CN101367870A - Bovine rotavirus recombinant VP6 protein antigen and method of preparing the same - Google Patents
Bovine rotavirus recombinant VP6 protein antigen and method of preparing the same Download PDFInfo
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- CN101367870A CN101367870A CNA2008101372773A CN200810137277A CN101367870A CN 101367870 A CN101367870 A CN 101367870A CN A2008101372773 A CNA2008101372773 A CN A2008101372773A CN 200810137277 A CN200810137277 A CN 200810137277A CN 101367870 A CN101367870 A CN 101367870A
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Abstract
The present invention relates to a bovine rotavirus recombinant protein antigen and a preparation method thereof, in particular to a bovine rotavirus recombinant VP6 protein antigen and a preparation method thereof. The bovine rotavirus recombinant VP6 protein antigen resolves the following problems: at present, the method for diagnosing bovine rotavirus diarrhea needs a great deal of time and labor and cannot rapidly diagnose bovine rotavirus diarrhea; the process is complex; and expensive molecular biology reagents and corresponding instruments need to be used. The bovine rotavirus recombinant VP6 protein antigen is expressed by colibacillus converted by recombinant vector plasmids. The preparation method includes the following steps: (1) RCR amplification of VP6 gene; (2) acquisition of recombinant plasmids; (3) acquisition of recombinant bacteria; (4) inducible expression of recombinant VP6 protein antigen; (5) purification. The present invention provides a diagnosis antigen for the serodiagnosis of bovine rotavirus diarrhea, and the diagnosis antigen has extremely strong specificity and accuracy. The diagnosis method, which uses the bovine rotavirus recombinant VP6 protein antigen and adopts fluorescent antibody test and serological tests such as enzyme linked immunosorbent assay, has the advantages of time and labor saving and rapidness and does not need expensive reagents and instruments.
Description
Technical field
The present invention relates to a kind of bovine rota recombinant protein antigen and preparation method thereof.
Background technology
(Bovine Rotavirus BRA) claims calf diarrhea virus (Calf diarrheavirus) again to bovine rota, belongs to Reoviridae (Reoviridae) rotavirus (Rotavirus).Bovine rota mainly infects the calf of 1～7 age in days, can cause the disorder of calf digestive tube, is feature with vomiting, diarrhoea, dehydration and acid base imbalance clinically, and death often takes place infected cattle, and case fatality rate can reach 50%.Since bovine rota have popular wide, sickness rate is high, harm is big, show oneself and develop into a kind of worldwide disease.
The traditional diagnosis method of bovine rota is often according to epidemiology and the clinical manifestation foundation as tentative diagnosis.But because the rotavirus of ox and bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) usually polyinfection, and on epidemiology and symptom, be difficult to difference, therefore must rely on virus separation and serology to test and could make a definite diagnosis at last.
Human Electronic Speculum such as nineteen sixty-eight U.S. Nebraska state Mebus have detected rotavirus from new-born calve diarrhoea ight soil, prove the cause of disease of new-born calve diarrhoea, and called after NCDV strain (Nebraska calfdiarrhea Virus, NCDV).Subsequently, the big sharp industry of Europe, the United States various countries and Australia, New Zealand and Japan etc. have all found the calf diarrhea that rotavirus causes.In addition, people find that gradually the new-born calve of half diarrhoea is relevant with rotavirus infection nearly.
Eqpidemic disease diagnosis fast and accurately is the important prerequisite of control calf rotavirus diarrhea.And China does not also have the effective prevention and treatment method of a cover at present.In clinical diagnosis suspicious pathological material of disease being carried out the virus separation is the most directly diagnostic method of this disease with evaluation, but isolated viral needs certain condition and considerable time, therefore, present viral isolation diagnostic method exists the drawback that time-consuming, effort again can not quick diagnosis.Adopting molecular biological RT-PCR method is the normal at present method of using of diagnosis calf rotavirus diarrhea, has high specificity and highly sensitive advantage; But this method needs viral nucleic acid to extract, transcriptive process,reversed and PCR reaction etc., and process is loaded down with trivial details, needs expensive molecular biology reagent and corresponding plant and instrument etc. simultaneously again.
Summary of the invention
The objective of the invention is does not have effective prevention and treatment method in order to solve at present to the calf rotavirus diarrhea, and adopt viral isolating diagnostic method to exist the drawback that time-consuming, effort again can not quick diagnosis, and adopt molecular biological RT-PCR procedure loaded down with trivial details, need problems such as expensive molecular biology reagent and corresponding plant and instrument again, and a kind of bovine rota reorganization VP6 proteantigen that provides and preparation method thereof.
Bovine rota reorganization VP6 proteantigen of the present invention is expressed by intestinal bacteria Rosetta (DE3) bacterial strain that transfer vector plasmid pProEXHTa transforms; Wherein gene clone has bovine rota NCDV strain VP6 gene among the transfer vector plasmid pProEXHTa.
The above-mentioned bovine rota reorganization of the present invention VP6 proteantigen prepares according to the following steps: one, extract bovine rota NCDV strain virus nucleic acid, carry out the synthetic of viral genome cDNA with the downstream primer P2 of bovine rota NCDV strain VP6 gene then, again the pcr amplification that carries out the VP6 gene with the upstream primer P1 and the downstream primer P2 of bovine rota NCDV strain VP6 gene; Two, the VP6 gene subclone that amplification is obtained to the pMD18-T carrier, again through BamHI and SalI double digestion rear clone in prokaryotic expression carrier plasmid pProEXHTa, obtain recombinant plasmid pPro-VP6; Three, with recombinant plasmid pPro-VP6 transformed into escherichia coli Rosetta (DE3), bacterium pPro-VP6/Rosetta (DE3) obtains recombinating; Four, the abduction delivering of reorganization VP6 proteantigen; Five, purifying promptly obtains bovine rota reorganization VP6 proteantigen.
The present invention the experiment proved that for serological method diagnosis calf rotavirus diarrhea provides diagnostic antigen bovine rota reorganization VP6 proteantigen of the present invention has extremely strong specificity and accuracy, can be discerned by antiserum(antisera).Therefore, use serological tests such as bovine rota reorganization VP6 proteantigen of the present invention, employing fluorescent antibody test and enzyme linked immunological absorption just can overcome the problem that virus is separated and the RT-PCR method exists, have that diagnostic method saves time, laborsaving, advantage fast, and do not need expensive molecular biology reagent and corresponding plant and instrument.
The present invention carries out bovine rota reorganization VP6 albumen with intestinal bacteria as transmitting antigenic carrier, having designed with coding bovine rota primary structure albumen VP6 protein gene segment is target gene, make up the proteic reorganization prokaryotic expression system of expression bovine rota VP6, overcome antigen toxin expelling, diffusing malicious potentially dangerous.
Bovine rota reorganization VP6 proteantigen simultaneously of the present invention has been established basic substance for control calf rotavirus diarrhea.
The present invention expresses bovine rota VP6 albumen recombination bacillus coli pPro-VP6/Rosetta (DE3) and belongs to Escherichia (Escherichia), be preserved in Chinese typical culture collection center (CCTCC), the preservation address is a Wuhan University, preservation date is on 09 02nd, 2008, and preserving number is CCTCC M208124.
Description of drawings
Fig. 1 is PCR qualification result figure in the embodiment eight; Fig. 2 is that the albumen of step 4 abduction delivering in the embodiment eight carries out SDS-PAGE qualification result figure; Fig. 3 is embodiment eight product Western-blot qualification result figure.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment bovine rota reorganization VP6 proteantigen is expressed by intestinal bacteria Rosetta (DE3) bacterial strain that transfer vector plasmid pProEXHTa transforms; Wherein gene clone has bovine rota NCDV strain VP6 gene among the transfer vector plasmid pProEXHTa.
Present embodiment bovine rota reorganization VP6 proteantigen protein molecular quality is about 50ku, and can excite stronger immunne response.
Embodiment two: the difference of present embodiment and embodiment one is: the gene order of the opening code-reading frame that bovine rota reorganization VP6 proteantigen is complete is shown in SEQ ID NO:1.Other is identical with embodiment one.
Embodiment three: present embodiment bovine rota reorganization VP6 proteantigen prepares according to the following steps: one, extract bovine rota NCDV strain virus nucleic acid, carry out the synthetic of viral genome cDNA with the downstream primer P2 of bovine rota NCDV strain VP6 gene then, again the pcr amplification that carries out the VP6 gene with the upstream primer P1 and the downstream primer P2 of bovine rota NCDV strain VP6 gene; Two, the VP6 gene subclone that amplification is obtained to the pMD18-T carrier, again through BamHI and SalI double digestion rear clone in prokaryotic expression carrier plasmid pProEXHTa, obtain recombinant plasmid pPro-VP6; Three, with recombinant plasmid pPro-VP6 transformed into escherichia coli Rosetta (DE3), bacterium pPro-VP6/Rosetta (DE3) obtains recombinating; Four, the abduction delivering of reorganization VP6 proteantigen; Five, purifying promptly obtains bovine rota reorganization VP6 proteantigen.
The medicine that uses in the present embodiment, reagent, enzyme, competent cell and plasmid etc. are all buied easily, if no particular requirement then concentration be that product marks concentration.Operation steps in the present embodiment is used operational manual referring to test kit.
Embodiment four: the difference of present embodiment and embodiment three is: the gene order of the upstream and downstream primer of bovine rota NCDV strain VP6 gene is in the step 1:
Upstream primer P1:5 '-GGATCCTTGGCTTTTAAACGAAGTCTT-3 ',
Downstream primer P2:5 '-GTCGACAATACCTAGACGCATCCTT-3 '.Other step and parameter are identical with embodiment three.
Embodiment five: the difference of present embodiment and embodiment three is: the amplification PCR reaction system of VP6 gene is 25 μ L in the step 1: water 14.5 μ L, cDNA2.5 μ L, 10 * Buffer2.5 μ L, dNTP 4.0 μ L, 10OD primer upstream 1.0 μ L, 10OD primer downstream 1.0 μ L and rTaq enzyme 0.25 μ L; The PCR reaction conditions: 95 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 48 ℃ of annealing 30s, 72 ℃ of extension 50s, 30 circulations, 72 ℃ are extended 5min.Other step and parameter are identical with embodiment three.
Embodiment six: the difference of present embodiment and embodiment three is: the abduction delivering of step 4 reorganization VP6 proteantigen carries out according to the following steps: a, the bacterium pPro-VP6/Rosetta (DE3) that will recombinate are inoculated in the LB liquid nutrient medium that penbritin concentration is 100 μ g/mL, place 37 ℃ of environment vibration shaking table overnight incubation; B, 10mL incubated overnight liquid is inoculated in the LB liquid nutrient medium that 500mL penbritin concentration is 100 μ g/mL, places 37 ℃, vibration shaking table environment to be cultured to OD
590Value reaches 0.5; C, to add the isopropyl-that concentration is 1mol/L (IPTG) final concentration of isopropyl-to the LB liquid nutrient medium again be 0.6mmol/L, places 37 ℃, vibration shaking table environment to continue to be cultured to OD then
590Value reaches 1.0.Other step and parameter are identical with embodiment three.
Embodiment seven: the difference of present embodiment and embodiment three is: the step 5 purifying carries out according to the following steps: a, with Ni
2+-NTA gel media adds chromatography column, washes post with deionized water then, washes post with Buffer A solution again; Nutrient solution behind b, the step 4 abduction delivering centrifugal 20min under the condition of 10000g/min, abandoning supernatant, the precipitation thalline stirs 1h with the dissolving of Buffer A solution, room temperature environment, continues to stir under 4 ℃ of conditions again to spend the night; C, under 4 ℃, the condition of 20000g/min centrifugal 40min, get supernatant liquor and join in the Ni-NTA chromatography column with the flow velocity of 0.5mL/min, with the flow velocity washing chromatography column of Buffer B, collect elutriant with 0.5mL/min, promptly finish purifying.Other step and parameter are identical with embodiment three.
Present embodiment Buffer A solution can the cracking thalline, dissolution precipitation.
Embodiment eight: present embodiment bovine rota reorganization VP6 proteantigen prepares according to the following steps: one, extract bovine rota NCDV strain virus nucleic acid, carry out the synthetic of viral genome cDNA with the downstream primer P2 of bovine rota NCDV strain VP6 gene then, again the pcr amplification that carries out the VP6 gene with the upstream primer P1 and the downstream primer P2 of bovine rota NCDV strain VP6 gene; Two, the VP6 gene subclone that amplification is obtained to the pMD18-T carrier, again through BamHI and SalI double digestion rear clone in prokaryotic expression carrier plasmid pProEXHTa, obtain recombinant plasmid pPro-VP6; Three, with recombinant plasmid pPro-VP6 transformed into escherichia coli Rosetta (DE3), bacterium pPro-VP6/Rosetta (DE3) obtains recombinating; Four, the abduction delivering of reorganization VP6 proteantigen; Five, purifying promptly obtains bovine rota reorganization VP6 proteantigen; Wherein the gene order of the upstream and downstream primer of bovine rota NCDV strain VP6 gene is in the step 1:
Upstream primer P1:5 '-GGATCCTTGGCTTTTAAACGAAGTCTT-3 ',
Downstream primer P2:5 '-GTCGACAATACCTAGACGCATCCTT-3 ';
The amplification PCR reaction system of VP6 gene is 25 μ L in the step 1: water 14.5 μ L, cDNA2.5 μ L, 10 * Buffer, 2.5 μ L, dNTP 4.0 μ L, 10OD primer upstream 1.0 μ L, 10OD primer downstream 1.0 μ L and rTaq enzyme 0.25 μ L; The PCR reaction conditions: 95 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 48 ℃ of annealing 30s, 72 ℃ of extension 50s, 30 circulations, 72 ℃ are extended 5min;
The abduction delivering of step 4 reorganization VP6 proteantigen carries out according to the following steps: a, the bacterium pPro-VP6/Rosetta (DE3) that will recombinate are inoculated in the LB liquid nutrient medium that penbritin concentration is 100 μ g/mL, place 37 ℃ of environment vibration shaking table overnight incubation; B, 10mL incubated overnight liquid is inoculated in the LB liquid nutrient medium that 500mL penbritin concentration is 100 μ g/mL, places 37 ℃, vibration shaking table environment to be cultured to OD
590Value reaches 0.5; C, to add the isopropyl-that concentration is 1mol/L (IPTG) final concentration of isopropyl-to the LB liquid nutrient medium again be 0.6mmol/L, places 37 ℃, vibration shaking table environment to continue to be cultured to OD then
590Value reaches 1.0;
The step 5 purifying carries out according to the following steps: a, with Ni
2+-NTA gel media adds chromatography column, washes post with deionized water then, washes post with Buffer A solution again; Nutrient solution behind b, the step 4 abduction delivering centrifugal 20min under the condition of 10000g/min, abandoning supernatant, the precipitation thalline stirs 1h with the dissolving of Buffer A solution, room temperature environment, continues to stir under 4 ℃ of conditions again to spend the night; C, under 4 ℃, the condition of 20000g/min centrifugal 40min, get supernatant liquor and join in the Ni-NTA chromatography column with the flow velocity of 0.5mL/min, with the flow velocity washing chromatography column of Buffer B, collect elutriant with 0.5mL/min, promptly finish purifying.
The medicine that uses in the present embodiment, reagent, enzyme, competent cell and plasmid etc. are all buied easily, if no particular requirement then concentration be that product marks concentration.Operation steps in the present embodiment is used operational manual referring to test kit.
Be connected with plasmid pProEXHTa fragment through the VP6 gene fragment (about 1.3kb) of BamHI and SalI double digestion in the present embodiment step 2 through BamHI and SalI double digestion; Express by step 3 transformed into escherichia coli Rosetta (DE3) then.Carry out positive recombinant plasmid single endonuclease digestion, double digestion and PCR and identify that qualification result as shown in Figure 1; " 1 " swimming lane standard specimen is the VP6 gene fragment (the about 1300bp of size) that pcr amplification obtains among Fig. 1, " 2 " swimming lane standard specimen is the gene fragment (the about 3900bp of size) that plasmid pProEXHTa obtains through the BamHI single endonuclease digestion, " 3 " swimming lane standard specimen is the gene fragment (size respectively about 2600bp and 1300bp) of plasmid pProEXHTa through BamHI and the acquisition of SalI double digestion, " 4 " and " 8 " swimming lane standard specimen is Marker, " 5 " swimming lane standard specimen is for being template with recombinant plasmid pPro-VP6, P1 and P2 are gene fragment (the about 1300bp of size)---the present embodiment target gene fragment that the primer PCR amplification obtains, " 6 " swimming lane standard specimen is the gene fragment (the about 6100bp of size) that recombinant plasmid pPro-VP6 obtains through the BamHI single endonuclease digestion, and " 7 " swimming lane standard specimen is the gene fragment (size respectively about 4800bp and 1300bp) of recombinant plasmid pPro-VP6 through BamHI and the acquisition of SalI double digestion.Qualification result explanation VP6 gene has been inserted in the expression vector plasmid, successfully obtains positive reorganization bacterium pPro-VP6/Rosetta (DE3) bacterial strain.
Albumen to present embodiment step 4 abduction delivering carries out the SDS-PAGE evaluation, and qualification result as shown in Figure 2; " 1 " swimming lane standard specimen is standard protein molecular weight (97kD-14kD) among Fig. 2, " 2 " swimming lane standard specimen is without IPTG inductive pPro-VP6/Rosetta (DE3) tropina, pPro-VP6/Rosetta (DE3) tropina of " 3 " swimming lane standard specimen for inducing 1h through IPTG, pPro-VP6/Rosetta (DE3) tropina of " 4 " swimming lane standard specimen for inducing 3h through IPTG, pPro-VP6/Rosetta (DE3) tropina of " 5 " swimming lane standard specimen for inducing 5h through IPTG, the about 51kD of protein band shown in the arrow among Fig. 2.
With the transfer printing NC film behind the SDS-PAGE electrophoresis of the product behind the present embodiment step 5 purifying, more respectively with the anti-NCDV hyper-immune serum in mouse source and sheep anti-mouse igg/HRP effect, Western-blot identifies that contrast and experiment is as shown in Figure 3; " 1 " swimming lane standard specimen is without IPTG inductive pPro-VP6/Rosetta (DE3) tropina among Fig. 3, " 2 " swimming lane standard specimen is that " 3 " swimming lane standard specimen contains the tropina of plasmid pProEXHTa for the IPTG inductive through IPTG inductive pPro-VP6/Rosetta (DE3) tropina; Experimental result is the reaction zone that expection does not all appear in " 1 " and " 3 " swimming lane, " 2 " swimming lane tangible reaction zone occurs in desired location (about molecular weight 50KD), experimental result illustration purpose albumen has obtained effective expression, expressed proteins can be discerned by antiserum(antisera), and the recombinant antigen that present embodiment obtains has good antigen reactivity.
Adopt viral isolation diagnostic and molecular biology RT-PCR multiple diagnostic method to verify, the result proves that the accuracy rate of diagnosis of the calf rotavirus diarrhea that bovine rota reorganization VP6 proteantigen that present embodiment obtains causes bovine rota is up to more than 90%.
Sequence table
＜110〉Northeast Agricultural University
＜120〉bovine rota reorganization VP6 proteantigen and preparation method thereof
<160>3
<210>1
<211>1269
<212>DNA
＜213〉bovine rota (bovine rotavirus, BRV)
<400>1
<210>2
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the upstream primer P1 of bovine rota NCDV strain VP6 gene.
<400>2
<210>3
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the downstream primer P2 of bovine rota NCDV strain VP6 gene.
<400>3
Claims (7)
1. bovine rota reorganization VP6 proteantigen is characterized in that intestinal bacteria Rosetta (DE3) the bacterial strain expression that bovine rota reorganization VP6 proteantigen is transformed by transfer vector plasmid pProEXHTa; Wherein gene clone has bovine rota NCDV strain VP6 gene among the transfer vector plasmid pProEXHTa.
2. bovine rota reorganization VP6 proteantigen according to claim 1 is characterized in that the gene order of the opening code-reading frame that bovine rota reorganization VP6 proteantigen is complete is as follows:
ATGGATGTCCTGTACTCCTTGTCAAAAACTCTTAAAGATGCTAGAGACAAAATTGTCGAAGGCACATTATACTCCAATGTAAGTGATCTAATTCAACAATTTAATCAAATGATAATTACTATGAATGGAAATGAGTTCCAAACTGGAGGAATTGGTAATCTACCGATTAGAAATTGGAATTTTGATTTTGGATTACTTGGAACAACTCTACTAAATTTAGATGCTAACTACGTCGAAACGGCCCGCAATACAATTGATTATTTTGTAGATTTTGTAGATAATGTATGTATGGACGAAATGGTTAGAGAATCACAAAGAAATGGAATTGCACCACAATCAGATTCACTTAGAAAGTTATCAGGCATTAAATTTAAAAGAATAAATTTTGACAATTCATCAGAATACATAGAGAACTGGAATTTGCAAAATAGAAGACAAAGaACGGGTTTTACATTTCATAAACCAAACATTTTcCCTTATTCAGCTTCATTCACGTTGAACAGATCACAACCGGCTCATGATAACTTGATGGGTACGATGTGGCTCAATGCGGGATCAGAAATTCAGGTCGCTGGATTCGACTACTCATGTGCAATAAACGCGCCAGCTAATACGCAACAATTTGAGCATATTGTACAGCTTCGAAGGGTGTTGACTACAGCTACAATAACTCTTTTACCAGATGCAGAAAGATTTAGTTTTCCAAGAGTGATTAATTCAGCTGACGGAGCGACTACATGGTACTTCAATCCAGTGATTCTTAGACCAAATAACGTTGAAATAGAGTTTCTACTAAACGGGCAGATAATAAATACTTACCAAGCAaGATTTGGAACGATCATAGCTAGAAATTTTGATACAATTAGATTGTCATTTCAGTTGATGAGACCACCAAATATGACACCAGCGGTAGCGGCGTTATTTCCAAATGCGCAGCCATTTGAACATCACGCAACAGTAGGACTCACGCTTAGAATTGAATCTGCAGTTTGTGAATCAGTACTTGCCGACGCAAGCGAAACAATGCTAGCAAATGTGACATCTGTTAGACAAGAATACGCGATACCAGTTGGACCAGTTTTTCCACCAGGTATGAATTGGACTGATTTGATCACTAACTATTCACCATCTAGAGAGGATAACTTGCAGCGTGTATTTACAGTGGCTTCCATTAGAAGCATGCTTGTCAAATGAGGACCAAGCTAACCACTTGGTATCCGACTTTGGTGAGTATGTAGCTACGTCAAGCTGTTTGAACTCTGTAAGTAA。
3. the preparation method of bovine rota reorganization VP6 proteantigen according to claim 1, it is characterized in that bovine rota reorganization VP6 proteantigen prepares according to the following steps: one, extract bovine rota NCDV strain virus nucleic acid, carry out the synthetic of viral genome cDNA with the downstream primer P2 of bovine rota NCDV strain VP6 gene then, again the pcr amplification that carries out the VP6 gene with the upstream primer P1 and the downstream primer P2 of bovine rota NCDV strain VP6 gene; Two, the VP6 gene subclone that amplification is obtained to the pMD18-T carrier, again through BamHI and SalI double digestion rear clone in prokaryotic expression carrier plasmid pProEXHTa, obtain recombinant plasmid pPro-VP6; Three, with recombinant plasmid pPro-VP6 transformed into escherichia coli Rosetta (DE3), bacterium pPro-VP6/Rosetta (DE3) obtains recombinating; Four, the abduction delivering of reorganization VP6 proteantigen; Five, purifying promptly obtains bovine rota reorganization VP6 proteantigen.
4. the preparation method of bovine rota reorganization VP6 proteantigen according to claim 3 is characterized in that the gene order of the upstream and downstream primer of bovine rota NCDV strain VP6 gene in the step 1 is:
Upstream primer P1:5 '-GGATCCTTGGCTTTTAAACGAAGTCTT-3 ',
Downstream primer P2:5 '-GTCGACAATACCTAGACGCATCCTT-3 '.
5. the preparation method of bovine rota reorganization VP6 proteantigen according to claim 3, the amplification PCR reaction system that it is characterized in that VP6 gene in the step 1 is 25 μ L: water 14.5 μ L, cDNA2.5 μ L, 10 * Buffer, 2.5 μ L, dNTP 4.0 μ L, 10OD primer upstream 1.0 μ L, 10OD primer downstream 1.0 μ L and rTaq enzyme 0.25 μ L; The PCR reaction conditions: 95 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 48 ℃ of annealing 30s, 72 ℃ of extension 50s, 30 circulations, 72 ℃ are extended 5min.
6. the preparation method of bovine rota reorganization VP6 proteantigen according to claim 3, the abduction delivering that it is characterized in that step 4 reorganization VP6 proteantigen carries out according to the following steps: a, the bacterium pPro-VP6/Rosetta (DE3) that will recombinate are inoculated in the LB liquid nutrient medium that penbritin concentration is 100 μ g/mL, place 37 ℃ of environment vibration shaking table overnight incubation; B, 10mL incubated overnight liquid is inoculated in the LB liquid nutrient medium that 500mL penbritin concentration is 100 μ g/mL, places 37 ℃, vibration shaking table environment to be cultured to OD
590Value reaches 0.5; C, to add concentration again be that isopropyl-final concentration of isopropyl-to the LB liquid nutrient medium of 1mol/L is 0.6mmol/L, places 37 ℃, vibration shaking table environment to continue to be cultured to OD then
590Value reaches 1.0.
7. the preparation method of bovine rota according to claim 3 reorganization VP6 proteantigen is characterized in that the step 5 purifying carries out according to the following steps: a, with Ni
2+-NTA gel media adds chromatography column, washes post with deionized water then, washes post with Buffer A solution again; Nutrient solution behind b, the step 4 abduction delivering centrifugal 20min under the condition of 10000g/min, abandoning supernatant, the precipitation thalline stirs 1h with the dissolving of Buffer A solution, room temperature environment, continues to stir under 4 ℃ of conditions again to spend the night; C, under 4 ℃, the condition of 20000g/min centrifugal 40min, get supernatant liquor and join in the Ni-NTA chromatography column with the flow velocity of 0.5mL/min, with the flow velocity washing chromatography column of Buffer B, collect elutriant with 0.5mL/min, promptly finish purifying.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101863966A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军第三军医大学 | Rotavirus-resistant drug acting target, building method and application method thereof |
| JP2012125220A (en) * | 2010-12-17 | 2012-07-05 | Univ Of Miyazaki | Method for prompt and simultaneous detection of group a bovine rotavirus and bovine coronavirus |
| CN108148814A (en) * | 2017-12-15 | 2018-06-12 | 东北农业大学 | A kind of double-antibody sandwich elisa diagnostic kit and its application for being used to detect bovine rota |
| CN108169492A (en) * | 2017-12-15 | 2018-06-15 | 东北农业大学 | It is a kind of to be used to detect colloidal gold immuno-chromatography test paper strip of bovine rota and its preparation method and application |
| CN108690126A (en) * | 2018-06-07 | 2018-10-23 | 西南民族大学 | A kind of yak source rotavirus recombination VP6 proteantigens and application |
| CN110903403A (en) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | Bovine rotavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibody |
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2008
- 2008-10-09 CN CNA2008101372773A patent/CN101367870A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101863966A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军第三军医大学 | Rotavirus-resistant drug acting target, building method and application method thereof |
| JP2012125220A (en) * | 2010-12-17 | 2012-07-05 | Univ Of Miyazaki | Method for prompt and simultaneous detection of group a bovine rotavirus and bovine coronavirus |
| CN108148814A (en) * | 2017-12-15 | 2018-06-12 | 东北农业大学 | A kind of double-antibody sandwich elisa diagnostic kit and its application for being used to detect bovine rota |
| CN108169492A (en) * | 2017-12-15 | 2018-06-15 | 东北农业大学 | It is a kind of to be used to detect colloidal gold immuno-chromatography test paper strip of bovine rota and its preparation method and application |
| CN108690126A (en) * | 2018-06-07 | 2018-10-23 | 西南民族大学 | A kind of yak source rotavirus recombination VP6 proteantigens and application |
| CN110903403A (en) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | Bovine rotavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibody |
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