CN101423833A - Method for preparing inhibin genetic engineering regulation antigen - Google Patents
Method for preparing inhibin genetic engineering regulation antigen Download PDFInfo
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- CN101423833A CN101423833A CNA2008102184771A CN200810218477A CN101423833A CN 101423833 A CN101423833 A CN 101423833A CN A2008102184771 A CNA2008102184771 A CN A2008102184771A CN 200810218477 A CN200810218477 A CN 200810218477A CN 101423833 A CN101423833 A CN 101423833A
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Abstract
The invention discloses a method for preparing an inhibin genetic engineering regulation antigen, which comprises the following steps: cloning an inhibin encoding gene; making use of the inhibin encoding gene to prepare an inhibin recombinant fusion protein; and making use of the inhibin recombinant fusion protein to prepare the inhibin genetic engineering regulation antigen. The method for preparing the inhibin genetic engineering regulation antigen has the advantages of simplicity, high purity, good immunogenicity, low price and the like; the inhibin genetic engineering regulation antigen prepared by the method does not have toxicity, pathogenicity and drug residue, is harmless to human and animals, has no adverse effect on consumers, can improve the ovulation rate and the litter size, improve utilization ratio of a superovulation embryo, promote the sexual maturation so as to improve the reproduction and the like.
Description
Technical field
The invention belongs to animal production traits regulation and control substance technical field, particularly a kind of preparation method of inhibin genetic engineering regulation antigen.
Background technology
Present livestock industry is in intensification and industrialized development process, because the application in genetic breeding technology, feed compounding technique, epidemic prevention and control technology, environmental health technology and modern enterprise technique of management and administration is greatly improved the production efficiency of livestock industry.But in each production link, the reproductive performance of animal does not but improve in the past decades.In domestic animal except that pig, animals such as the ox of the overwhelming majority and sheep are only arranged one piece of ovum and can only produce a son when oestrusing, when producing a meat animals, must support a kind dam simultaneously, cause the production cost of beef raising and mutton sheep industry high, the raising of production efficiency and economic benefit is subjected to extremely restriction.At present, how to improve litter size is the target that herding production and herding institute are pursued with obtaining the more good embryo always.
Litter size is few and to obtain utilizing the embryo in the embryo transfer process be because ovarian follicle excretory Gonadostatin less, suppresses hypophysis secretion FSH, and little ovarian follicle can not normal growth, and ovary can only be discharged an ovum under field conditions (factors), and single tire appears in cattle and sheep.And when super row also because heavy dose of injection FSH causes the ovum utilization ratio of discharging very low.
The method that is used at present improve litter size and obtain the more good embryo can be passed through two kinds of methods: use medicine and internal secretion immunoregulation.Using conventional medicine is domestic animal injection FSH or the similar medicine of FSH, and the antagonism statin plays a role, and according to present result: the embryo's utilization ratio that obtains in super row's process is very low, not have the potentiality of excavation.Then can obtain more can utilize the embryo by the internal secretion immunoregulation, in the follicular development process of animal, follicular cell is reactionless to FSH before not expressing fsh receptor, and its division growth and functional development are subjected to the promotion of another paracrine factor activin (Activin) of ovarian follicle excretory.Activin has the effect of the FSH of promotion excretory, also is the hormone of statin antagonism simultaneously.When immunosuppressant promotes hypophysis secretion FSH, also will remove the antagonistic action of statin, thereby promote little follicular development to make its surface have fsh receptor and become the ovarian follicle that FSH is had reaction follicular cell surface active element acceptor.Therefore re-using FSH on the basis of immunosuppressant carries out superovulation, and the not developable ovarian follicle of these scripts also can continue to grow, and last mature ovulation, handles more embryo in dam breeding back generation than common super row.
Summary of the invention
The objective of the invention is to overcome the shortcoming that exists in the prior art, provide that a kind of cost is low, the preparation method of the simple inhibin genetic engineering regulation antigen of technology, described inhibin genetic engineering regulation antigen is used to improve animal number of eggs ovulated, litter size and embryo production.
Purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method of inhibin genetic engineering regulation antigen is provided, comprises the steps:
(1) clone's statin encoding gene;
(2) utilize the statin encoding gene, the recombination fusion protein of preparation statin;
(3) utilize this statin recombination fusion protein to prepare the genetic engineering regulation antigen of statin.
The described clone's statin of step (1) encoding gene may further comprise the steps:
(a) according to the pig statin subunit gene sequence of delivering among the Genbank (Genbank number: NM_214189), design specificity upstream primer I1 and downstream primer I2;
(b) take guanidinium isothiocyanate-chloroform-Virahol method from the pig ovary tissue of the blue pool, to extract total geneome RNA, obtain cDNA by reverse transcription;
Desirable blue pool pig ovary tissue, place homogenate in the 1ml guanidinium isothiocyanate phenol solution (Trizol liquid is purchased the company in Invitrogen), remove protein with chloroform then, take out supernatant liquor after centrifugal 15 minutes, in supernatant liquor, add the Virahol total geneome RNA of centrifugation again.The concentration that total RNA is made into 1g/L is dissolved in the water that contains DEPC, and (RT) obtains cDNA by reverse transcription.
(c) with primer I 1, I2, dNTP and the synthetic cDNA of reverse transcription as template, under the catalysis of archaeal dna polymerase by the PCR reaction amplification specific band that to obtain a length be 1095bp, i.e. the code cDNA fragment of pig statin.
Described upstream primer I1 of step (a) and downstream primer I2 are:
I1：5’-ATGTGGCCTCAGCTGCTCCTCTTGC-3’；
I2：5’-TTAGATGCAGGCACAGTGCTGGGTG-3’。
The recombination fusion protein of the described preparation statin of step (2) is meant and utilizes IPTG to induce intestinal bacteria E.coli BL21 (DE3) strain to express the preparation recombination fusion protein; May further comprise the steps:
(d) according to the cDNA base sequence of statin mature peptide and the multiple clone site base sequence of colibacillus expression plasmid pRSET A, synthetic upstream primer Mf of design and downstream primer Mr;
(e) utilize the coded cDNA sequence of the statin that described primer Mf, Mr and above-mentioned post transcription cloning obtain to be template, amplify the cDNA cloned sequence of statin mature peptide by the PCR reaction;
(f) behind the cDNA cloned sequence of described statin mature peptide process Bgl II and the EcoR I double digestion, be cloned between the Bgl II and EcoRI two restriction enzyme sites of expression plasmid pRSET A, be built into recombinant expression plasmid pInhibin SCAU;
(g) described recombinant expression plasmid pInhibin SCAU is transformed into intestinal bacteria E.coli BL21 (DE3) strain, the IPTG that adds 0.05mmol/L～0.2mmol/L in nutrient solution induces, and obtains the statin recombination fusion protein that recombinant bacteria is expressed.
Described upstream primer Mf of step (d) and downstream primer Mr are:
Mf:5 '-AAT
TCTCCACCGCCCCTCTGCCC-3 ', wherein square frame part A GATCT is a Bgl II restriction enzyme site, setting-out part TC is a base of keeping reading frame;
Mr:5 '-ATC
AGATGCAGGCACAGTGCTGGG-3 ', wherein square frame part GAATTC is an EcoR I restriction enzyme site.
The described induction time of step (g) is 4～6 hours.
Step (2) also comprises the purification step of described statin recombination fusion protein.
Described purifying is the intestinal bacteria E.coli BL21 (DE3) with the plain recombination fusion protein of 6～8Mol urea cracking expression inhibiting; Adopt Ni-NTA gel-purified resin that the solution that contains the statin recombination fusion protein is carried out chromatography; The wash-out purifying plain recombination fusion protein that is inhibited.
The described genetic engineering regulation antigen that utilizes the statin recombination fusion protein to prepare statin of step (3) may further comprise the steps:
(h) be that the statin recombination fusion protein of 2～6mg/mL is dissolved in the aqueous solution that contains 2～4% tween-80s and makes water with concentration;
(i) No. 10 lightweight white oils and Si Ben 80 are mixed, add aluminum stearate again, mix and make oil phase;
(j) water and oil phase are mixed, and stir and make water in oil homogeneous emulsion, promptly get inhibin genetic engineering regulation antigen.
The volume ratio of described No. 10 lightweight white oils of step (i) and Si Ben 80 is 94%:6%, and the concentration of described aluminum stearate is 2% (w:v); The volume ratio of described water of step (j) and oil phase is 4～5:5～6.
The present invention adopts the approach of " active immunity ", clones the gene of statin with engineered method, and makes genetic engineering regulation antigen with the statin recombination fusion protein of expression in escherichia coli.Before handling, superovulation carries out active immunity, the 0th day this recombinant protein of first immunisation, the 28th day booster immunization for the first time, the 56th day booster immunization for the second time.Each intramuscular injection.Just can utilize the antibody of the immunity system long-term production anti-" statin " of animal self, this antibody can in and statin promote hypophysis excretory FSH, remove the biological action that statin suppresses hypophysis secretion FSH, thereby improve ovulation rate, obtain more can utilizing the embryo.
The present invention compared with prior art has following advantage and effect:
Advantages such as (1) inhibin genetic engineering regulation antigen preparation method of the present invention is simple, purity is high, the good price of immunogenicity is cheap.
(2) the inhibin genetic engineering regulation antigen nontoxicity of the present invention's preparation, no pathogenicity, drug residue free to the person poultry harmless, has no adverse effects to the human consumer.
(3) inhibin genetic engineering regulation antigen for preparing of the inventive method can improve animal ovulation rate, litter size, raising superovulation embryo utilization ratio, promote sexual maturity to improve breeding etc.
Description of drawings
The structure schema of the colibacillus expression plasmid of Fig. 1 statin reorganization fusion gene
The electrophoretogram of the inhibin gene coding region behind Fig. 2 post transcription cloning
Fig. 3 pig statin recombinant expression plasmid carries out the PCR evaluation and enzyme is cut the electrophoretogram of evaluation
The expression of Fig. 4 pig statin recombination fusion protein
The immunoblotting of Fig. 5 pig statin recombination fusion protein is identified
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is done further detailed description, but embodiments of the present invention are not limited thereto.
Present embodiment adopts and clones the genetic engineering regulation antigen that pig statin code cDNA prepares statin from blue pool pig ovary tissue.Need to prove, up to the present the mature peptide amino acid residue sequence of the statin of the different animals of being found is very similar, therefore except adopting the pig inhibin gene, can also adopt the inhibin gene of other animal to prepare the genetic engineering regulation antigen of statin.
Preparation process specifically may further comprise the steps:
(1) clone pig statin encoding gene;
(2) utilize pig statin encoding gene, the recombination fusion protein of preparation pig statin;
(3) the expressed pig statin recombination fusion protein of purifying;
(4) pig statin recombination fusion protein behind the purifying and mineral oil adjuvant are mixed and made into the pig inhibin genetic engineering regulation antigen.
The concrete operations step is as follows:
(1) clone of pig statin encoding gene cDNA:
A, according to the pig inhibin gene sequence of being delivered among the GenBank (Genbank number: NM_214189), design the specificity upstream primer I1 and the downstream primer I2 of this gene.Upstream primer I1 (5 '-ATGTGGCCTCAGCTGCTCCTCTTGC-3 ') corresponding to the 1st～25 Nucleotide in pig inhibin gene coding region; Downstream primer I2 (5 '-TTAGATGCAGGCACAGTGCTGGGTG-3 ') corresponding to the 1071st～1095 Nucleotide in pig inhibin gene coding region.Described pig inhibin gene total length 1095bp contains complete statin α subunit gene coding region sequence.
B, get blue pool pig ovary tissue, place 1ml guanidinium isothiocyanate phenol solution (Trizol liquid, purchase company in Invitrogen) in homogenate, remove protein with chloroform then, take out supernatant liquor after centrifugal 15 minutes, add the Virahol total geneome RNA of centrifugation again in supernatant liquor, the concentration that total RNA is made into 1g/L is dissolved in the water that contains DEPC, and (RT) obtains cDNA by reverse transcription.
C, usefulness primer I 1, I2, dNTP and the synthetic cDNA of reverse transcription are as template, under the catalysis of archaeal dna polymerase by the PCR reaction amplification specific band that to obtain a length be 1095bp, be the code cDNA fragment of pig statin, the electrophoretogram of the inhibin gene coding region behind the post transcription cloning as shown in Figure 2.
D, pig statin code cDNA fragment that amplification is obtained and cloned plasmids PMD18-T carrier (purchase give birth in Shanghai worker company) are connected, then the recombinant plasmid transformed that structure is obtained go into that bacillus coli DH 5 strain (purchasing the company in Invitrogen) is duplicated, preservation forever and carry out nucleotide sequencing.SEQ ID NO:1 is the nucleotide sequence of the pig statin cDNA that obtains with this method post transcription cloning in the sequence table, and wherein capitalization part is distinguished corresponding I1 and I2 primer; This consecutive nucleotides is the coded cDNA sequence of pig statin mature peptide, therefore the pig inhibin gene nucleotide sequence of this amplification and the homology of other domestic animal can prepare inhibin genetic engineering regulation antigen with pig statin code cDNA fully more than 90%.
(2) recombination fusion protein of preparation pig statin: utilize the cDNA base sequence of pig statin mature peptide, can carry out the expression of pig statin recombination fusion protein by prokaryotic expression system E.coli BL21 (DE3) strain (purchasing company) in Invitrogen:
A, according to the nucleotide sequence of the resulting pig statin cDNA of order-checking, by the cDNA sequence of its mature peptide, synthetic upstream primer Mf of design and downstream primer Mr, wherein,
Mf(5’-AAT
TCTCCACCGCCCCTCTGCCC-3’)
Mf is corresponding to the 691st～708 nucleotide sequence of the coding region of sequence pig inhibin gene shown in the SEQ ID NO:1, and square frame partly contains Bgl II restriction enzyme site, and setting-out partly is a base of keeping reading frame;
Mr5’-ATC
AGATGCAGGCACAGTGCTGGG-3)
Mr is corresponding to the 1073rd～1093 nucleotide sequence of the coding region of sequence pig inhibin gene shown in the SEQ ID NO:1, and square frame partly contains EcoR I restriction enzyme site.
B, utilize primer Mf, Mr, once more amplifying length through the PCR reaction as template and dNTP with pig statin coding region cDNA fragment under the catalysis of Pfu archaeal dna polymerase is the cDNA cloned sequence that 423bp, two ends have the pig statin mature peptide of Bgl II and EcoR I restriction enzyme site, is used to insert expression plasmid pRSET A (purchasing the company in Invitrogen).
Accompanying drawing 1 has been explained the structure flow process of the colibacillus expression plasmid that contains pig statin reorganization fusion gene.Wherein 1 represents pig statin code cDNA, 2 expression primer Mf, Mr amplifies pig statin mature peptide cDNA cloned sequence, 3 expression Bgl II+EcoR I double digestions, 4 expression T4 ligase enzymes connect, 5 expression promotors (T7promoter), the mark (His tag) of 6 continuous Histidines of 6 expressions, 7 expression polyclone restriction enzyme sites (multiple cloing site), 8 expression f1 transcribe and open initial point (f1 origin), 9 expression ampicillin resistance genes (Ampicillin), 10 transcribing of expression plasmid pUC are opened beginning site (pUC ori), 11 expression pRSET A (2897bp), 12 expression Bgl II+EcoRI double digestions, 13 expression plasmid pRSET A Bgl II+EcoR I double digestions, 14 expression Bgl II restriction enzyme sites, 15 expression EcoR I restriction enzyme sites, 16 expression plasmid pInhibinSCAU.Utilize primer Mf and Mr the amplification obtain pig statin mature peptide cDNA cloned sequence, with this fragment through Bgl II and EcoR I double digestion; To have the polyclone restriction enzyme site, can load and use BglII and EcoR I double digestion equally at expression in escherichia coli expression of exogenous gene plasmid pRSET A; Connect pig statin mature peptide cDNA cloned sequence and expression plasmid pRSET A carrier with the T4 ligase enzyme then, be built into expression plasmid pInhibin SCAU.Pig statin reorganization fusion gene pInhibin SCAU carried out PCR identifies and enzyme is cut evaluation, all from plasmid, obtained the purpose fragment, pig statin recombinant expression plasmid carries out PCR identifies, enzyme is cut evaluation electrophoretogram as shown in Figure 3, wherein in the swimming lane 1 and 2 between 250 and 500bp between band be statin α subunit mature peptide cDNA fragment.To the pInhibin SCAU evaluation of checking order, the result shows and wherein contains pig statin mature peptide gene, and reading frame is correct, and gene order is shown in SEQ ID NO:2.The 1st～121 site Nucleotide is from the leader sequence of plasmid pRSET A in the sequence shown in the SEQID NO:2, and the 13rd～30 site Nucleotide is from 6 Histidine sequence labels that are used for purifying of plasmid pRSET A; The 116th～141 corresponding with primer Mf, and the 505th～532 corresponding with primer Mr; The 116th～121 site " AGATCT " is a Bgl II restriction enzyme site, (the 122nd～123 site) two base TC are artificial bases that are used to keep reading frame of inserting after the Bgl II restriction enzyme site, the 527th～532 site " GAATTC " is the EcoRI restriction enzyme site, before the EcoR I restriction enzyme site is terminator codon TGA (the 526th～528 site), part between Bgl II and the terminator codon is a statin mature peptide cDNA sequence (the 124th～525 site), and the part before the mature peptide cDNA sequence is the nucleotide sequence that comes from plasmid.
C, with this recombinant expression plasmid pInhibin SCAU and Transformed E coli.BL21 (DE3) strain, the IPTG that adds 0.05mmol/L～0.2mmol/L in nutrient solution induces and expressed pig statin recombination fusion protein in 4～6 hours, expression is expressed statin α subunit recombination fusion protein near the band of 20kD in the swimming lane 2 wherein as shown in Figure 4.This pig statin recombination fusion protein can be by the anti-ox α-Yi Zhisu of rabbit antibody specific recognition, and the immunoblotting qualification result of pig statin recombination fusion protein is seen accompanying drawing 5.
(3) purifying of pig statin recombination fusion protein:
Utilize the aminoterminal of the pig statin recombination fusion protein of escherichia coli expression to have the mark (His-tag) of 6 continuous Histidines, special affine the combination can take place with nickel ion in this His-Tag mark.Therefore, adopt the cracking of 8M urea soln to express the bacterium of pig statin recombination fusion protein, with lysate process Ni-NTA resin gel chromatography, can be purified into wherein pig statin recombination fusion protein and with recombinant protein, and this albumen also can be by the anti-ox of rabbit-statin antibody specific recognition, qualification result is seen accompanying drawing 5, and wherein the protein band in the swimming lane 1 is purified statin α subunit recombination fusion protein.To contain this proteic solution flush away urea wherein of dialysing one by one then.
(4) prepare the pig inhibin genetic engineering regulation antigen with pig statin recombination fusion protein: the pig statin recombination fusion protein after above-mentioned expression, purifying and the dialysis is dissolved in the aqueous solution that contains 2～4% Tween 80 by the concentration of 2～6mg/mL makes water; Again with No. 10 lightweight white oils and Si Ben 80 by 94% and 6% mixed, add 2% aluminum stearate again and mix and make oil phase; Water and oil phase are pressed the mixed of 4～5:5～6, and under stirrer, make water in oil homogeneous emulsion, i.e. pig inhibin genetic engineering regulation antigen.
Test case 1: to pig reorganization statin Expression of Fusion Protein and evaluation
With plasmid pInhibin SCAU and Transformed E coli.BL21 (DE3) strain, OD is cultivated in 37 ℃ of joltings in LB liquid nutrient medium (containing penbritin 100 μ g/mL)
600After reaching more than 0.6, adding IPTG concentration is after 0.01mol/L cultivates 1h, to give expression to the reorganization pig statin that conforms to theoretical molecular 20kD, and high expression level amount accounts for tropina amount about 32%, and expression as shown in Figure 4.Expressed proteins can be deviate from a purifying band with the 50%Ni-NTA resin wash under the sex change condition, and molecular weight conforms to expection.(western-blot), a specific reaction band has appearred in the result, and qualification result is seen accompanying drawing 5 through immunoblotting assay to utilize the anti-ox α-Yi Zhisu of rabbit antibody.These presentation of results should contain the homologous amino acid sequence of pig statin mature peptide in the recombinant protein of expressing.
Test case 2: immune effect test
The pig inhibin genetic engineering regulation antigen immune cattle of the present invention preparation is to the influence of superovulation: to the milk cow in normal oestrus cycle is arranged, active immunity before superovulation, the 0th day this recombinant protein of first immunisation 1ml/ head, concentration is 1mg/ml, the 28th day booster immunization 1ml/ head for the first time, concentration is 0.5mg/ml, the 56th day booster immunization 1ml/ head for the second time, and concentration is 0.5mg/ml.Each intramuscular injection.Employings in 15 days successive subtraction method on the four that booster immunization begins in the second time carries out superovulation to be handled, and immune group obtains to utilize 12.33 of embryo numbers as a result, is higher than 8.33 of control group, increase rate 32%.
These test-results explanations, the immunosuppressant genetic engineering regulation antigen can improve milk cow available embryo's number in embryo transfer.
SEQUENCE?LISTING
＜110〉Agricultural University Of South China
＜120〉a kind of preparation method of inhibin genetic engineering regulation antigen
<130>
<160>2
<170>PatentIn?version?3.2
<210>1
<211>1095
<212>DNA
＜213〉artificial sequence
<400>1
<210>2
<211>532
<212>DNA
＜213〉artificial sequence
<400>2
Claims (10)
1, a kind of preparation method of inhibin genetic engineering regulation antigen is characterized in that comprising the steps:
(1) clone's statin encoding gene;
(2) utilize the statin encoding gene, the recombination fusion protein of preparation statin;
(3) utilize this statin recombination fusion protein to prepare the genetic engineering regulation antigen of statin.
2, the preparation method of inhibin genetic engineering regulation antigen according to claim 1 is characterized in that the described clone's statin of step (1) encoding gene may further comprise the steps:
(a) according to existing inhibin gene sequence, design specificity upstream primer I1 and downstream primer I2;
(b) take guanidinium isothiocyanate-chloroform-Virahol method from the pig ovary tissue of the blue pool, to extract total geneome RNA, obtain cDNA by reverse transcription;
(c) synthesize cDNA as template with primer I 1, I2, dNTP and reverse transcription, under the catalysis of archaeal dna polymerase, react the code cDNA fragment of amplification pig statin by PCR.
3, the preparation method of inhibin genetic engineering regulation antigen according to claim 2 is characterized in that described upstream primer I1 of step (a) and downstream primer I2 are:
I1：5’-ATGTGGCCTCAGCTGCTCCTCTTGC-3’；
I2：5’-TTAGATGCAGGCACAGTGCTGGGTG-3’。
4, the preparation method of inhibin genetic engineering regulation antigen according to claim 1 is characterized in that the recombination fusion protein of the described preparation statin of step (2) is meant that utilizing IPTG to induce e. coli strains to express prepares recombination fusion protein; May further comprise the steps:
(d) according to the cDNA base sequence of statin mature peptide and the multiple clone site base sequence of colibacillus expression plasmid pRSET A, synthetic upstream primer Mf of design and downstream primer Mr;
(e) utilize the coded cDNA sequence of the statin that described primer Mf, Mr and above-mentioned post transcription cloning obtain to be template, amplify the cDNA cloned sequence of statin mature peptide by the PCR reaction;
(f) behind the cDNA cloned sequence of described statin mature peptide process Bgl II and the EcoR I double digestion, be cloned between the Bgl II and EcoR I two restriction enzyme sites of expression plasmid pRSET A, be built into recombinant expression plasmid pInhibin SCAU;
(g) described recombinant expression plasmid pInhibin SCAU is transformed into e. coli strains, the IPTG that adds 0.05mmol/L～0.2mmol/L in nutrient solution induces, and obtains the statin recombination fusion protein that recombinant bacteria is expressed.
5, the preparation method of inhibin genetic engineering regulation antigen according to claim 4 is characterized in that described upstream primer Mf of step (d) and downstream primer Mr are:
Mf:5 '-AATAGATCTTCTCCACCGCCCCTCTGCCC-3 ', wherein square frame partly is a Bgl II restriction enzyme site, setting-out partly is a base of keeping reading frame;
Mr:5 '-ATCGAATTCAGATGCAGGCACAGTGCTGGG-3 ', wherein square frame partly is an EcoR I restriction enzyme site.
6, the preparation method of inhibin genetic engineering regulation antigen according to claim 4 is characterized in that the described induction time of step (g) is 4～6 hours.
7, the preparation method of inhibin genetic engineering regulation antigen according to claim 4 is characterized in that also comprising the purification step of described statin recombination fusion protein.
8, the preparation method of inhibin genetic engineering regulation antigen according to claim 7 is characterized in that described purifying is the intestinal bacteria with the plain recombination fusion protein of 6～8Mol urea cracking expression inhibiting; Adopt Ni-NTA gel-purified resin that the solution that contains the statin recombination fusion protein is carried out chromatography; The wash-out purifying plain recombination fusion protein that is inhibited.
9, the preparation method of inhibin genetic engineering regulation antigen according to claim 1 is characterized in that the described genetic engineering regulation antigen that utilizes the statin recombination fusion protein to prepare statin of step (3) may further comprise the steps:
(h) be that the statin recombination fusion protein of 2～6mg/mL is dissolved in the aqueous solution that contains 2～4% tween-80s and makes water with concentration;
(i) No. 10 lightweight white oils and Si Ben 80 are mixed, add aluminum stearate again, mix and make oil phase;
(j) water and oil phase are mixed, and stir and make water in oil homogeneous emulsion, promptly get inhibin genetic engineering regulation antigen.
10, the preparation method of inhibin genetic engineering regulation antigen according to claim 9 is characterized in that the volume ratio of described No. 10 lightweight white oils of step (i) and Si Ben 80 is 94%:6%, and the concentration of described aluminum stearate is 2%; The volume ratio of described water of step (j) and oil phase is 4～5:5～6.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102166348A (en) * | 2010-12-31 | 2011-08-31 | 华南农业大学 | Application of inhibin recombinant fusion protein to preparing medicines for promoting oestrus and hybridization of sows |
| CN103041381A (en) * | 2013-01-09 | 2013-04-17 | 江苏省农业科学院 | Recombinant inhibin vaccine and preparation method and application of recombinant inhibin vaccine |
| CN104012466A (en) * | 2014-06-10 | 2014-09-03 | 江苏省农业科学院 | Method for promoting oestrus of cattle and increasing hybridization conception rate of cattle |
| WO2023109803A1 (en) * | 2021-12-15 | 2023-06-22 | 宁波三生生物科技股份有限公司 | Antibody against inhibin and use thereof |
| CN116375844A (en) * | 2023-02-22 | 2023-07-04 | 四川农业大学 | Inhibin antigen peptide, vaccine and preparation method thereof |
-
2008
- 2008-10-21 CN CNA2008102184771A patent/CN101423833A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102166348A (en) * | 2010-12-31 | 2011-08-31 | 华南农业大学 | Application of inhibin recombinant fusion protein to preparing medicines for promoting oestrus and hybridization of sows |
| CN102166348B (en) * | 2010-12-31 | 2013-01-09 | 华南农业大学 | Application of inhibin recombinant fusion protein to preparing medicines for promoting oestrus and hybridization of sows |
| CN103041381A (en) * | 2013-01-09 | 2013-04-17 | 江苏省农业科学院 | Recombinant inhibin vaccine and preparation method and application of recombinant inhibin vaccine |
| CN104012466A (en) * | 2014-06-10 | 2014-09-03 | 江苏省农业科学院 | Method for promoting oestrus of cattle and increasing hybridization conception rate of cattle |
| CN104012466B (en) * | 2014-06-10 | 2016-01-20 | 江苏省农业科学院 | Promote that ox oestruses and improves the method for ox conception rate |
| WO2023109803A1 (en) * | 2021-12-15 | 2023-06-22 | 宁波三生生物科技股份有限公司 | Antibody against inhibin and use thereof |
| CN116375844A (en) * | 2023-02-22 | 2023-07-04 | 四川农业大学 | Inhibin antigen peptide, vaccine and preparation method thereof |
| CN116375844B (en) * | 2023-02-22 | 2024-03-29 | 四川农业大学 | Inhibin antigen peptide, vaccine and preparation method thereof |
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