贵州地区羊口疮病毒B2L基因克隆及原核表达载体构建

摘要

用H2L基因特异性引物对羊口疮临床疑似病料进行鉴定,采用PCR扩增出阳性样品羊口疮病毒结构蛋白B2L基因,回收纯化PCR产物,克隆至pMD18-T,测序结果表明插入的片段为目的基因,全长1137 bp.将目的基因定向克隆至pET-32a构建了B2L基因原核表达载体pET-32a-B2L,经PCR鉴定、酶切及进一步测序证明表达载体构建成功,为下一步的表达及基因工程疫苗的研究奠定了良好基础.%To identify suspected goats orf case and construction of prokaryotic expression vector of B2L gene of orf virus (ORFV),clinical and suspect samples of orf could be amplified by a pair of specific primers, then the structural protein B2L gene of orf virus was amplified by PCR method using specific primers. The B2L gene was ligated with pMD18-T vector and the sequencing results showed that the full-length of the inserted gene fragment was 1137 kb. Cutting the target fragment, which was cloned into pET-32a vector and construction of prokaryotic expression vector pET-32a-B2L,the recombinant plasimid was identified by restriction enzyme and PCR,sequence analysis indicated that to be successful construction of pET-32a-B2L expression vector and it has laid a good foundation for the expression and genetic engineering vaccine research.