猪繁殖与呼吸综合征病毒ORF5基因的克隆及杆状病毒表达载体的构建

Cloning of ORF5 Gene of PRRS Virus and Construction of Its Baculovirus Expression Vector

根据猪繁殖与呼吸综合症病毒已知序列(ch-1a,AY032626)设计包含完整ORF5序列的1对引物,采用RT-PCR方法扩增PRRSV甘肃LZH株的ORF5基因,将其克隆到杆状病毒载体pFastBacHTA上,经酶切鉴定、PCR鉴定筛选出阳性重组转移载体pFastBacHTA-ORF5,并对阳性质粒进行测序及序列分析;将pFastBacHTA-ORF5转化到DH10Bac感受态细胞中,与Bacmid发生位点特异性转座作用,获得重组转座子rBacmid-ORF5;并成功构建了PRRSV LZH株ORF5基因的杆状病毒载体,为基因工程疫苗的研制奠定了基础。

According to the published genome sequences of PRRSV, one pair of specific primers were designed to amplify ORF5 gene of LZH strain of GANSU province by RT-PCR. The amplified fragment was cloned into pFastBacHTA donor plasmid. The recombinant pFastBacHTA was identified by digestion with endonuclease enzyme, PCR amplification and sequencing. Then the purified plasmid was transformed into DH10Bac-host cells, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, rBacmid-ORFS. The baculovirus expression vector was successfully contructed, which would be useful for production of gene vaccination in future.