the recombinant SPG protein is shown as SEQ ID NO: 4.
The method for Ni metal chelating column chromatography comprises the following steps:
(1) Ni packing balance, namely, using BufferA (500 mmol/L NaCl,20mmol/L Tris0, finally using HCl to adjust the pH to 8.0 and 4% (v/v) Buffer B (500 mmol/L NaCl,20mmol/L Tris,500 mmol/LImidazole), finally using HCl to adjust the pH to 8.0, flushing Ni column (His-tagbindresinNi +) after mixing, flushing more than 20 column volumes until the base line is flushed, and zeroing the base line;
(2) Loading, namely loading the filtered supernatant at a flow rate of 2mL/min, flushing the Ni+ column by using the balance buffer solution in the step (1) after loading, and collecting flow-through liquids, which are named as flow-through 1 and flow-through 2, respectively;
(3) Washing impurities, namely washing a Ni column with BufferA +10% (v/v) BufferB at a flow rate of 2mL/min, collecting impurity washing solutions which are respectively named as impurity washing 1 and impurity washing 2, and carrying out impurity washing N until the impurity washing solutions are washed until the base line of the whole ultraviolet absorption peak is leveled;
(4) Eluting by flushing Ni column with BufferB at flow rate of 3mL/min, collecting eluting peak when OD280 is increased to 0.02Au, stopping collecting when OD280 returns to the vicinity of baseline, respectively named eluting 1, eluting 2, eluting N, temporarily storing the eluting peak at 4deg.C, and storing in-20deg.C refrigerator if necessary;
(5) And regenerating Ni filler, namely flushing AKATA the prepared liquid chromatograph system and the Ni column by using ultrapure water with the flow rate of 1mL/min and 20 percent ethanol respectively, taking down the Ni+ affinity column, and storing in a refrigerator at the temperature of 4 ℃.
Taking solutions (1: pre-induction bacterial sludge, 2: post-induction bacterial sludge, 3: post-bacterial supernatant, 4: post-bacterial precipitation, 5: purified recombinant SPG protein flow-through, 6: purified recombinant SPG protein first-time impurity washing liquid, 7: purified recombinant SPG protein last-time impurity washing liquid, 8: purified recombinant SPG protein first-time eluent and 9: purified SPG protein second-time eluent) in different components, adding 100 μl each into 25 μl of 5× Loadingbuffer, heating in 100 ℃ water bath for 10min, performing SDS-PAGE electrophoresis detection, and measuring protein content by BCA method for 20 μl of each component, and storing the rest sample at-20deg.C for later use.
Collecting all eluates, ultrafiltering, centrifuging, concentrating, collecting target protein, performing SDS-PAGE electrophoresis, dyeing with coomassie brilliant blue, gray scanning to obtain purified recombinant SPG with purity of 91.2+ -1.72%, quantifying protein BCA, determining the concentration of recombinant SPG protein of 3.18+ -0.34 μg/μl, performing biological repeated experiments for 3 times, and quantifying BCA protein.
The recombinant SPG protein referred to in this example is recombinant Streptococcus G protein (Streptococcus ProteinG, SPG for short).
The recombinant SPG protein prepared in this example was lyophilized to a concentration of 3.18.+ -. 0.34. Mu.g/. Mu.L, and the lyophilized SPG protein powder was dissolved in PBS buffer having a pH=8.0 of Na 2SO4 at a final concentration of 0.2moL/L to give a recombinant SPG protein solution having a final concentration of 4mg/mL to 16mg/mL for use in the preparation of serum antibody affinity separation materials of examples 3 to 6.
Example 3
The preparation method of the serum antibody affinity separation material of the embodiment comprises the following steps:
S1, washing an agarose matrix, namely placing agarose (Sepharose 6 FF) in a sand core funnel, pumping and washing the agarose matrix with ultrapure water for 20 times, and filtering the agarose matrix to obtain pumped agarose;
sepharose 6FF is commercially available from the biotechnology company of west amps Lan Xiao;
S2, epoxy activation reaction, namely adding epoxy chloropropane solution into the pumped agarose obtained in the step S1, preheating for 30min in a shaking table with the temperature of 25 ℃ and the rotating speed of 220rpm/min, then continuously reacting for 4h on the shaking table with the temperature of 25 ℃ and the rotating speed of 220rpm/min, detecting the pH value for 1 time every 1h, regulating the pH value to an alkaline condition by NaOH dry powder, and pumping and washing for 10 times by ultrapure water after the reaction is finished to obtain activated agarose gel, wherein the mass volume ratio of the pumped agarose to the epoxy chloropropane solution is 1g to 2.1mL, and the pH value of the activated agarose gel is 7.4;
Weighing 1g of the obtained activated agarose gel as an experimental group, weighing 1g of untreated Sepharose 6FF as a control group, respectively adding 20mL of 1.3mol/L sodium thiosulfate solution and 10 mu L of neutral red indicator, placing in a 37 ℃ incubator for 30min, shaking uniformly every 10min, titrating with 1mol/L HCl until deep pink appears after the reaction is finished, and recording the HCl consumption;
The density of matrix-coupled epoxy groups is positively correlated to the amount of small molecule ligands bound thereto. The invention adopts orthogonal experiments to screen out the reaction condition with highest epoxy group density by taking the high epoxy group activation density as the target. Namely, after washing Sepharose 6FF with ddH 2 O, soaking in 100% DMSO solution for 15min, adding reaction solution with the volume ratio of DMSO to ECH of 65% to 35% into agarose matrix according to the mass volume ratio of matrix to reaction solution of 1:2 (g/mL), and maintaining the alkaline (pH > 8.0) state of the reaction solution by taking NaOH powder as a catalyst to finally obtain an activated matrix with the epoxy group density of 111.42 +/-7.72 mu mol/g, wherein the experimental result is the best experimental result determined by multiple experiments.
S3, preparation of serum antibody affinity separation materials:
coupling the activated agarose gel obtained in the step S2 with recombinant SPG protein to obtain a serum antibody affinity separation material, wherein the specific method comprises the following steps:
Coupling recombinant SPG protein, namely, purifying the obtained recombinant SPG protein by a Ni metal chelating chromatographic column, replacing a system by a SuperdexG gel filtration column, collecting protein solution by using a PBS buffer solution with the pH value of Na 2SO4 with the final concentration of 0.2 moL/L=8.0, performing vacuum freeze drying to obtain frozen SPG protein powder, adding a PBS buffer solution with the pH value of Na 2SO4 with the final concentration of 0.2 moL/L=8.0 for dissolving to obtain a recombinant SPG protein solution with the concentration of 4mg/mL, adding the recombinant SPG protein solution into activated agarose gel obtained in S2, coupling for 24 hours at the temperature of 16 ℃ and the rotating speed of 70rpm/min, adding NaCl solution with the concentration of 0.1moL/L, and performing double-evaporation water washing to obtain a recombinant SPG affinity medium, namely the serum antibody affinity separation material;
the preparation method of the recombinant SPG protein in this example is the same as that in example 2.
In this example, the epoxy group ring-opening reaction can be directly completed by using the amino group of the recombinant SPG protein.
The serum antibody affinity separation material prepared in this example was used to adsorb antibody IgG (IgG is immunoglobulin G, immunoglobulinG), and the SPG affinity matrix prepared in this example was purified in one step, and the adsorption capacity, purity and protein recovery rate were 14.78±1.34mg/mL,83.9±1.75% and 88.35 ±5.23% respectively by 3 repeated experiments.
Example 4
The preparation method of the serum antibody affinity separation material of the embodiment comprises the following steps:
S1, washing an agarose matrix, namely placing agarose (Sepharose 6 FF) in a sand core funnel, pumping and washing the agarose matrix with ultrapure water for 10 times, and filtering the agarose matrix to obtain pumped agarose;
S2, epoxy activation reaction, namely adding epoxy chloropropane solution into the pumped agarose obtained in the step S1, preheating for 30min in a shaking table with the temperature of 60 ℃ and the rotating speed of 170rpm/min, then continuously reacting for 4h on the shaking table with the temperature of 60 ℃ and the rotating speed of 170rpm/min, detecting the pH value for 1 time every 1h, regulating the pH value to an alkaline condition by using 1mol/LNaOH solution, and pumping and washing for 10 times by using ultrapure water after the reaction is finished to obtain activated agarose gel, wherein the mass volume ratio of the pumped agarose to the epoxy chloropropane solution is 1g to 2.1mL, and the pH value of the activated agarose gel is 7.4;
s3, preparation of serum antibody affinity separation materials:
coupling the activated agarose gel obtained in the step S2 with recombinant SPG protein to obtain a serum antibody affinity separation material, wherein the specific method comprises the following steps:
Coupling recombinant SPG protein, namely, purifying the obtained recombinant SPG protein by a Ni metal chelating chromatographic column, replacing a system by a SuperdexG gel filtration column, collecting protein solution by using a PBS buffer with the pH value of Na 2SO4 with the final concentration of 0.2 moL/L=8.0, performing vacuum freeze drying to obtain frozen SPG protein powder, adding a PBS buffer with the pH value of Na 2SO4 with the final concentration of 0.2 moL/L=8.0 for dissolving to obtain a recombinant SPG protein solution with the concentration of 16mg/mL, adding the recombinant SPG protein solution into activated agarose gel obtained in S2, coupling for 24 hours at the temperature of 37 ℃ and the rotating speed of 180rpm/min, adding a NaCl solution with the concentration of 2.0moL/L, and performing double-evaporation water washing to obtain a recombinant SPG affinity medium, namely the serum antibody affinity separation material;
In the embodiment, experiments of different coupling times of the recombinant SPG protein with the concentration of 10.2mg/mL in the step S3 are also carried out, namely 5mL of prepared serum antibody affinity separation materials are taken at 2h, 8h, 24h, 48h and 72h of coupling of the recombinant SPG protein, and are used for evaluating the separation effect of the affinity matrix on serum samples at different time points.
And (3) taking serum antibody affinity separation materials prepared by recombinant SPG proteins in different time periods to purify bovine serum antibodies. Briefly, 1mL of each of the affinity matrices of the coupling SPG proteins of the 2h, 8h, 24h, 48h and 72h is taken out and immediately filled into a 10mL chromatographic column to determine the sedimentation volume until the experiment is needed, ddH 2 O and PBS of 10 column volumes are respectively balanced by using ultrapure water and PBS of 10 column volumes, 5mL of calf serum of 10mg/mL and PBS of 1mL are balanced, gel is repeatedly blown by a Pasteur pipette after being incubated for 15min, all the obtained fluid is collected, 10 times of PBS of 10 column volumes is used for washing media, the first (first tube) and the last (last tube) of the purified recombinant SPG proteins are collected, gly-HCl solution of which the concentration is 0.05mol/L, pH of 2.5 is used for repeatedly washing media for two times, all the eluted samples are collected, the eluent of the purified bovine serum antibody IgG is obtained by using ultrapure water of 10 times of 10 column volumes, the washing media is pumped to dryness, and the volume fraction is kept at room temperature to 20% ethanol.
The recombinant SPG protein is quantitatively detected by a BCA method, SDS-PAGE electrophoresis detection is carried out, the effect of the recombinant SPG protein affinity medium on specific adsorption of antibody IgG at different time is analyzed, and the SDS-PAGE electrophoresis detection result is shown as shown in figure 2 (1, 30 times diluted calf serum is original, 2, 24h medium flow through liquid coupling, 24h medium first impurity washing liquid coupling, 24h affinity medium last impurity washing liquid coupling, 2h affinity medium eluent coupling, 8h affinity medium eluent coupling, 7, 24h affinity medium eluent coupling, 48h affinity medium eluent coupling and 72h medium eluent coupling).
On SDS-PAGE electrophoresis, it is clear that the coupling 24h affinity medium gave the best separation effect on calf serum samples, and the highest antibody adsorption (BCA protein quantification) was obtained.
The serum antibody affinity separation material prepared in this example is used for adsorbing antibody IgG, the recombinant SPG protein is coupled with the epoxy activated matrix for 24 hours in this example, the prepared SPG affinity matrix is purified in one step, and the adsorption capacity, purity and protein recovery rate are respectively 15.47+/-1.63 mg/mL,88.92 +/-2.17% and 87.95 +/-4.68% serum antibody IgG through 3 repeated experiments.
Example 5
The preparation method of the serum antibody affinity separation material of the embodiment comprises the following steps:
S1, washing an agarose matrix, namely placing agarose (Sepharose 6 FF) in a sand core funnel, pumping and washing the agarose matrix with ultrapure water for 10 times, and filtering the agarose matrix to obtain pumped agarose;
s2, epoxy activation reaction, namely adding epichlorohydrin solution into the pumped agarose obtained in the S1, preheating for 30min in a shaking table with the temperature of 40 ℃ and the rotating speed of 200rpm/min, then continuously reacting for 4h on the shaking table with the temperature of 40 ℃ and the rotating speed of 200rpm/min, detecting pH for 1 time every 1h, regulating pH to 12.0 by using NaOH solution with the concentration of 8mol/L, and pumping and washing for 10 times by using ultrapure water after the reaction is finished to obtain activated agarose gel, wherein the mass volume ratio of the pumped agarose to the epichlorohydrin solution is 1g:2.1mL, and the pH value of the activated agarose gel is 7.4;
s3, preparation of serum antibody affinity separation materials:
the activated agarose gel obtained in the step S2 is subjected to amination reaction and indirect arm connection to obtain a serum antibody affinity separation material, and the specific method comprises the following steps:
An amination reaction, namely adding an amination reagent into the activated agarose gel obtained in the step S2, adjusting the pH value to 12.0, and performing a reaction for 12 hours at the temperature of 50 ℃ and the rotating speed of 200rpm/min, and then performing suction washing with ultrapure water for 10 times to obtain the aminated agarose, wherein the amination reagent is 1,6 hexamethylenediamine or hydrazine hydrate;
Suspending the aminated agarose in 4 ℃ ultrapure water, adding an acetone solution of cyanuric chloride pre-cooled for 3 hours at the temperature of minus 20 ℃, carrying out ice bath reaction for 5.5 hours under the condition of 170rpm/min, detecting the pH value every 0.5 hour in the reaction, regulating the pH value to 8.0 by using a saturated Na 2CO3 solution, and carrying out suction washing for 10 times by using ultrapure water after the reaction is finished, and carrying out suction filtration to obtain the agarose after the indirect arm connection, wherein the dosage ratio of the aminated agarose to the cyanuric chloride acetone solution is 1g to 2.1mL, and the dosage ratio of cyanuric chloride to the acetone solution of cyanuric chloride is 0.03g to 1mL;
Coupling agarose connected with the indirect arm and purified recombinant SPG protein solution with the concentration of 10.2mg/mL for 24 hours at the temperature of 25 ℃ and the rotating speed of 200rpm/min to obtain recombinant SPG affinity medium, namely the serum antibody affinity separation material, wherein the dosage ratio of the agarose connected with the indirect arm and the recombinant SPG protein solution is 1g:2.0mL.
In this example, calf serum antibodies were purified respectively on the epoxy-activated Sepharose6FF matrix, the affinity medium obtained by coupling 1,6 hexamethylenediamine-cyanuric chloride with recombinant SPG protein 24, and the epoxy-activated Sepharose6FF matrix, the affinity medium obtained by coupling hydrazine hydrate-cyanuric chloride with recombinant SPG protein 24. Namely, taking 1mL of recombinant SPG protein affinity matrix coupled for 24h respectively, taking out the recombinant SPG protein affinity matrix, immediately loading the recombinant SPG protein affinity matrix into a10 mL chromatographic column to determine the sedimentation volume until the experiment is required, balancing the affinity matrix by 10 column volumes of ddH 2 O and PBS respectively by using 10 times of column volumes of ultrapure water and PBS, taking 10mg/mL calf serum 5mL and 1mL PBS for balancing, repeatedly blowing gel by using a Pasteur pipette, incubating for 15min, collecting all obtained fluid, separating 10 times of cleaning medium by using 10 times of column volumes of PBS, collecting the first (first tube) and last (last tube) of the purified recombinant SPG protein, separating two times of repeated cleaning medium by using 1mL of Gly-HCl solution with the concentration of 0.05mol/L, pH of 2.5, collecting all eluted samples, obtaining the eluent of the purified bovine serum antibody IgG, pumping by using 10 times of ultrapure water with the volume of 10 times of the cleaning medium, and storing the eluent in 20% ethanol at room temperature.
The recombinant SPG protein is quantitatively detected by a BCA method, SDS-PAGE electrophoresis detection is carried out, the effect of the recombinant SPG protein affinity medium on specific adsorption of antibody IgG at different time is analyzed, and the result of the SDS-PAGE electrophoresis detection is shown in figure 3:
1, diluting calf serum which is 35 times as it is;
2, coupling an affinity matrix of recombinant SPG protein by the method, and purifying a flow-through liquid when the bovine serum antibody is purified by the affinity matrix obtained by coupling hydrazine hydrate-cyanuric chloride with the recombinant SPG protein for 24 hours;
3, purifying the last impurity washing liquid when the bovine serum antibody is purified by using an affinity matrix obtained by coupling hydrazine hydrate-cyanuric chloride with recombinant SPG protein for 24 hours, wherein M is protein Marker;
4 and 5 are respectively eluent 1 and eluent 2 obtained by eluting a conjugate by Gly-HCl with pH of 2.5 when the bovine serum antibody is purified by using an affinity matrix of hydrazine hydrate-cyanuric chloride and recombinant SPG protein coupled for 24 hours;
Coupling an affinity matrix of the recombinant SPG protein by the method, and carrying out affinity separation on calf serum by coupling hydrazine hydrate-cyanuric chloride with the recombinant SPG protein for 24 hours to obtain a flow-through liquid;
7, coupling an affinity matrix of the recombinant SPG protein by the method described in the example 5, and performing affinity separation on calf serum by coupling 1,6 hexamethylenediamine-cyanuric chloride with the recombinant SPG protein for 24 hours to obtain a washing liquid;
8 and 9 are Gly-HCl eluent 1 and eluent 2 with pH of 2.5 when calf serum is separated by coupling 1,6 hexamethylenediamine-cyanuric chloride with recombinant SPG protein for 24h matrix affinity.
The preparation method of the recombinant SPG protein in this example is the same as that in example 2.
In this example, sepharose6FF is activated by epichlorohydrin under strong alkaline condition, the activated medium is washed and then reacts with ammonia water, dimethyl sulfoxide is used to wash the medium with water, then cyanuric chloride is connected under alkaline condition, finally, the purified recombinant SPG protein amino is directly substituted with chlorine atom of cyanuric chloride to form covalent bond, the amino-containing compound reacted with epoxy group in this example includes but is not limited to ammonia water, and the amino-containing compound includes hydrazine hydrate, 1, 10-diaminodecane, 1, 4-butanediamine, 1, 6-hexanediamine and other series of double amino-containing compounds.
The serum antibody affinity separation material prepared in this example is used for adsorbing antibody IgG, and the recombinant SPG protein is coupled with hydrazine hydrate-trichlorocyano after being activated by epoxy for 24 hours, and the prepared SPG affinity matrix is purified in one step, so that the adsorption capacity, purity and protein recovery rate are respectively 15.28+/-2.12 mg/mL,87.78 +/-4.32% and 84.36+/-6.23% of serum antibody IgG. The experimental data are obtained by analyzing three times of separation and purification data of calf serum by using the recombinant SPG protein affinity matrix, and the prepared SPG affinity matrix is purified by coupling the recombinant SPG protein with 1,6 hexamethylenediamine-trichlorocyano substance for 24 hours after the recombinant SPG protein is activated by epoxy, and the adsorption capacity, the purity and the protein recovery rate are respectively 13.14+/-1.69 mg/mL,84.25 +/-5.16% and 83.27+/-5.47% of serum antibody IgG through 3 times of repeated experiments.
Example 6
The preparation method of the serum antibody affinity separation material of the embodiment comprises the following steps:
s1, washing an agarose matrix, namely placing agarose into a sand core funnel, pumping and washing the agarose matrix with ultrapure water for 20 times, and performing pumping filtration to obtain pumped agarose;
S2, epoxy activation reaction, namely adding epoxy chloropropane solution into the pumped agarose obtained in the step S1, preheating for 30min in a shaking table with the temperature of 60 ℃ and the rotating speed of 220rpm/min, then continuously reacting for 4h on the shaking table with the temperature of 60 ℃ and the rotating speed of 170rpm/min, detecting the pH value for 1 time every 1h, regulating the pH value to 13.0 by using a NaOH solution with the concentration of 7mol/L, pumping and washing for 10 times by using ultrapure water after the reaction is finished to obtain activated agarose gel, wherein the mass volume ratio of the pumped agarose to the epoxy chloropropane solution is 1g to 2.1mL, and the pH value of the activated agarose gel is about 7.4;
s3, preparation of serum antibody affinity separation materials:
the activated agarose gel obtained in the step S2 is subjected to amination reaction and indirect arm connection to obtain a serum antibody affinity separation material, and the specific method comprises the following steps:
Adding an amination reagent into the activated agarose gel obtained in the step S2, regulating the pH value to 13.0, reacting for 12-16 hours at the temperature of 60 ℃ and the rotating speed of 170rpm/min, and then pumping and washing with ultrapure water for 20 times to obtain the aminated agarose, wherein the amination reagent is ammonia water;
Suspending the aminated agarose in 4 ℃ ultrapure water, adding an acetone solution of cyanuric chloride pre-cooled for 3 hours at the temperature of minus 20 ℃, carrying out ice bath reaction for 5.5 hours under the condition of 170rpm/min, detecting the pH value every 0.5 hour in the reaction, regulating the pH value to 8.0 by using a saturated Na 2CO3 solution, and carrying out suction washing for 10 times by using ultrapure water after the reaction is finished, and carrying out suction filtration to obtain the agarose after the indirect arm connection, wherein the dosage ratio of the aminated agarose to the cyanuric chloride acetone solution is 1g to 2.1mL, and the dosage ratio of cyanuric chloride to the acetone solution of cyanuric chloride is 0.03g to 1mL;
Coupling the agarose connected with the indirect arm and the purified recombinant SPG protein solution with the concentration of 10mg/mL for 24 hours at the temperature of 25 ℃ and the rotating speed of 200rpm/min to obtain a recombinant SPG affinity medium, namely the serum antibody affinity separation material, wherein the dosage ratio of the agarose connected with the indirect arm to the recombinant SPG protein solution is 1g:2.0mL.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention. Any simple modification, variation and equivalent variation of the above embodiments according to the technical substance of the invention still fall within the scope of the technical solution of the invention.
Claims (7)
1. A preparation method of a serum antibody affinity separation material is characterized by comprising the following steps:
s1, washing an agarose matrix, namely placing agarose into a sand core funnel, pumping and washing the agarose matrix with ultrapure water for 10-20 times, and performing pumping filtration to obtain pumped agarose;
S2, epoxy activation reaction, namely adding an epoxy chloropropane solution into the pumped agarose obtained in the step S1, preheating for 30min in a shaking table with the temperature of 25-60 ℃ and the rotating speed of 170-220 rpm/min, then continuing to react on the shaking table with the temperature of 25-60 ℃ and the rotating speed of 170-220 rpm/min for 4h, detecting the pH value for 1 time every 1h, regulating the pH value to an alkaline condition by using NaOH dry powder or NaOH solution with the concentration of 1-8 mol/L, and performing pumping washing for 10 times after the reaction is finished to obtain activated agarose gel;
s3, preparation of serum antibody affinity separation materials:
coupling the activated agarose gel obtained in the step S2 with recombinant SPG protein to obtain a serum antibody affinity separation material, wherein the specific method comprises the following steps:
Coupling recombinant SPG protein, namely, subjecting the recombinant SPG protein obtained by purification of a Ni metal chelating chromatographic column to Superdex G75 gel filtration column to replace a system, collecting protein solution by using PBS buffer with the pH of 2SO4 with the final concentration of 0.2moL/L and 8.0, performing vacuum freeze drying to obtain frozen SPG protein powder, adding PBS buffer with the pH of 2SO4 with the final concentration of 0.2moL/L and 8.0 for dissolving to obtain recombinant SPG protein solution with the concentration of 4-16 mg/mL, adding the recombinant SPG protein solution into activated agarose gel obtained in S2, performing coupling for 24 hours under the conditions of the temperature of 16-37 ℃ and the rotating speed of 70-180 rpm/min, then adding NaCl solution with the concentration of 0.1-2.0 moL/L, and performing double-evaporation water washing to obtain recombinant SPG affinity medium, namely serum antibody affinity separation material;
Or coupling the activated agarose gel obtained in the step S2 with cyanuric chloride after amination reaction, and then carrying out substitution reaction with amino residues on recombinant SPG protein to finally obtain the serum antibody affinity separation material, wherein the specific method comprises the following steps of:
An amination reaction, namely adding an amination reagent into the activated agarose gel obtained in the step S2, adjusting the pH value to 12.0-13.0, reacting for 12-16 hours under the conditions of 50-60 ℃ and 170-200 rpm/min, and then pumping and washing with ultrapure water for 10-20 times to obtain the aminated agarose;
Suspending the aminated agarose in ultrapure water with the temperature of 0-4 ℃, adding an acetone solution of cyanuric chloride pre-cooled for 3 hours at the temperature of-20 ℃, carrying out ice bath reaction for 5.5 hours under the condition of the rotating speed of 170rpm/min, detecting the pH value every 0.5 hour in the reaction, regulating the pH value to 7.0-8.0 by using a saturated Na 2CO3 solution, carrying out suction washing for 10 times by using ultrapure water after the reaction is finished, and carrying out suction filtration to obtain the agarose after the connection of the indirect arm, wherein the dosage ratio of cyanuric chloride to the acetone solution of cyanuric chloride in the acetone solution of cyanuric chloride is 0.03 g/1 mL;
Coupling agarose connected with the indirect arm with purified recombinant SPG protein solution with concentration of 4 mg/mL-16 mg/mL for 24h at the temperature of 25 ℃ and the rotating speed of 200rpm/min to obtain recombinant SPG affinity medium, namely the serum antibody affinity separation material.
2. The method for preparing a serum antibody affinity separation material according to claim 1, wherein the mass-to-volume ratio of the pumped agarose to epichlorohydrin solution in S2 is 1g:2.1ml.
3. The method for producing a serum antibody affinity separation material according to claim 1, wherein when the activated agarose gel obtained in S2 is subjected to an amination reaction and an indirect arm connection to obtain a serum antibody affinity separation material, the amination reagent in S3 comprises ammonia, hydrazine hydrate or 1, 6-hexamethylenediamine.
4. The method for producing a serum antibody affinity separation material according to claim 1, wherein when the activated agarose gel obtained in S2 is subjected to an amination reaction and an indirect arm connection to obtain a serum antibody affinity separation material, the ratio of the amount of the aminated agarose to the amount of the cyanuric chloride acetone solution in S3 is 1g to 2.1ml.
5. The method for preparing a serum antibody affinity separation material according to claim 1, wherein when the activated agarose gel obtained in S2 is subjected to an amination reaction and an indirect arm connection, the ratio of the agarose after the indirect arm connection to the recombinant SPG protein solution in S3 is 1 g to 0.7ml.
6. The method for preparing a serum antibody affinity separation material according to claim 1, wherein the method for preparing recombinant SPG protein in the recombinant SPG protein solution in S3 comprises the steps of:
S301, preparing a gene for encoding the recombinant SPG protein by using a chemical synthesis method, performing codon optimization to obtain a nucleotide sequence of the recombinant SPG protein, and then taking the gene sequence as a template, designing a primer sequence for PCR amplification to obtain a PCR product, wherein the nucleotide sequence of the gene of the recombinant SPG protein is shown as SEQ ID NO. 4;
the PCR amplification reaction system comprises 10 mu L of 5 XPS buffer, 4 mu L of dNTP with the concentration of 2.5mmol/L, 1 mu L of upstream primer with the concentration of 10 mu mol/L, 1 mu L of downstream primer with the concentration of 10 mu mol/L, 1.0 mu L of DNA polymerase with the concentration of 2.5U/mu L, 1 mu L of genome DNA to be detected and double distilled water to be supplemented to 50 mu L, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3;
the PCR amplification reaction procedure comprises 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 30s,65 ℃ annealing for 30s,72 ℃ extension for 80s, 32 cycles, 72 ℃ continuous extension for 10min, and 4 ℃ preservation;
S302, carrying out electrophoresis on the PCR product in S301, cutting gel, recovering a band at about 500bp to obtain a purified target gene, and carrying out double enzyme digestion on the purified target gene and an expression vector pET-28a by NdeI/XhoI restriction enzyme to obtain a target gene double enzyme digestion product and an expression vector pET-28a double enzyme digestion product respectively;
The enzyme digestion reaction system comprises 6 mu L of purified target gene or expression vector pET-28a, 10× FASTDIGEST BUFFER 16 mu L, FASTDIGEST NDEI mu L, FASTDIGEST XHOI mu L and ddH 2 O36 mu L;
S303, respectively recovering the target gene double enzyme digestion product obtained in the S302 and the expression vector pET-28a double enzyme digestion product by using a AxyPrepTM DNA Gel Extraction Kit glue recovery kit to respectively obtain a double enzyme digestion post-glue recovery SPG gene and a double enzyme digestion pET-28a glue recovery product, and carrying out a connection reaction on the double enzyme digestion post-glue recovery SPG gene and the double enzyme digestion pET-28a glue recovery product for 1.5 hours under the condition that the temperature is 22 ℃ to obtain a connection product;
Transforming the connection product into competent cells E.coli DH5 alpha by a heat shock method, screening positive clones by agar plate LB with kana antibiotics with the concentration of 1mM, culturing by LB liquid culture medium, verifying positive clones by colony PCR, and obtaining the recombinant clone strain with correct sequencing result as pET-28a-SPG-DH5a strain;
The system of the ligation reaction is 5X Ligase Reaction Buffer mu L, SPG gene is recovered by double enzyme digestion, product is recovered by double enzyme digestion pET-28a, 2 mu L of T4 DNA ligase with concentration of 5U/mu L and double distilled water is recovered by double enzyme digestion;
S304, expressing recombinant SPG protein, namely extracting plasmid in logarithmic phase of pET28a-SPG-DH5a strain obtained in S303 by using a plasmid extraction kit, converting expression host bacteria E.coli BL21 (DE 3) competent cells by heat shock, inoculating positive monoclonal colony screened by a solid LB plate to LB liquid culture medium of kana antibiotics with the pre-concentration of 1mM, culturing overnight at 37 ℃ and the rotating speed of 180rpm/min, transferring to new LB liquid culture medium of kana antibiotics with the pre-concentration of 1mM with the volume of 500mL, culturing continuously at 37 ℃ and the rotating speed of 180rpm/min until the light absorption value of bacterial liquid reaches 0.4-0.6 at the wavelength of 600nm, adding isopropyl-beta-D-thiogalactoside solution, obtaining 1mL after induction for 8 hours, centrifuging at the rotating speed of 10000rpm/min, collecting bacterial mud, detecting that the bacterial mud contains SDS-containing target protein by 10 ℃ and diluting with the phosphate buffer solution of 10 ℃ and carrying out heat dilution on the bacterial mud to obtain the target protein by detecting the SDS-containing the target protein by using the liquid of Loading Buffer ℃ after the bacterial mud;
S305, purifying recombinant SPG protein, namely, after three stages of activation, transfer and enrichment culture of the expression host bacteria containing the target protein obtained in S304, re-suspending the expression host bacteria containing the target protein in phosphate buffer with the concentration of 10mmol/L according to the mass-volume ratio of 1g to 20mL, ultrasonically crushing bacterial cell walls under the ice bath condition of 0-4 ℃, centrifuging for 30min at the rotating speed of 8000rpm/min, collecting supernatant, purifying by Ni metal chelating column chromatography to obtain the recombinant SPG protein, wherein the ultrasonic condition is that firstly ultrasonic bacteria breaking is carried out for 2S, then intermittent for 3S, the ultrasonic bacteria breaking and intermittent operation are repeated for 40min, the power is 55W, and the nucleotide sequence of the recombinant SPG protein is shown as SEQ ID NO: 4.
7. Use of a serum antibody affinity separation material prepared according to any one of claims 1-6, characterized in that said serum antibody affinity separation material is used for adsorption of antibody IgG.
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