CN101935665B - Preparation method and application of recombinant protein A gene and expression product thereof - Google Patents
Preparation method and application of recombinant protein A gene and expression product thereof Download PDFInfo
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- CN101935665B CN101935665B CN 201010245004 CN201010245004A CN101935665B CN 101935665 B CN101935665 B CN 101935665B CN 201010245004 CN201010245004 CN 201010245004 CN 201010245004 A CN201010245004 A CN 201010245004A CN 101935665 B CN101935665 B CN 101935665B
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Abstract
The invention provides a recombinant protein A gene with a nucleotide sequence expressed as SEQ ID No: 1 and recombinant protein A coded by the gene. The invention also provides a recombinant expression vector PET32a-P for expressing the recombinant protein A gene, escherichia coli BL21/DE3 transformed by the recombinant expression vector and a method for preparing the recombinant protein A. The recombinant protein A provided by the invention is used for antibody detection, separation and purification, and the recombinant protein A and a chromatographic medium vector can form a compatible chromatographic medium. The recombinant protein A produced by the invention has the advantages of strong protein binding specificity, high affinity and great improvement on purification efficiency; and compared with the commercial ProteinA Sepharose 4Fast Flow, the recombinant protein A has obviously improved dynamic adsorption capacity.
Description
The present invention for denomination of invention is: the preparation method of a kind of A gene of recombined protein and expression product thereof and purposes, application number is: the dividing an application of the Chinese patent application of 200710085148.X.
Technical field
The invention belongs to biological technical field, specifically, relate to a kind of A gene of recombined protein, and contain the carrier of this recombination, bacterial strain and the A gene of recombined protein expressed products of conversion, and its production and use.
Background technology
(Staphylococal Protein A SPA) is a kind of protein that separates from the aureus cell wall to SP.1940, Vevwey found to contain a kind of material in some streptococcus aureus, in double diffusion test, can form precipitation with normal human serum.Jensen (1959) has also found similar phenomenon, with its called after A antigen.
Lofkvist in 1963 etc. have separated A antigen, and prove that it is a kind of protein, and have any different with sugar; Grov (1960) is called for short SPA albumin A (ProteinA) with its called after SP.The gene of coding SPA was cloned and at expression in escherichia coli (Duggleby in nineteen eighty-three, C.J and Jones, SA:cloning and expression of the Staphylococcus aureus protein A gene in Escherichiacoli.Nucl Acids Res.11 (1983) 3065-3076; Lofdahl, S., Guss, B., et al; Gene forStaphylococcal protein A.Proc.Natl.Acid.Sci.USA 80 (1983) 697-701).To discovering of SPA structure and function, the SPA molecule comprises A, B, C, D, five homeodomains of E, each structural domain have can with the ability of the autonomous combination of IgG.The SPA gene has 1600bp.
Since have on the SP 5 structural domains can with the Fc district combination of IgG, have the ability of being combined with the Fc of most of Mammals IgG section.As a kind of affinity ligand, albumin A is fixed on above the agarose, and 5 structural domains above it just can the free combination with the Fc of IgG, and the fixing albumin A of a part can be combined with bimolecular IgG, its C end of recombinant protein A has a Gelucystine, and is stronger in conjunction with the ability of IgG.Recombinant protein A is widely used in separation and purification and the detection of monoclonal antibody or polyclonal antibody.When use contains the affinity chromatography technology of proteinA, there are problems such as avidity is low, purification efficiency is low, therefore when this affinity chromatography technology of use, need a kind of improved ProteinA, realize higher avidity, improve the effect of purifying.
Summary of the invention
One of technical issues that need to address of the present invention provide a kind of gene order of new recombinant protein A, to overcome the deficiencies in the prior art.
Two of the technical issues that need to address of the present invention provide the aminoacid sequence of this recombinant protein A, to overcome the deficiencies in the prior art.
Three of the technical issues that need to address of the present invention provide the carrier that contains this A gene of recombined protein.
Four of the technical issues that need to address of the present invention provide the expression vector transformed host cell that is contained A gene of recombined protein.
Five of the technical issues that need to address of the present invention provide the preparation method of this recombinant protein A.
Six of the technical issues that need to address of the present invention provide the purposes of this recombinant protein A.
Seven of the technical issues that need to address of the present invention provide a kind of affinity chromatography medium of being made up of this recombinant protein A and chromatography media carrier.
Technical scheme of the present invention is as follows:
A gene of recombined protein provided by the invention is that the A gene of recombined protein monomer with 3 synthetic is connected in series, 5 ' hold Ala-Asp to be mutated into Val-Asp, to form AccI restriction enzyme site (GTAGAC), link to each other with the AccI restriction enzyme site between each A gene of recombined protein monomer, wherein first A gene of recombined protein monomer front end adds the NcoI restriction enzyme site that links to each other with expression vector, 6 His (are used for affinity chromatography, be combined with the nickel post, reduce purification step) and the EK restriction enzyme site (His and DDDDK are cut, form correct Protein A), terminal terminator codon TAA and the BamH I restriction enzyme site that links to each other with expression vector pET32a of adding of last A gene of recombined protein monomer.This gene is made of 621 Nucleotide, 180 amino acid of encoding.This gene order and amino acid sequence coded thereof are seen sequence table SEQ ID No.1 and SEQ ID No.2.
The present invention has also made up the carrier that contains above-mentioned A gene of recombined protein, these construction of carrier are according to ordinary method, to cut between the corresponding restriction enzyme site that inserts respective carrier in the back by the A gene of recombined protein monomeric enzyme of synthetic, with Acc I endonuclease digestion, connect into the A gene of recombined protein expression vector that comprises a plurality of polyphone A gene of recombined protein monomers again.
The recombinant expression vector that the A gene of recombined protein that the above-mentioned coli expression carrier that contains A gene of recombined protein is preferentially synthesized by the present invention and coli expression carrier PET32 are built into, called after pET32a-P.
The present invention also provides the method for utilizing the above-mentioned intestinal bacteria recombinant strain that contains the intestinal bacteria recombinant expression vector to produce recombinant protein A, this method is bacterial classification to be carried out cultivation and fermentation make its highly effective expressing recombinant protein A, regather thalline, obtain the recombinant protein A product through separation and purification.
The coli strain of the above-mentioned energy of the present invention express recombinant protein A is preferably the bacterial strain that is obtained by the expression vector pET32a-P transformed into escherichia coli BL21 (DE3) that contains A gene of recombined protein, called after BL21-pET32a.
The recombinant protein A that the present invention produces is expressed in the Escherichia coli cell pericentral siphon, is conducive to the separation and purification of expression product.Utilize the present invention to express the production recombinant protein A, have the expression efficiency height, expression amount big (accounting for the 60-80% of bacterium total soluble protein), is easy to advantages such as purifying at expression time short (only 3-5 hour).
The present invention adopts escherichia expression system to produce recombinant protein A, also has (1 day) with short production cycle, and the characteristics that production cost is low are conducive to the commercial application of genetically engineered recombinant protein A.
The expression product recombinant protein A of A gene of recombined protein of the present invention is used for becoming the separation and purification that affinity chromatography medium is used for antibody with various chromatography media carrier as couplings such as: sepharose, dextran, Mierocrystalline cellulose, hydroxyl high molecular polymer, silica gel.
The recombinant protein A that the present invention produces has the protein binding high specificity, and the advantage that avidity height, purification efficiency improve is greatly compared with commercially available ProteinA Sepharose4Fast Flow, and its dynamic adsorption carrying capacity obviously improves.
Fig. 1 is the recombinant expression vector pET32a-P that the present invention makes up, and is to be connected into the proteinA gene in carrier pET32a.
Fig. 2: the SDS-PAGE gel electrophoresis result of thalline, wherein swimming lane 1 and 5 is marker; Swimming lane 2 is empty carriers; Swimming lane 3 is the supernatants behind the broken bacterium of the BL21-pET32a through inducing; Swimming lane 4 is the precipitations behind the broken bacterium of the BL21-pET32a through inducing.
Fig. 3: recombinant protein A sepharose 5S CP separation and purification IgG2aSDS-PAGE electrophoresis result from mouse ascites: swimming lane 1 is standard protein Marker, swimming lane 2 is ascites, and swimming lane 3 is the elution peak of recombinant protein A sepharose 5S CP affinity media of the present invention.
Fig. 4: the recombinant protein A dextrane gel is separation and purification IgG2aSDS-PAGE electrophoresis result from mouse ascites: swimming lane 1 is standard protein Marker, swimming lane 2 is ascites, the elution peak of the pillar that swimming lane 3 is adorned for recombinant protein A dextrane gel affinity media of the present invention.In the swimming lane 3, heavy chain and light chain that upper and lower two bands are respectively IgG2a.
Synthesizing of embodiment 1 A gene of recombined protein monomer
Method by chemosynthesis, the sequence of the synthetic repeated fragment of A gene of recombined protein of design, its sequence comprises that length is 168bp, front end adds the NcoI restriction enzyme site that links to each other with expression vector, 6 His (are used for affinity chromatography, be combined with the nickel post, reduce purification step) and the EK restriction enzyme site, and the AccI restriction enzyme site that is used for being connected into a plurality of repeated fragments.In addition, also comprise terminator codon TAA, and the clone is total to 9bp with BamH I restriction enzyme joint.Also must be by the method for chemosynthesis, the sequence of the synthetic repeated fragment of A gene of recombined protein of design, its sequence comprises that length is 168bp, two ends are the AccI restriction enzyme site, are used for making up the sequence that contains a plurality of repeated fragments.
With restricted type restriction endonuclease NcoI and BamH I above-mentioned A gene of recombined protein monomer is downcut, be connected with the carrier pET32a that cuts through NcoI and BamHI enzyme, transformed into escherichia coli, screening has the transformant of amicillin resistance, extract through plasmid, enzyme is cut and is identified that back proof A gene of recombined protein monomer is cloned among the pET32a.
Above-mentioned pET32a carrier and the A gene of recombined protein monomer that contains an A gene of recombined protein monomer cut with the AccI enzyme, connect after reclaiming respective segments, transformed into escherichia coli, screening has the transformant of amicillin resistance, extract through plasmid, enzyme is cut the pET32a-P carrier that the screening acquisition contains the recombinant protein A of 3 protein A gene monomers.
The structure of the coli strain BL21-pET32a of embodiment 4 express recombinant protein A
Use CaCl
2Method transforms BL21 (DE3) with pET32a-P, screens transformant at the LB flat board that contains penbritin, detects and the restriction analysis acquisition contains the sub-BL21-pET32a of recombinant conversion of pET32a through plasmid.
1) culture of strains fermentation
Picking colibacillus engineering BL21-pET32a, be inoculated in the LB substratum, inoculum size 1～2%V/V, in 30 ℃ of overnight incubation, next day under aseptic condition with above-mentioned cultured seed substratum by 1: 10-1: 5 are inoculated on the fermention medium, 30 ℃ ferment to O.D600 and reach 0.4～0.8, are warming up to 42 ℃ and induce, centrifugal receipts bacterium after 3～5 hours;
The cell that takes a morsel adds 2 * sample-loading buffer, do the SDS-PAGE gel electrophoresis by standard, result such as Figure of description 2, supernatant behind the broken bacterium of BL21-pET32a through inducing a new protein band (seeing Fig. 2 swimming lane 3) occurs in the position of 20kD, transform in the precipitation behind the broken bacterium of BL21 (DE3) bacterium of PET32 empty carrier and the BL21-pET32a through inducing and this band (seeing Fig. 2 swimming lane 2 and 4) do not occur, prove that the egg of recombinating induced soluble-expression from A in BL21-pET32a.
2) purifying of the recombinant protein A of Biao Daing
Above-mentioned collection thalline is suspended with NaCl ten phosphate buffered saline buffers (pH7.0-8.0), ultrasonication, 4 ℃ are centrifugal, collect supernatant, get crude extract.
Show through the SDS-PAGE electrophoresis detection, it is fine to extract the recombinant protein A effect that is expressed in the Escherichia coli cell pericentral siphon with this law, slightly carrying the most of recombinant protein A in back is slightly carried, the amount that remains in thalline is atomic: crude extract is carried out Sephacryl 5200 molecular sieve purification, collect the recombinant protein A that characteristic peak (second elution peak) is purifying, its purity can reach more than 90%.
Embodiment 6 ELISA methods detect the test of recombinant protein A and antibody binding activity
1) bag quilt: every hole bag is by recombinant protein A sample 100u l, 37 ℃, 1h in 96 orifice plates;
2) sealing: every hole is with 1% the BSA sealing of 100u l, 37 ℃, 1h;
3) add primary antibodie: every hole adds about 100ug human IgG antibody, 37 ℃, 1h;
4) adding two resists: every hole adds about 100u l1: 1000 horseradish peroxidase-labeled antibody, 37 ℃, 1h;
5) colour developing.
Detect through ELISA that to show that recombinant protein A that the present invention extracts is combined activity with the human IgG antibody strong, wrap in every hole can be obtained tangible detection signal by the 4.5ng recombinant protein A, and the result shows that recombinant protein A provided by the invention can be used for detection of antibodies.
The preparation of embodiment 7 recombinant protein A sepharose 5S CP
1, react in water medium with epoxy chloropropane, sodium hydroxide and agarose (5% cross-linked agarose gel), reaction is 2-3 hour under 30-60 ℃ temperature, has reacted back the cleaning to neutrality with distilled water and has drained;
2, above-mentioned product and recombinant protein A of the present invention were reacted 15-20 hour under 5-25 ℃ of temperature, reacted the back cleaning and drained again, namely obtain recombinant protein A sepharose 5S CP
Recombinant protein A sepharose 5S CP has following characteristic:
1) characteristics: group comes off few, and binding specificity is strong;
2). aglucon density: ≈ 6mg recombinant protein A/ml;
3) absorption carrying capacity: 3～30mg mouse IgG2a/ml;
4) granular size of affinity media: 30-180 μ m;
5) Peak Flow Rate: 250cm/h
6) pH scope: 2-11;
7) storage temperature: 4-8 ℃;
8) preserve liquid: 20% ethanol.
If the adsorptive power between antibody purified to be separated and affinity chromatography medium is more weak, then can suitably improve pH value and the salt concn of adsorption-buffering liquid, can obtain separating effect preferably.
Embodiment 8 recombinant protein A sepharose 5S CP separation and purification IgG2a from mouse ascites
1, recombinant protein A sepharose 5S CP dress post, 1.6 * 20cm, column volume are 10ml;
2, with buffer A (20mM phosphate buffered saline buffer, pH7.4, i.e. the PBS solution of pH7.4.Preparation: 0.2M NaH
2PO
419ml, 0.2M Na
2HPO
481ml, NaCl 9g adds water to 1000ml.) a balance 5-10 bed volume, flow velocity is 1ml/min;
3, the 2ml mouse ascites is diluted to 20ml with buffer A, 0.45 μ m membrane filtration, last sample.Flow velocity is 1ml/min;
4, wash 5-10 bed volume again with buffer A, flow velocity is 1ml/min;
5, with buffer B (20mM citrate buffer solution, pH4.0.Preparation: citric acid 2.1g adds water 950ml, transfers to pH 4.0 with 5M NaOH, adds water to 1000ml) wash-out, flow velocity is 1ml/min, collects elution peak;
6, wash 10 column volumes with pure water stream, wash 10 column volumes with 20% ethanol stream again, flow velocity is 2ml/min, and pillar places 4-8 ℃ of environment to preserve;
7, IgG2a and the reference substance with separation and purification carries out the SDS-PAGE electrophoretic analysis simultaneously; (should be in order to damping fluid C (0.5M acetate buffer solution, pH3.0 after the every use several times of pillar.Preparation: the 0.5M acetum transfers to pH 3.0 with solid NaOH) stream washes 1 time, in order to will adsorb more firm albumen removal.) result:
The SDS-PAGE electrophoretic analysis the results are shown in Figure 3, and swimming lane 1 is standard protein Marker, and swimming lane 2 is ascites, the elution peak of the pillar that swimming lane 3 is adorned for recombinant protein A sepharose 5S CP affinity media of the present invention.In the swimming lane 3, heavy chain and light chain that upper and lower two bands are respectively IgG2a.Test-results has shown that recombinant protein A sepharose 5S CP affinity chromatography medium of the present invention can obtain purity greater than 95% IgG2a once going on foot.
The preparation of embodiment 9 recombinant protein A dextrane geles
1, react in water medium with epoxy chloropropane, sodium hydroxide and dextrane gel, reaction is 2-3 hour under 30-60 ℃ temperature, has reacted back the cleaning to neutrality with distilled water and has drained;
2, above-mentioned product and recombinant protein A of the present invention were reacted 15-20 hour under 5-25 ℃ of temperature, reacted the back cleaning and drained again, namely obtain the recombinant protein A dextrane gel.
The recombinant protein A dextrane gel has following characteristic:
1) characteristics: group comes off few, and binding specificity is strong;
2) aglucon density: ≈ 6mg recombinant protein A/ml;
3) absorption carrying capacity: 3～30mg mouse IgG2a/ml;
4) granular size of affinity media: 20-130 μ m;
5) Peak Flow Rate: 200cm/h
6) pH scope: 2-11;
7) storage temperature: 4-8 ℃;
8) preserve liquid: 20% ethanol.
If the adsorptive power between antibody purified to be separated and affinity chromatography medium is more weak, then can suitably improve pH value and the salt concn of adsorption-buffering liquid, can obtain separating effect preferably.
Embodiment 10 recombinant protein A dextrane geles separation and purification IgG2a from mouse ascites
1, recombinant protein A dextrane gel dress post, 1.6 * 20cm, column volume are 10ml;
2, with buffer A (20mM phosphate buffered saline buffer, pH7.4, i.e. the PBS solution of pH7.4.Preparation: 0.2M NaH
2PO
419ml, 0.2M Na
2HP0481ml, NaCl 9g adds water to 1000ml.) a balance 5-10 bed volume, flow velocity is 1ml/min;
3, the 2ml mouse ascites is diluted to 20ml with buffer A, 0.45 μ m membrane filtration, last sample.Flow velocity is 1ml/min;
4, wash 5-10 bed volume again with buffer A, flow velocity is 1ml/min;
5, with buffer B (20mM citrate buffer solution, pH4.0.Preparation: citric acid 2.1g adds water 950ml, transfers to pH 4.0 with 5M NaOH, adds water to 1000ml) wash-out, flow velocity is 1ml/min, collects elution peak;
6, wash 10 column volumes with pure water stream, wash 10 column volumes with 20% ethanol stream again, flow velocity is 2ml/min, and pillar places 4-8 ℃ of environment to preserve;
7, IgG2a and the reference substance with separation and purification carries out the SDS-PAGE electrophoretic analysis simultaneously;
(should be in order to damping fluid C (0.5M acetate buffer solution, pH3.0 after the every use several times of pillar.Preparation: the 0.5M acetum transfers to pH 3.0 with solid NaOH) stream washes 1 time, in order to will adsorb more firm albumen removal.) result:
The SDS-PAGE electrophoretic analysis the results are shown in Figure 4, and swimming lane 1 is standard protein Marker, and swimming lane 2 is ascites, the elution peak of the pillar that swimming lane 3 is adorned for recombinant protein A dextrane gel affinity media of the present invention.In the swimming lane 3, heavy chain and light chain that upper and lower two bands are respectively IgG2a, test-results has shown that recombinant protein A dextrane gel affinity chromatography medium of the present invention can obtain purity greater than 95% IgG2a once going on foot.
The mensuration of embodiment 11 recombinant protein A sepharoses, dextrane gel and the human IgG1's dynamic adsorption of commercially available ProteinA Sepharose 4Fast Flow carrying capacity relatively
1, with buffer A (20mM phosphate buffered saline buffer, pH7.4, i.e. the PBS solution of pH7.4.Preparation: 0.2M NaH
2PO
419ml, 0.2M Na
2HP0
481ml, NaCl 9g adds water to 1000ml.) a balance 5-10 bed volume, flow velocity is 1ml/min;
2, concentration known human IgG1 (obtaining by disclosed embodiment among the Chinese invention patent 01132225.X) goes up sample, and flow velocity is 1ml/min, stops when penetrating to 10%;
3, per sample concentration, go up the dynamic adsorption carrying capacity that sample volume and column volume calculate 10% each gel when penetrating.
Can judge that according to result's (seeing Table 1) the recombinant protein A sepharose that this patent provides and recombinant protein A dextrane gel all are higher than commercially available ProteinA Sepharose 4FastFlow to human IgG1's dynamic adsorption carrying capacity.
Table 1: each gel IgG1 dynamic adsorption carrying capacity relatively
Claims (6)
1. the preparation method of a recombinant protein A affinity chromatography medium is characterized in that, described preparation method may further comprise the steps:
(1) synthesizes the A gene of recombined protein monomer by the method design of chemosynthesis, 5 ' hold Ala-Asp to be mutated into Val-Asp, to form the AccI restriction enzyme site, link to each other with the AccI restriction enzyme site between each A gene of recombined protein monomer, first A gene of recombined protein monomer front end adds the NcoI restriction enzyme site that links to each other with expression vector, 6 His and EK restriction enzyme site, terminal terminator codon TAA and the BamH I restriction enzyme site that links to each other with expression vector pET32a of adding of last A gene of recombined protein monomer;
(2) the A gene of recombined protein monomeric enzyme of step (1) is cut between the corresponding restriction enzyme site that inserts carrier pET32a in the back, again with Acc I endonuclease digestion, connected into the A gene of recombined protein expression vector pET32a-P that comprises 3 polyphone A gene of recombined protein monomers;
(3) with the expression vector pET32a-P transformed into escherichia coli BL21 (DE3) of step (2), screening, enzyme are cut, and obtain bacterial strain BL21-pET32a;
(4) the bacterial strain BL21-pET32a with step (3) carries out cultivation and fermentation, makes its highly effective expressing recombinant protein A, regathers thalline, obtains the recombinant protein A product through separation, purifying;
(5) react in water medium with epoxy chloropropane, sodium hydroxide and agarose or dextrane gel, the recombinant protein A that reaction product and step (4) obtain reacted 15-20 hour under 5-25 ℃ of temperature, react the back cleaning and drained again, obtained the recombinant protein A gel;
The aminoacid sequence of described recombinant protein A is shown in SEQ ID NO.1.
2. preparation method as claimed in claim 1 is characterized in that, described step (5) sepharose is 5% cross-linked agarose gel.
3. as the recombinant protein A affinity chromatography medium of each described preparation method's acquisition among the claim 1-2, wherein the aminoacid sequence of recombinant protein A is shown in SEQ ID NO.1.
4. recombinant protein A affinity chromatography medium as claimed in claim 3, wherein the nucleotide sequence of recombinant protein A is shown in SEQ ID NO.2.
5. as the purposes of the recombinant protein A affinity chromatography medium of each described preparation method's acquisition among the claim 1-2, it is characterized in that described recombinant protein A affinity chromatography medium is used for the antibody separation and purification.
6. as the purposes of each described recombinant protein A affinity chromatography medium among the claim 3-4, it is characterized in that described recombinant protein A affinity chromatography medium is used for the antibody separation and purification.
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| US9546208B2 (en) * | 2014-01-03 | 2017-01-17 | Bio-Rad Laboratories, Inc. | Removal of impurities from protein A eluates |
| CN115947791A (en) * | 2021-05-11 | 2023-04-11 | 博格隆(浙江)生物技术有限公司 | Preparation method of recombinant Protein A Protein and affinity chromatography medium |
| CN116854788B (en) * | 2023-08-31 | 2023-11-07 | 江苏百英生物科技有限公司 | Recombinant protein A and application thereof |
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| WO1995022389A1 (en) * | 1994-02-22 | 1995-08-24 | Smithkline Beecham Corporation | Antibody purification |
| CN1432578A (en) * | 2003-02-26 | 2003-07-30 | 本元正阳基因技术股份有限公司 | Recombinant protein A gene and prepn and application of its expression product |
| CN1524957A (en) * | 2003-02-26 | 2004-09-01 | 本元正阳基因技术股份有限公司 | Recombinant protein a gene ,its expression products and use |
| CN1212884C (en) * | 2001-01-17 | 2005-08-03 | 浙江科锐生物科技有限公司 | Affinity adsorption medium and its preparing medium |
| WO2006024497A1 (en) * | 2004-08-30 | 2006-03-09 | Lonza Biologics Plc. | Affinity- plus ion exchange- chromatography for purifying antibodies |
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| WO1995022389A1 (en) * | 1994-02-22 | 1995-08-24 | Smithkline Beecham Corporation | Antibody purification |
| CN1212884C (en) * | 2001-01-17 | 2005-08-03 | 浙江科锐生物科技有限公司 | Affinity adsorption medium and its preparing medium |
| CN1432578A (en) * | 2003-02-26 | 2003-07-30 | 本元正阳基因技术股份有限公司 | Recombinant protein A gene and prepn and application of its expression product |
| CN1524957A (en) * | 2003-02-26 | 2004-09-01 | 本元正阳基因技术股份有限公司 | Recombinant protein a gene ,its expression products and use |
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