CN1524957A - Recombinant protein a gene ,its expression products and use - Google Patents
Recombinant protein a gene ,its expression products and use Download PDFInfo
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- CN1524957A CN1524957A CNA031499821A CN03149982A CN1524957A CN 1524957 A CN1524957 A CN 1524957A CN A031499821 A CNA031499821 A CN A031499821A CN 03149982 A CN03149982 A CN 03149982A CN 1524957 A CN1524957 A CN 1524957A
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Abstract
The invention belongs to the field of biological engineering, wherein the designed chemical synthesizing combination protein A (rPA) structural element gene group serial is repeatedly inserted into the thermal evoked type bacillus coli, the bacillus coli expression carrier pBV20 containing the genes and transported escherichia coli DH5 alpha (pBVA20) recombination strain, method for preparing the recombination protein A by using the strain are constructed. The strain has the advantages of high expression capacity, low cost, and easy purification of expression product yield.
Description
Technical field the invention belongs to technical field of bioengineering.Be specifically related to a kind of A gene of recombined protein, contain the bacterial strain that the carrier of A gene of recombined protein and this carrier transform and the preparation and the application of A gene of recombined protein expression product.
(Staphylococal Protein A is a kind of from the isolating protein of aureus cell wall SPA) to the background technology SP.As far back as 1940, Vevwey found to contain a kind of material in some streptococcus aureus, can form precipitation with normal human serum in double diffusion test.Jensen (1959) also finds similar phenomenon, and with its called after A antigen.Separated A antigen up to Lofkvist in 1963 etc., and proved that it is a kind of protein, and had any different with sugar, Grov (1960) is called for short SPA or albumin A (Protein A) with its called after SP.Because some immunological characteristics of SPA, it has caused numerous immunologists' interest, and develops into a kind of useful instrument on the immunology.
(Duggleby is cloned and expressed to the gene of coding SPA in nineteen eighty-three in E.coli, 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 26S Proteasome Structure and Function, the SPA molecule comprises A, B, C, D, five homeodomains of E, each structural domain all have can with the autonomous bonded ability of IgG.The SPA gene has 1600bp, and SPA gene order and amino acid sequence coded thereof are seen sequence table SEQ IDNo.3 and SEQ ID No.4.
Because SP has the Fc section bonded ability with most of Mammals IgG, therefore be widely used in the separation and purification and the detection of monoclonal antibody or polyclonal antibody, but the purity of using SP to carry out the antibody of separation and purification has much room for improvement.The main source of SPA is to extract from s. aureus protein, and SPA accounts for 1.7% of bacterial protein.Because streptococcus aureus is a kind of pathogenic bacterium, simultaneously, there is certain difficulty in the separation and purification of its tropina, therefore, obtaining recombinant protein A by gene engineering method becomes a kind of important alternative approach, for the scale operation albumin A provides approach new, safety.
Summary of the invention the invention provides: 1. A gene of recombined protein; 2. make up the carrier, particularly expression vector that contains this gene; 3. the microorganism strains by this gene transformation is provided; 4. the method by these transformed bacteria plant height efficient expressions, production recombinant protein A is provided; 5. the application of expression product.
The A gene of recombined protein of invention is to be connected in series with 3 artificial synthetic A gene of recombined protein monomers, link to each other with Acc I restriction enzyme site between each A gene of recombined protein monomer, wherein first A gene of recombined protein monomer front end adds GAATTCATATGGTG, comprise initiator codon ATG and with expression vector pBV220 (Zhang ZQ, Yao LH, HouYD.Construction and application of a high level expression vector containing P RP L promoter.BingduXuebao, 1990; 6:111-116) continuous EcoR I restriction enzyme site, the terminal GTAGACTAAGGATCC that adds of last A gene of recombined protein monomer is comprising ending codon TAA and the BamH I restriction enzyme site that links to each other with expression vector pBV220.The essential characteristic of this gene is as follows:
A. sequence signature:
Type: nucleic acid
Chain number: two strands
B. molecule type: DNA
C. originate: the A gene of recombined protein monomer of synthetic is connected in series
D. the structure of this gene is connected in series by 3 A gene of recombined protein monomers.This gene is made of 551 Nucleotide,
178 amino acid of encoding.This gene order and amino acid sequence coded thereof are seen sequence table SEQ ID No.1, SEQID 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, again with Acc I endonuclease digestion, connect into and comprise the monomeric A gene of recombined protein expression vector of a plurality of polyphone A gene of recombined protein.
The recombinant expression vector that the above-mentioned coli expression carrier that contains A gene of recombined protein preferentially is built into by synthetic A gene of recombined protein of the present invention and coli expression carrier pBV220, called after pBVA20, its building process is as shown in Figure 1.
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 that bacterial classification is carried out cultivation and fermentation and makes its highly effective expressing recombinant protein A through thermal induction, 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 pBVA20 transformed into escherichia coli DH5 α that contains A gene of recombined protein, called after Escherichia coli DH5 α/pBVA20 is called for short DH5 α/pBVA20.
The concrete grammar that utilizes intestinal bacteria recombinant bacterial strain DH5 α/pBVA20 to produce recombinant protein A is:
1. the preparation of seed liquor: in triangular flask, inoculum size 1-2%V/V is in 30 ℃ of overnight incubation with bacterial classification inoculation; Work as O.D
600Get final product inoculation fermentation when reaching 3-3.5; Above-mentioned used seed culture medium is: the LB substratum adds the 100-200mg/l penbritin;
The fermentation: under aseptic condition with above-mentioned cultured seed substratum by 1: 20-1: 5 are inoculated on the fermention medium, and 30 ℃ ferment to O.D
600When reaching 0.4-0.8, be warming up to 42C and induce, induce 4 ℃ 8000 rev/mins centrifugal 10 minutes receipts bacterium after 3～5 hours;
3. the separation and purification of recombinant protein A:
1) slightly carry:
Above-mentioned collection thalline is suspended with NaCl+ phosphate buffered saline buffer (7.0-8.0), ultrasonication, 4 ℃ 8000 rev/mins centrifugal 10 minutes, collect supernatant, crude extract.
Show that 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 carry the most of recombinant protein A in back and slightly carried that the amount that remains in thalline is atomic;
2) purifying
Crude extract is carried out Sephacryl S200 molecular sieve (Pharmacia Corp's production) purifying, with PBS (Ph 7.4) is that damping fluid is with Sephacryl S200 molecular sieve on the crude extract, collect the recombinant protein A that characteristic peak (second elution peak) is purifying, its purity can reach more than 90%.
The recombinant protein A that the present invention produces is expressed in the Escherichia coli cell pericentral siphon, helps the separation and purification of expression product.Utilize the present invention to express the production recombinant protein A, has expression amount efficient height, expression amount big (accounting for the 60-80% of bacterium total soluble protein), expression time short (only 3-5 hour), be easy to advantages such as purifying, and the experiment proved that it is close that the expressed recombinant protein A of the present invention and wild-type protein A combine activity, and and the bonding force between antibody slightly a little less than.
The present invention adopts the temperature control type escherichia expression system to produce recombinant protein A, also has (1 day) with short production cycle, consumes lowly, can reduce cost effectively, helps 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 is compared with the staphylococcus aureus protein A of wild-type, the avidity of itself and antibodies slightly a little less than, make the elution requirement milder after the antibodies, help keeping the activity of antibody, make the antibody separation and purification respond well, antibody purity can one the step greater than 95%.
Above-mentioned affinity chromatography medium can be prepared by following steps: 1. react in water medium with epoxy chloropropane, sodium hydroxide and respective media, reaction is 2-4 hour under 30-60 ℃ temperature, cleans to neutrality after having reacted and drains; 2. under 4-25 ℃ of temperature, react to react in 16-20 hour afterwards to clean with recombinant protein A of the present invention again and drain again; Obtain above-mentioned affinity chromatography medium after the cleaning.
It is few that above step gained affinity chromatography medium good stability, group come off, and long service life is easy to use, can be from ascites or nutrient solution direct separation and purification antibody.Above-mentioned affinity chromatography medium also can prepare with cyanogen bromide-activated method and additive method.
Description of drawings
Fig. 1 is the synoptic diagram that recombinant expression vector pBVA20 makes up, and wherein, PR, PL are phage promoter, the transcription termination sequence that T1, T2 are.
Fig. 2 is the SDS-PAGE electrophorogram of recombinant protein A expression product.Wherein, M is standard molecular weight contrast, and swimming lane 1 is 42 ℃ and induces the DH5 α/pBV220 thalline that contains the pBV220 empty carrier, and swimming lane 2 is DH5 α/pBVA20 thalline of 42 ℃ of abduction deliverings, and swimming lane 3 is DH5 α/pBVA20 yeast culture supernatants of 42 ℃ of abduction deliverings.
Fig. 3 is the SDS-PAGE electrophorogram of recombinant protein A expression product substep purifying.Wherein, M is the standard molecular weight contrast, swimming lane 1 is DH5 α (pBVA20) thalline of abduction delivering, and swimming lane 2, swimming lane 3 are recombinant protein As that thalline is slightly carried the back preliminary purification, and swimming lane 4 is that thalline is slightly carried after the recombinant protein A behind the molecular sieve Sephacryl S200 purifying.
Fig. 4 is the separation and purification color atlas that recombinant protein A sepharose 6B FF separates the IgG2a in the ascites;
Fig. 5 is the SDS-PAGE electrophoresis result figure that recombinant protein A sepharose 6B FF separates the IgG2a in the ascites, wherein swimming lane 1:mark; Swimming lane 2: ascites; Swimming lane 3:IgG2a;
Fig. 6 is the separation and purification color atlas that the recombinant protein A dextrane gel separates the IgG2a in the ascites;
Fig. 7 is the SDS-PAGE electrophoresis result figure that the recombinant protein A dextrane gel separates the IgG2a in the ascites, wherein swimming lane 1:mark; Swimming lane 2: ascites; Swimming lane 3:IgG2a;
Fig. 8 is the separation and purification color atlas that the recombinant protein A cellulose gel separates the IgG2a in the ascites;
Fig. 9 is the SDS-PAGE electrophoresis result figure that the recombinant protein A cellulose gel separates the IgG2a in the ascites, wherein swimming lane 1:mark; Swimming lane 2: ascites; Swimming lane 3:IgG2a;
Figure 10 is the separation and purification color atlas that the recombinant protein A polyvinyl alcohol gel separates the IgG2a in the ascites;
Figure 11 is the SDS-PAGE electrophoresis result figure that the recombinant protein A polyvinyl alcohol gel separates the IgG2a in the ascites, wherein swimming lane 1:mark; Swimming lane 2: ascites; Swimming lane 3:IgG2a
Embodiment is described in further detail the present invention by the following examples in conjunction with the accompanying drawings, and this does not limit the scope of the invention.
Embodiment 1 A gene of recombined protein is monomeric synthetic:
By the method for chemosynthesis, synthetic monomeric two chains of A gene of recombined protein of design, annealing obtains the A gene of recombined protein sequence monomer.Its sequence comprises that length is 174bp, and two ends are Acc I restriction enzyme sites, is used for the tumor-necrosis factor glycoproteins district that a plurality of A gene of recombined protein monomers make up.In addition, also comprise initiator codon ATG, the second codon GTG, terminator codon TAA, and the clone is total to 29bp with EcoR I and BamH I restriction enzyme joint.Said process is seen Figure of description Fig. 1.
Contain a monomeric pBV220 carrier of A gene of recombined protein and the A gene of recombined protein monomer is cut with the AccI enzyme with above-mentioned, connect after reclaiming respective segments, transformed into escherichia coli, screening has the transformant of amicillin resistance, extract through plasmid, enzyme is cut the pBVA20 carrier that screening obtains to contain A gene of recombined protein.Said process is seen Fig. 1 of Figure of description.
The structure of coli strain DH5 α/pBVA20 of embodiment 4 energy highly effective expressing recombinant protein A:
Use CaCl
2Method is pBVA20 Transformed E .coli DH5 α, screens transformant containing on the LB flat board of penbritin, detects and the restriction analysis acquisition contains the sub-DH5 α/pBVA20 of recombinant conversion of pBVA20 through plasmid.Said process is seen Fig. 1 of Figure of description.
Embodiment 5 utilizes colibacillus engineering E.coli DH5 α/pBVA20 to produce recombinant protein A:
1) culture of strains fermentation
Picking colibacillus engineering E.coli DH5 α/pBVA20 is inoculated in the LB substratum, and inoculum size 1-2%V/V is in 30 ℃ of overnight incubation; Next day under aseptic condition with above-mentioned cultured seed substratum by 1: 20-1: 5 are inoculated on the fermention medium, and 30 ℃ ferment to O.D
600When reaching 0.4-0.8, be warming up to 42 ℃ and induce, induce centrifugal receipts bacterium after 3～5 hours;
The cell that takes a morsel adds 2 * sample-loading buffer, boil after 5 minutes and do the SDS-PAGE gel electrophoresis by standard, result such as Figure of description Fig. 2, in the position of 20kD a new protein band (seeing Fig. 2 swimming lane 1 and swimming lane 3) appears through inductive E.coli DH5 α/pBVA20 thalline and supernatant, (DH5 α/pBV220 is the DH5 α bacterium that has transformed the pBV220 empty carrier to DH5 α/pBV220, see Fig. 2 swimming lane 1) this band does not appear, prove recombinant protein A abduction delivering in E.coli DH5 α/pBVA20.
2) purifying of the recombinant protein A of Biao Daing
Above-mentioned collection thalline is suspended with NaCl+ phosphate buffered saline buffer (pH7.0-8.0), ultrasonication, 4 ℃ are centrifugal, collect supernatant, get crude extract.Show that 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 carry the most of recombinant protein A in back and slightly carried that the amount that remains in thalline is atomic; Crude extract is carried out Sephacryl S200 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 recombinant protein As are used for antibody test, and Elisa detects the recombinant protein A of expression and the experiment of antibodies:
1) bag quilt: 96 orifice plates, every hole bag was by recombinant protein A sample 100 μ l, 37 ℃, 1 hour.
2) sealing: every hole was with the BSA sealing of 100 μ l 1%, 37 ℃, 1 hour.
3) adding one resists: every hole adds about 100 μ g people antibody, 37 ℃, 1 hour.
4) adding two resists: every hole adds about 100 μ l 1:1000 horseradish traget antibodies, 37 ℃, 1 hour.
5) colour developing.
Detect through Elisa and to show that the recombinant protein A that the present invention extracts has the strong activity that combine with people's antibody, to combine activity close with wild-type protein A, and wrap in every hole can be obtained obvious detection signal by the 5ng recombinant protein A.The result shows that recombinant protein A can be used for detection of antibodies.
The preparation of embodiment 7 recombinant protein A sepharose 6B FF:
1. react in water medium with epoxy chloropropane, sodium hydroxide and agarose (6% cross-linked agarose gel), reaction is 2-4 hour under 30-60 ℃ temperature, has reacted back the cleaning to neutrality with distilled water and has drained;
2. under 4-25 ℃ of temperature, react to react in 16-20 hour afterwards to clean with recombinant protein A of the present invention again and drain again; Recombinant protein A sepharose 6B FF has following characteristic:
1. characteristics: group comes off few, and binding specificity is strong
2. matrix: 6% cross-linked agarose gel
3. aglucon: recombinant protein A
4. aglucon density: ≈ 6mg recombinant protein A/ml
5. absorption carrying capacity: 2-5mg mouse IgG2a/ml
6. the granular size of affinity media: 50-160 μ m
7. Peak Flow Rate: 200cm/h
8.pH scope: 3-10, the pH scope can arrive 2-11 during cleaning on the throne
9. use temperature: normal temperature
10. storage temperature :+4～8 ℃
11. preservation liquid: 20% ethanol
If the adsorptive power between antibody purified to be separated and affinity chromatography medium is more weak, then can suitably improve the pH value and the salt concn of adsorption-buffering liquid, can obtain separating effect preferably.
Embodiment 8 recombinant protein A sepharose 6B FF separation and purification IgG2a from mouse ascites:
1, recombinant protein A sepharose 6B FF dress post, 1.6 * 20cm, column volume are 10ml;
2, with 5-10 bed volume of buffer A balance, 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, use the buffer B 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+and 4 ~ 8 ℃ of environment preserve
7, IgG2a and the reference substance with separation and purification carries out the SDS-PAGE electrophoretic analysis simultaneously.
Remarks: should wash 1 time in order to damping fluid C stream after the every use several times of pillar, remove so that will adsorb more firm albumen.
Damping fluid is formed:
Buffer A: 20mM phosphate buffered saline buffer, pH7.4, i.e. the PBS solution of pH7.4.Preparation: 0.2M NaH2PO419ml, 0.2M Na2HPO4 81ml, NaCl9g adds water to 1000ml.
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.
Damping fluid C:0.5M acetate buffer solution, pH3.0.Preparation: the 0.5M acetum transfers to pH 3.0 with solid NaOH.
Damping fluid D:1M Tris-HCl damping fluid, pH9.0.Preparation: Tris salt 121.14g, add water to 950ml, concentrated hydrochloric acid is transferred pH to 9.0, adds water to 1000ml.
The result:
The separation and purification color atlas is seen Fig. 4; The SDS-PAGE electrophoretic analysis the results are shown in Figure 5, and swimming lane 1 is standard protein Marker, and swimming lane 2 is an ascites, the elution peak of the pillar that swimming lane 3 is adorned for recombinant protein A sepharose 6B FF 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 6B FF 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 dextran, reaction is 2-4 hour under 30-55 ℃ temperature, has reacted back the cleaning to neutrality with distilled water and has drained;
2. under 4-25 ℃ of temperature, react to react in 16-20 hour afterwards to clean with recombinant protein A of the present invention again and drain again;
3 distilled water obtain the recombinant protein A dextrane gel after cleaning, and it has the identical characteristic with recombinant protein A sepharose 6B FF.
Embodiment 10 recombinant protein A dextrane geles separation and purification IgG2a from mouse ascites:
With step, the identical damping fluid of application that recombinant protein A sepharose 6B FF is identical among the embodiment 8, with recombinant protein A dextrane gel separation and purification IgG2a from mouse ascites;
The result: the separation and purification color atlas is seen Fig. 6; The SDS-PAGE electrophoretic analysis the results are shown in Figure 7, and swimming lane 1 is standard protein Marker, and swimming lane 2 is an 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 preparation of embodiment 11 recombinant protein As and Mierocrystalline cellulose link coupled affinity chromatography medium recombinant protein A cellulose gel:
1. react in water medium with epoxy chloropropane, sodium hydroxide and Mierocrystalline cellulose, reaction is 2-4 hour under 30-60 ℃ temperature, has reacted back the cleaning to neutrality with distilled water and has drained;
2. under 4-25 ℃ of temperature, react to react in 16-20 hour afterwards to clean with recombinant protein A of the present invention again and drain again;
3. distilled water obtains the recombinant protein A cellulose gel after cleaning, and it has the identical characteristic with recombinant protein A sepharose 6B FF.
Embodiment 12 recombinant protein As and Mierocrystalline cellulose link coupled affinity chromatography medium recombinant protein A cellulose gel separation and purification IgG2a from mouse ascites:
With the step that recombinant protein A sepharose 6B FF is identical among the embodiment 8, identical experiment condition, the identical damping fluid of application, with recombinant protein A cellulose gel separation and purification IgG2a from mouse ascites;
The result: the separation and purification color atlas is seen Fig. 8; The SDS-PAGE electrophoretic analysis the results are shown in Figure 9, and swimming lane 1 is standard protein Marker, and swimming lane 2 is an ascites, the elution peak of the pillar that swimming lane 3 is adorned for recombinant protein A cellulose 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 cellulose gel affinity chromatography medium of the present invention can obtain purity greater than 95% IgG2a once going on foot.
The preparation of embodiment 13 recombinant protein As and hydroxyl carrier link coupled affinity chromatography mediums such as high molecular polymer:
1. react in water medium with epoxy chloropropane, sodium hydroxide and hydroxyl high molecular polymer polyvinyl alcohol, reaction is 2-4 hour under 30-60 ℃ temperature, react afterwards to clean to neutrality with distilled water and has drained;
2. under 4-25 ℃ of temperature, react to react in 16-20 hour afterwards to clean with recombinant protein A of the present invention again and drain again;
3. distilled water obtains the recombinant protein A polyvinyl alcohol gel after cleaning, and it has the identical characteristic with recombinant protein A sepharose 6B FF.
Embodiment 14 recombinant protein As and hydroxyl high molecular polymer link coupled affinity chromatography Jie separation and purification IgG2a from mouse ascites:
With the step that recombinant protein A sepharose 6B FF is identical among the embodiment 8, identical experiment condition, the identical damping fluid of application, with recombinant protein A polyvinyl alcohol gel separation and purification IgG2a from mouse ascites;
The result: the separation and purification color atlas is seen Figure 10; The SDS-PAGE electrophoretic analysis the results are shown in Figure 11, and swimming lane 1 is standard protein Marker, and swimming lane 2 is an ascites, the elution peak of the pillar that swimming lane 3 is adorned for recombinant protein A cellulose 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 cellulose gel affinity chromatography medium of the present invention can obtain purity greater than 95% IgG2a once going on foot.Explanation to the noun in the specification sheets:
1. SP: be called for short SPA or albumin A (ProteinA).
2.pBVA20: for containing the pBV220 of the described target protein A of patent of the present invention.
3.pBV220: a kind of temperature control type coli expression carrier.
4.E.coliDH5 α: coli strain DH5 α.
5.DH5 α/pBV220: for having transformed the Escherichia coli DH5 α bacterium of pBV220; DH5 α is the abbreviation of Escherichia coli DH5 α.
6.BSA: bovine serum albumin.
7. A gene of recombined protein monomer: promptly constitute the gene fundamental unit of A gene of recombined protein, relatively independent structure is arranged.
Sequence table
(1) general information:
Denomination of invention: the preparation of A gene of recombined protein and expression product thereof and application
Sequence number: 4
(2) information of SEQ ID No1:
(I) sequence signature:
(A) type: nucleic acid
(B) length: 551 bases
(C) chain number: two strands
(II) molecule type: DNA
(III) sequence description:
GAATTCATAT?GGTGGTAGAC?AACAAATTCA?ACAAAGAACA?ACAAAACGCG?TTCTATGAGA
TCTTACATTT?ACCTAACTTA?AACGAAGAAC?AACGAAACGC?CTTCATCCAA?AGTTTAAAAG
ATGACCCAAG?CCAAAGCGCT?AACCTTTTAG?CAGAAGCTAA?AAAGCTAAAT?GATGCTCAGG
CGCCGAAAGT?AGACAACAAA?TTCAACAAAG?AACAACAAAA?CGCGTTCTAT?GAGATCTTAC
ATTTACCTAA?CTTAAACGAA?GAACAACGAA?ACGCCTTCAT?CCAAAGTTTA?AAAGATGACC
CAAGCCAAAG?CGCTAACCTT?TTAGCAGAAG?CTAAAAAGCT?AAATGATGCT?CAGGCGCCGA
AAGTAGACAA?CAAATTCAAC?AAAGAACAAC?AAAACGCGTT?CTATGAGATC?TTACATTTAC
CTAACTTAAA?CGAAGAACAA?CGAAACGCCT?TCATCCAAAG?TTTAAAAGAT?GACCCAAGCC
AAAGCGCTAA?CCTTTTAGCA?GAAGCTAAAA?AGCTAAATGA?TGCTCAGGCG?CCGAAAGTAG
ACTAAGGATC?C
(3) information of SEQ ID No2:
(I) sequence signature:
(A) type: amino acid
(B) length: 178 amino acid
(II) molecule type: protein
(III) sequence description:
Met?Val?Val?Asp?Asn?Lys?Phe?Asn?Lys?Glu?Gln?Gln?Asn?Ala?Phe?Tyr?Glu
Ile?Leu?His?Leu?Pro?Asn?Leu?Asn?Glu?Glu?Gln?Arg?Asn?Ala?Phe?Ile?Gln
Ser?Leu?Lys?Asp?Asp?Pro?Ser?Gln?Ser?Ala?Asn?Leu?Leu?Ala?Glu?Ala?Lys
Lys?Leu?Asn?Asp?Ala?Gln?Ala?Pro?Lys?Val?Asp?Asn?Lys?Phe?Asn?Lys?Glu
Gln?Gln?Asn?Ala?Phe?Tyr?Glu?Ile?Leu?His?Leu?Pro?Asn?Leu?Asn?Glu?Glu
Gln?Arg?Asn?Ala?Phe?Ile?Gln?Ser?Leu?Lys?Asp?Asp?Pro?Ser?Gln?Ser?Ala
Asn?Leu?Leu?Ala?Glu?Ala?Lys?Lys?Leu?Asn?Asp?Ala?Gln?Ala?Pro?Lys?Val
Asp?Asn?Lys?Phe?Asn?Lys?Glu?Gln?Gln?Asn?Ala?Phe?Tyr?Glu?Ile?Leu?His
Leu?Pro?Asn?Leu?Asn?Glu?Glu?Gln?Arg?Asn?Ala?Phe?Ile?Gln?Ser?Leu?Lys
Asp?Asp?Pro?Ser?Gln?Ser?Ala?Asn?Leu?Leu?Ala?Glu?Ala?Lys?Lys?Leu?Asn
Asp?Ala?Gln?Ala?Pro?Lys?Val?Asp
(4) information of SEQ ID No3:
(I) sequence signature:
(A) type: nucleic acid
(B) length: 1600 bases
(C) chain number: two strands
(II) molecule type: DNA
(III) sequence description:
tgattttgcg?gttttaagcc?ttttacttcc?tgaataaatc?tttcagcaaa?atatttattt
tataagttgt?aaaacttaca?tttaaattta?attataaata?tagattttag?tattgcaata
cataattcgt?tatattatga?tgactttaca?aatacataca?gggggtatta?atttgaaaaa
gaaaaacatt?tattcaattc?gtaaactagg?tgtaggtatt?gcatctgtaa?ctttaggtac
attacttata?tctggtggcg?taacacctgc?tgcaaatgct?gcgcaacacg?atgaagctca
acaaaatgct?ttttatcaag?tgttaaatat?gcctaactta?aacgctgatc?aacgtaatgg
ttttatccaa?agccttaaag?atgatccaag?ccaaagtgct?aacgttttag?gtgaagctca
aaaacttaat?gactctcaag?ctccaaaagc?tgatgcgcaa?caaaataact?tcaacaaaga
tcaacaaagc?gccttctatg?aaatcttgaa?catgcctaac?ttaaacgaag?cgcaacgtaa
cggcttcatt?caaagtctta?aagacgaccc?aagccaaagc?actaatgttt?taggtgaagc
taaaaaatta?aacgaatctc?aagcaccgaa?agctgataac?aatttcaaca?aagaacaaca
aaatgctttc?tatgaaatct?tgaatatgcc?taacttaaac?gaagaacaac?gcaatggttt
catccaaagc?ttaaaagatg?acccaagcca?aagtgctaac?ctattgtcag?aagctaaaaa
gttaaatgaa?tctcaagcac?cgaaagcgga?taacaaattc?aacaaagaac?aacaaaatgc
tttctatgaa?atcttacatt?tacctaactt?aaacgaagaa?caacgtaacg?gcttcatcca
aagccttaaa?gacgatcctt?cagtgagcaa?agaaatttta?gcagaagcta?aaaagctaaa
cgatgctcaa?gcaccaaaag?aggaagacaa?caaaaaacct?ggtaaagaag?acggcaacaa
acctggcaaa?gaagacggca?acaagcctgg?taaagaagac?aacaaaaaac?ctggtaaaga
agacggcaac?aagcctggta?aagaagacaa?caacaaacct?ggcaaagaag?acggcaacaa
gcctggtaaa?gaagacaaca?acaagcctgg?taaagaagac?ggcaacaagc?ctggtaaaga
agacggcaac?aaacctggta?aagaagacgg?caacggagta?catgtcgtta?aacctggtga
tacagtaaat?gacattgcaa?aagcaaacgg?cactactgct?gacaaaattg?ctgcagataa
caaattagct?gataaaaaca?tgatcaaacc?tggtcaagaa?cttgttgttg?ataagaagca
accagcaaac?catgcagatg?ctaacaaagc?tcaagcatta?ccagaaactg?gtgaagaaaa
tccattcatc?ggtacaactg?tatttggtgg?attatcatta?gccttaggtg?cagcgttatt
agctggacgt?cgtcgcgaac?tataaaaaca?aacaatacac?aacgatagat?atcattttat
ccaaaccaat?tttaacttat?atacgttgat?taacacattc
(5) information of SEQ ID No4:
(I) sequence signature:
(A) type: amino acid
(B) length: 450 amino acid
(II) molecule type: protein
(III) sequence description:
MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVLNMPNLNADQR
NGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEAQR
NGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGF
IQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQS
LKDDPSVSKEILAEAKKLNDAQAPKEEDNKKPGKEDGNKPGKEDGNKPGKEDNKKPGKEDG
NKPGKEDNNKPGKEDGNKPGKEDNNKPGKEDGNKPGKEDGNKPGKEDGNGVHVVKPGDTVN
DIAKANGTTADKIAADNKLADKNMIKPGQELVVDKKQPANHADANKAQALPETGEENPFIG
TTVFGGLSLALGAALLAGRRREL
Claims (10)
1. A gene of recombined protein, it has the described base sequence of SEQ ID No.1 in the sequence table.
2. a recombinant protein A is made up of the described aminoacid sequence of SEQ ID No.2 in the sequence table.
3. recombinant vectors, it contains the described A gene of recombined protein of claim 1.
4. the recombinant vectors pBVA20 that contains the described A gene of recombined protein of claim 1.
5. the conversion microorganism that contains the described recombinant vectors of claim 3, wherein this recombinant vectors contains the described A gene of recombined protein of claim 1.
6. a method for preparing the expression product of A gene of recombined protein is characterized in that, the process for preparing the expression product of described A gene of recombined protein is: make up the expression vector that contains A gene of recombined protein earlier; The expression vector that will contain A gene of recombined protein again changes over to and forms the intestinal bacteria recombinant strain in the bacillus coli DH 5 alpha; Through strain culturing, induce to make its highly effective expressing recombinant protein A, obtain recombinant protein A through separation and purification again.
7. an affinity chromatography medium is characterized by, and it is made up of recombinant protein A and chromatography media carrier.
8. A gene of recombined protein according to claim 1 is characterized in that, the recombinant protein A of described A gene of recombined protein coding is used for antibody test, separation, purifying.
9. recombinant protein A according to claim 2, the avidity of itself and antibodies slightly a little less than, make the elution requirement milder after the antibodies, help keeping the activity of antibody, thereby reach good antibody separation and purification effect.
10. affinity chromatography medium according to claim 7 is characterized by, and described chromatography media carrier can be sepharose or dextran or Mierocrystalline cellulose or silica gel or hydroxyl high molecular polymer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031499821A CN1524957A (en) | 2003-02-26 | 2003-08-01 | Recombinant protein a gene ,its expression products and use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03105360 CN1432578A (en) | 2003-02-26 | 2003-02-26 | Recombinant protein A gene and prepn and application of its expression product |
| CN03105360.2 | 2003-02-26 | ||
| CNA031499821A CN1524957A (en) | 2003-02-26 | 2003-08-01 | Recombinant protein a gene ,its expression products and use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1524957A true CN1524957A (en) | 2004-09-01 |
Family
ID=34314735
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031499821A Pending CN1524957A (en) | 2003-02-26 | 2003-08-01 | Recombinant protein a gene ,its expression products and use |
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| Country | Link |
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| CN (1) | CN1524957A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050464B (en) * | 2006-03-17 | 2011-07-06 | 上海抗体药物国家工程研究中心有限公司 | Method for preparing A gene of recombined protein, and expressed products and application |
| CN101339197B (en) * | 2008-07-30 | 2012-07-04 | 广州康盛生物科技有限公司 | Staphylococal protein A quantitative determination reagent kit and quantitative determination method |
| CN101935665B (en) * | 2006-03-17 | 2013-08-28 | 上海抗体药物国家工程研究中心有限公司 | Preparation method and application of recombinant protein A gene and expression product thereof |
| CN103333911A (en) * | 2013-06-11 | 2013-10-02 | 大连理工大学 | Method for producing protein A by utilizing recombinant pichia pastoris |
| CN101760466B (en) * | 2008-12-24 | 2015-12-09 | 本元正阳基因技术有限公司 | The preparation of A gene of recombined protein and expression product thereof |
| CN109311948A (en) * | 2016-05-11 | 2019-02-05 | 通用电气医疗集团生物工艺研发股份公司 | The method of cleaning and/or disinfection isolation medium |
| CN109721645A (en) * | 2017-12-29 | 2019-05-07 | 兆生生物科技南京有限公司 | A kind of albumin A of gene mutation and its application |
| CN109810957A (en) * | 2019-02-19 | 2019-05-28 | 自然资源部第三海洋研究所 | Recombinate Gluc |
-
2003
- 2003-08-01 CN CNA031499821A patent/CN1524957A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050464B (en) * | 2006-03-17 | 2011-07-06 | 上海抗体药物国家工程研究中心有限公司 | Method for preparing A gene of recombined protein, and expressed products and application |
| CN101935665B (en) * | 2006-03-17 | 2013-08-28 | 上海抗体药物国家工程研究中心有限公司 | Preparation method and application of recombinant protein A gene and expression product thereof |
| CN101339197B (en) * | 2008-07-30 | 2012-07-04 | 广州康盛生物科技有限公司 | Staphylococal protein A quantitative determination reagent kit and quantitative determination method |
| CN101760466B (en) * | 2008-12-24 | 2015-12-09 | 本元正阳基因技术有限公司 | The preparation of A gene of recombined protein and expression product thereof |
| CN103333911A (en) * | 2013-06-11 | 2013-10-02 | 大连理工大学 | Method for producing protein A by utilizing recombinant pichia pastoris |
| CN109311948A (en) * | 2016-05-11 | 2019-02-05 | 通用电气医疗集团生物工艺研发股份公司 | The method of cleaning and/or disinfection isolation medium |
| CN109311948B (en) * | 2016-05-11 | 2022-09-16 | 思拓凡生物工艺研发有限公司 | Method for cleaning and/or disinfecting a separation matrix |
| CN109721645A (en) * | 2017-12-29 | 2019-05-07 | 兆生生物科技南京有限公司 | A kind of albumin A of gene mutation and its application |
| CN109810957A (en) * | 2019-02-19 | 2019-05-28 | 自然资源部第三海洋研究所 | Recombinate Gluc |
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