Preparation method of beta2-glycoprotein antigen

Abstract

The invention relates to the technical field of biology, and discloses a preparation method of a beta2-glycoprotein antigen, which comprises the following steps: S1, designing plasmids: S11, selecting a carrier: selecting a self-developed signal peptide-containing plasmid as the carrier; s12, gene cloning: determining a DNA sequence to be inserted, performing codon optimization, and constructing by using a seamless cloning technology; s13, synthesizing or obtaining DNA fragments, wherein the DNA fragments are obtained through artificial synthesis; s14, drawing a plasmid atlas: drawing the plasmid atlas by using software such as SnapGene so as to ensure that all elements are correctly assembled; s2, preparing plasmids: extracting plasmids without endotoxin; and transforming the synthesized plasmids into host cells TOP10. The expression level of beta2-glycoprotein is improved by adopting self-researched signal peptide and an expression process, the prepared antigen can be used for any type of immunoassay, and the purity is as high as 95% or above; the yield is high and is 200mg / L; compared with similar products, the method has the advantages of better consistency and repeatability and easier standardized production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for preparing a beta 2-glycoprotein antigen. Background Art

[0002] β2-glycoprotein is the antigen that pathogenic antibodies target in patients with antiphospholipid antibody syndrome (APS) and is widely used in laboratory diagnostics. These tests use plasma β2-glycoprotein, which is difficult to purify in plasma and may be oxidized and denatured during the purification process and cause conformational changes in β2GPI, thereby affecting antigen activity. Recombinant β2-glycoprotein has always been difficult to prepare due to its complex conformation and numerous disulfide bonds.

[0003] Therefore, we proposed a method for preparing β2-glycoprotein antigen to solve the problem. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention provides a method for preparing a β2-glycoprotein antigen, which solves the problems in the above-mentioned background technology.

[0006] (II) Technical solution

[0007] To achieve the above object, the present invention provides the following technical solution: a method for preparing a β2-glycoprotein antigen, comprising the following steps: S1: Design plasmid S11: Select vector: select a self-developed plasmid containing a signal peptide as the vector; S12: Gene cloning: determine the DNA sequence to be inserted, perform codon optimization, and construct using seamless cloning technology; S13: Synthesize or obtain DNA fragments: obtained by artificial synthesis; S14: Plasmid map: Use software such as SnapGene to draw a plasmid map to ensure that all components are assembled correctly; S2: Preparation of plasmids Extraction of endotoxin-free plasmid: Transform the synthetic plasmid into host cell TOP10, and culture overnight to extract the endotoxin-free plasmid; S3: Protein expression S31: Transfection: The above plasmids were introduced into 293 cells using PEI chemical transfection method; S32: Culture: Add the culture medium of the transfected cells to the feed to promote protein expression; S4: Protein purification S41: Collecting the supernatant: Centrifuge the cells cultured for 6 days to allow the cells to settle at the bottom of the tube, carefully aspirate the supernatant on the upper layer to avoid aspirating the cell precipitate, dialyze the collected supernatant to remove small molecule impurities, and dialyze it into a suitable buffer; S42: Purification: The collected supernatant is centrifuged to remove possible impurities and precipitates. The supernatant is passed through a Ni column. The target protein will bind to the Ni column and be eluted using a buffer containing gradient imidazole. Proteins with different binding strengths will be eluted at different imidazole concentrations. The eluted samples are analyzed by SDS-PAGE electrophoresis to determine the expression of the target protein.

[0008] Preferably, the preparation steps of the signal peptide plasmid are: S111: Design and synthesis: Design and synthesize relevant fragments according to the sequences of target genes and signal peptides; S112: Construction of plasmid: Connect the target gene and signal peptide fragment to a suitable plasmid vector; S113: Transformation and culture: Transform the constructed plasmid into competent cells and culture them; S114: Plasmid extraction: Extract the plasmid containing the signal peptide from the cultured cells.

[0009] Preferably, the specific method of artificially synthesizing DNA in step S13 is: S131: Design sequence: Determine the DNA sequence to be synthesized according to the requirements; S132: Chemical synthesis: DNA fragments are synthesized step by step through a series of chemical reactions; S133: Purification: removal of impurities and by-products produced during the synthesis process; S134: Ligation and assembly: connect the synthesized fragments into a complete DNA molecule.

[0010] Preferably, the specific steps of endotoxin-free plasmid extraction are: S21: Transformation: Transform the synthesized plasmid into host cell TOP10; S22: Overnight culture: Allow cells to grow and express plasmid; S23: Collect cells: collect cells by centrifugation or other methods; S24: Lyse cells: Use appropriate methods to lyse cells and release plasmids; S25: Endotoxin removal: Specific methods are used to remove endotoxins; S26: Purify the plasmid: Purify the plasmid by chromatography, precipitation, etc.

[0011] (III) Beneficial effects

[0012] Compared with the prior art, the present invention provides a method for preparing a β2-glycoprotein antigen, which has the following beneficial effects: The present invention uses a self-developed signal peptide and expression process to improve the expression level of β2-glycoprotein. The prepared antigen can be used for any type of immunoassay, and the purity is better than 95%; the yield is high, 200 mg / L; it is not easily affected by the source, and has better consistency, repeatability and easier standardized production than similar products. DETAILED DESCRIPTION

[0013] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] A method for preparing a β2-glycoprotein antigen comprises the following steps: S1: Design plasmid S11: Select vector: select a self-developed plasmid containing a signal peptide as the vector; Here, the steps for preparing the signal peptide plasmid are: S111: Design and synthesis: Design and synthesize relevant fragments according to the sequences of target genes and signal peptides; S112: Construction of plasmid: Connect the target gene and signal peptide fragment to a suitable plasmid vector; S113: Transformation and culture: Transform the constructed plasmid into competent cells and culture them; S114: Plasmid extraction: Extract the plasmid containing the signal peptide from the cultured cells.

[0015] S12: Gene cloning: determine the DNA sequence to be inserted, perform codon optimization, and construct using seamless cloning technology; In this step, the signal peptide helps to direct the target protein to a specific organelle or extracellular environment, which may aid in the correct folding and expression of the protein and improve the expression level. It can be designed and optimized for specific cell types or physiological conditions.

[0016] S13: Synthesize or obtain DNA fragments: obtained by artificial synthesis; Here, the specific method of artificially synthesizing DNA is: S131: Design sequence: Determine the DNA sequence to be synthesized according to the requirements; S132: Chemical synthesis: DNA fragments are synthesized step by step through a series of chemical reactions; S133: Purification: removal of impurities and by-products produced during the synthesis process; S134: Ligation and assembly: connect the synthesized fragments into a complete DNA molecule; S14: Plasmid map: Use software such as SnapGene to draw a plasmid map to ensure that all components are assembled correctly; S2: Preparation of plasmids Extraction of endotoxin-free plasmid: Transform the synthetic plasmid into host cell TOP10, and culture overnight to extract the endotoxin-free plasmid; The specific steps are as follows: S21: transformation: transforming the synthesized plasmid into the host cell TOP10; S22: Overnight culture: Allow cells to grow and express plasmid; S23: Collect cells: collect cells by centrifugation or other methods; S24: Lyse cells: Use appropriate methods to lyse cells and release plasmids; S25: Endotoxin removal: Specific methods are used to remove endotoxins; S26: Purify plasmid: Purify plasmid by chromatography, precipitation, etc. S3: Protein expression S31: Transfection: The above plasmids were introduced into 293 cells using PEI chemical transfection method; S32: Culture: Add the culture medium of the transfected cells to the feed to promote protein expression; S4: Protein purification S41: Collecting the supernatant: Centrifuge the cells cultured for 6 days to allow the cells to settle at the bottom of the tube, carefully aspirate the supernatant on the upper layer to avoid aspirating the cell precipitate, dialyze the collected supernatant to remove small molecule impurities, and dialyze it into a suitable buffer; S42: Purification: The collected supernatant is centrifuged to remove possible impurities and precipitates. The supernatant is passed through a Ni column. The target protein will bind to the Ni column and be eluted using a buffer containing gradient imidazole. Proteins with different binding strengths will be eluted at different imidazole concentrations. The eluted samples are analyzed by SDS-PAGE electrophoresis to determine the expression of the target protein.

[0017] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a β2-glycoprotein antigen, characterized in that: The following steps are involved: S1: Design plasmid S11: Select vector: select a self-developed plasmid containing a signal peptide as the vector; S12: Gene cloning: determine the DNA sequence to be inserted, perform codon optimization, and construct using seamless cloning technology; S13: Synthesize or obtain DNA fragments: obtained by artificial synthesis; S14: Plasmid map: Use software such as SnapGene to draw a plasmid map to ensure that all components are assembled correctly; S2: Preparation of plasmids Extraction of endotoxin-free plasmid: Transform the synthetic plasmid into host cell TOP10, and culture overnight to extract the endotoxin-free plasmid; S3: Protein expression S31: Transfection: The above plasmids were introduced into 293 cells using PEI chemical transfection method; S32: Culture: Add the culture medium of the transfected cells to the feed to promote protein expression; S4: Protein purification S41: Collecting the supernatant: Centrifuge the cells cultured for 6 days to allow the cells to settle at the bottom of the tube, carefully aspirate the supernatant on the upper layer to avoid aspirating the cell precipitate, dialyze the collected supernatant to remove small molecule impurities, and dialyze it into a suitable buffer; S42： Purification: The collected supernatant is centrifuged to remove possible impurities and precipitates. The supernatant is passed through a Ni column. The target protein will bind to the Ni column and be eluted using a buffer containing gradient imidazole. Proteins with different binding strengths will be eluted at different imidazole concentrations. The eluted samples are analyzed by SDS-PAGE electrophoresis to determine the expression of the target protein.

2. The method for preparing a β2-glycoprotein antigen according to claim 1, characterized in that: The preparation steps of signal peptide plasmid are as follows: S111: Design and synthesis: Design and synthesize relevant fragments according to the sequences of target genes and signal peptides; S112: Construction of plasmid: Connect the target gene and signal peptide fragment to a suitable plasmid vector; S113: Transformation and culture: Transform the constructed plasmid into competent cells and culture them; S114: Plasmid extraction: Extract the plasmid containing the signal peptide from the cultured cells.

3. The method for preparing a β2-glycoprotein antigen according to claim 1, characterized in that: The specific method of artificially synthesizing DNA in step S13 is: S131: Design sequence: Determine the DNA sequence to be synthesized according to the requirements; S132: Chemical synthesis: DNA fragments are synthesized step by step through a series of chemical reactions; S133: Purification: removal of impurities and by-products produced during the synthesis process; S134: Ligation and assembly: connect the synthesized fragments into a complete DNA molecule.

4. The method for preparing a β2-glycoprotein antigen according to claim 1, characterized in that: The specific steps for endotoxin-free plasmid extraction are: S21: Transformation: Transform the synthesized plasmid into host cell TOP10; S22: Overnight culture: Allow cells to grow and express plasmid; S23: Collect cells: collect cells by centrifugation or other methods; S24: Lyse cells: Use appropriate methods to lyse cells and release plasmids; S25: Endotoxin removal: Specific methods are used to remove endotoxins; S26: Purify the plasmid: Purify the plasmid by chromatography, precipitation, etc.