BVDV自体蛋白酶P20和核心蛋白P14基因的克隆及原核表达
摘要
牛病毒性腹泻-粘膜病病毒（Bovine viral diarrhea-mucosal disease virus，BVDV）是黄病毒科、瘟病毒属（Flaviviridae Pestivirus）的代表种，主要引起牛的病毒性腹泻-粘膜病（BVD-MD），表现为腹泻，急、慢性粘膜病，持续性感染与免疫耐受，免疫抑制，母畜流产、死胎和畸胎等。该病呈世界范围分布，严重危害养牛业的发展。
BVDV P20和P14基因位于基因组的5’端，分别编码病毒的非结构蛋白——自体蛋白酶蛋白和结构蛋白核心蛋白，核心蛋白P14具有抗原性，而自体蛋白酶P20的抗原性尚未见报道。为了辅助基因工程亚单位疫苗的应用，本研究克隆BVDV P20和P14基因，并将其在原核系统中表达，旨在为基因工程诊断抗原的研究奠定基础，同时为P20蛋白抗原性的阐述作一必要铺垫。
根据GenBank中BVDV Oregon C24V株基因组序列设计二对引物，应用RT-巢式PCR（RT-nested PCR）方法体外扩增BVDV Osloss-Like株P20基因，将其克隆至pMD18-T载体，序列测定及同源性分析表明，BVDV Osloss-Like株P20基因与BVDV VEDEVAC（AJ585412）、Osloss（M96687）、Oregon C24V（AF091605）、SD1（M96751）及NADL（AJ133738）株核苷酸同源性分别为99.21%、93.65%、80.95%、79.96%、78.97%。根据这一结果，参考GenBank上发表的BVDV Osloss株基因组序列设计一对引物，扩增P14基因，并进行序列测定及同源性分析，结果表明BVDV Osloss-Like株P14基因与BVDV VEDEVAC（AJ585412）、Osloss（M96687）、SD1（M96751）、Oregon C24V（AF091605）及NADL（AJ133738）株核苷酸同源性分别为98.37%、94.77%、82.53%、79.41%、78.76%。系统发生分析结果表明BVDV Osolss-Like株P20和P14基因与VEDEVAC株亲源关系较近。
利用DNA重组技术，将BVDV P20和P14基因各分别亚克隆至原核表达载体pGEX-6P-1和pPROEX-HTb，构建P20和P14基因重组原核表达载体pGEX-6P-P20、pPROEX-HTb-P20、pGEX-6P-P14及pPROEX-HTb-P14。将上述重组质粒转化至工程菌，在IPTG诱导下重组质粒pGEX-6P-P20和pPROEX-HTb-P20得到高效表达，重组质粒pGEX-6P-P14表达量低，而重组质粒pPROEX-HTb-P14未见有明显表达。因此分别对重组质粒pGEX-6P-P14及pPROEX-HTb-P14的表达条件进行探索，根据结果推测BVDV Osloss-Like株P14基因表达的蛋白可能是一种毒性蛋白，其对大肠杆菌BL21（DE3）pLysS和DH5α具有毒性。
Bovine viral diarrhea-mucosal disease virus (BVDV) represented the prototype member of the pestivirus genus, family Flaviviridae, mainly caused bovine viral diarrhea-mucosal disease. Bovine infected by BVDV showed diarrhea, acute and chronic mucosal diaease, persistent infection and immunotolerance, immunosupression, pregnant cow abortion, dead fetus and abnormal fetus. BVDV was a worldwide pathogen in cattle which had not been controlled by classical vaccination，and had seriously endangered the cattle herds.
BVDV P20 and P14 gene located in the 5’ end of the genome, encoded nonstructural protein of autoprotease and structural protein of core protein respectively. Core protein had antigenicity, while the antigenicity of autoprotease protein had not been reported. In order to meet the need of the application of genetically engineered subunit vaccine, BVDV P20 and P14 gene were cloned and expressed in prokaryotic expression system, whose aim was to lay a foundation for genetically engineered diagnostic antigen and to make a necessary preparation for elucidating the antigenicity of protein P20.
Two sets of primers were disigned, according to the genomic sequence of BVDV Oregon C24V(AF091605) strain published in GenBank, for amplifying P20 gene of BVDV Osloss-Like strain in vitro by the method of RT-nested PCR. The product of PCR of P20 gene was cloned into pMD18-T vector for sequencing. Homologous analysis showed that the homology of nucleotide sequence of P20 gene between Osloss-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), Oregon C24V(AF091605), SD1(M96751), and NADL(AJ133738) strain was 99.21%, 93.65%, 80.95%, 79.96% and 78.97% respectively. On the base of the result of homology analysis of P20 gene, another set of primer was designed according to the gneomic sequence of BVDV Osloss strain published in GenBank, P14 gene was amplified specially with this set of primer. Sequencing and homologous analysis showed that the homology of nucleotide sequence of P14 gene between Osloss-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), SD1(M96751), Oregon C24V(AF091605), NADL(AJ133738) strain was 98.37%, 94.77%, 82.53%, 79.41% and 78.76% respectively. Phylogenetic analysis showed that the biogenesis of P20 and P14 gene between BVDV Osloss-Like strain and VEDEVAC strain was closer than between Osloss-Like and other strains.
By recombinant DNA techniques, both P20 and P14 gene of BVDV Osloss-Like strain
were subcloned into prokaryotic expression vector pGEX-6P-1 and pPROEX-HTb respectively to construct recombinant expression vector of pGEX-6P-P20, pPROEX-HTb-P20, pGEX-6P-P14 and pPROEX-HTb-P14. Then the recombinant expression plasmid above all were transformed into engineering bacteria of E.coli respectively, recombinant expression plasmid of pGEX-6P-P20 and pPROEX-HTb-P20 highly expressed after induced with IPTG, while the expression amount of recombinant expression plasmid of pGEX-6P-P14 was very low after induced with IPTG, and the expression of pPROEX-HTb-P14 was hardly detected. In order to achieve the expression of P14 gene in E.coli, differrnt expression conditions were performed on recombinant expression plasmid of pGEX-6P-P14 and pPROEX-HTb-P14. It was assumed that the protein expressed by P14 gene of BVDV Osloss-Like strain may be a kind of toxic protein and was toxic to E.coli of BL21(DE3)pLysS and DH5α.
Postgraduate: Li Huixin
Specialty: Preventive Veterinary Science
Supervisor: Prof.Wang Junwei
BVDV P20和P14基因位于基因组的5’端，分别编码病毒的非结构蛋白——自体蛋白酶蛋白和结构蛋白核心蛋白，核心蛋白P14具有抗原性，而自体蛋白酶P20的抗原性尚未见报道。为了辅助基因工程亚单位疫苗的应用，本研究克隆BVDV P20和P14基因，并将其在原核系统中表达，旨在为基因工程诊断抗原的研究奠定基础，同时为P20蛋白抗原性的阐述作一必要铺垫。
根据GenBank中BVDV Oregon C24V株基因组序列设计二对引物，应用RT-巢式PCR（RT-nested PCR）方法体外扩增BVDV Osloss-Like株P20基因，将其克隆至pMD18-T载体，序列测定及同源性分析表明，BVDV Osloss-Like株P20基因与BVDV VEDEVAC（AJ585412）、Osloss（M96687）、Oregon C24V（AF091605）、SD1（M96751）及NADL（AJ133738）株核苷酸同源性分别为99.21%、93.65%、80.95%、79.96%、78.97%。根据这一结果，参考GenBank上发表的BVDV Osloss株基因组序列设计一对引物，扩增P14基因，并进行序列测定及同源性分析，结果表明BVDV Osloss-Like株P14基因与BVDV VEDEVAC（AJ585412）、Osloss（M96687）、SD1（M96751）、Oregon C24V（AF091605）及NADL（AJ133738）株核苷酸同源性分别为98.37%、94.77%、82.53%、79.41%、78.76%。系统发生分析结果表明BVDV Osolss-Like株P20和P14基因与VEDEVAC株亲源关系较近。
利用DNA重组技术，将BVDV P20和P14基因各分别亚克隆至原核表达载体pGEX-6P-1和pPROEX-HTb，构建P20和P14基因重组原核表达载体pGEX-6P-P20、pPROEX-HTb-P20、pGEX-6P-P14及pPROEX-HTb-P14。将上述重组质粒转化至工程菌，在IPTG诱导下重组质粒pGEX-6P-P20和pPROEX-HTb-P20得到高效表达，重组质粒pGEX-6P-P14表达量低，而重组质粒pPROEX-HTb-P14未见有明显表达。因此分别对重组质粒pGEX-6P-P14及pPROEX-HTb-P14的表达条件进行探索，根据结果推测BVDV Osloss-Like株P14基因表达的蛋白可能是一种毒性蛋白，其对大肠杆菌BL21（DE3）pLysS和DH5α具有毒性。
Bovine viral diarrhea-mucosal disease virus (BVDV) represented the prototype member of the pestivirus genus, family Flaviviridae, mainly caused bovine viral diarrhea-mucosal disease. Bovine infected by BVDV showed diarrhea, acute and chronic mucosal diaease, persistent infection and immunotolerance, immunosupression, pregnant cow abortion, dead fetus and abnormal fetus. BVDV was a worldwide pathogen in cattle which had not been controlled by classical vaccination，and had seriously endangered the cattle herds.
BVDV P20 and P14 gene located in the 5’ end of the genome, encoded nonstructural protein of autoprotease and structural protein of core protein respectively. Core protein had antigenicity, while the antigenicity of autoprotease protein had not been reported. In order to meet the need of the application of genetically engineered subunit vaccine, BVDV P20 and P14 gene were cloned and expressed in prokaryotic expression system, whose aim was to lay a foundation for genetically engineered diagnostic antigen and to make a necessary preparation for elucidating the antigenicity of protein P20.
Two sets of primers were disigned, according to the genomic sequence of BVDV Oregon C24V(AF091605) strain published in GenBank, for amplifying P20 gene of BVDV Osloss-Like strain in vitro by the method of RT-nested PCR. The product of PCR of P20 gene was cloned into pMD18-T vector for sequencing. Homologous analysis showed that the homology of nucleotide sequence of P20 gene between Osloss-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), Oregon C24V(AF091605), SD1(M96751), and NADL(AJ133738) strain was 99.21%, 93.65%, 80.95%, 79.96% and 78.97% respectively. On the base of the result of homology analysis of P20 gene, another set of primer was designed according to the gneomic sequence of BVDV Osloss strain published in GenBank, P14 gene was amplified specially with this set of primer. Sequencing and homologous analysis showed that the homology of nucleotide sequence of P14 gene between Osloss-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), SD1(M96751), Oregon C24V(AF091605), NADL(AJ133738) strain was 98.37%, 94.77%, 82.53%, 79.41% and 78.76% respectively. Phylogenetic analysis showed that the biogenesis of P20 and P14 gene between BVDV Osloss-Like strain and VEDEVAC strain was closer than between Osloss-Like and other strains.
By recombinant DNA techniques, both P20 and P14 gene of BVDV Osloss-Like strain
were subcloned into prokaryotic expression vector pGEX-6P-1 and pPROEX-HTb respectively to construct recombinant expression vector of pGEX-6P-P20, pPROEX-HTb-P20, pGEX-6P-P14 and pPROEX-HTb-P14. Then the recombinant expression plasmid above all were transformed into engineering bacteria of E.coli respectively, recombinant expression plasmid of pGEX-6P-P20 and pPROEX-HTb-P20 highly expressed after induced with IPTG, while the expression amount of recombinant expression plasmid of pGEX-6P-P14 was very low after induced with IPTG, and the expression of pPROEX-HTb-P14 was hardly detected. In order to achieve the expression of P14 gene in E.coli, differrnt expression conditions were performed on recombinant expression plasmid of pGEX-6P-P14 and pPROEX-HTb-P14. It was assumed that the protein expressed by P14 gene of BVDV Osloss-Like strain may be a kind of toxic protein and was toxic to E.coli of BL21(DE3)pLysS and DH5α.
Postgraduate: Li Huixin
Specialty: Preventive Veterinary Science
Supervisor: Prof.Wang Junwei
引文
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