羊传染性脓疱病毒CEV112基因的克隆与原核表达

为了克隆羊传染性脓疱病毒CEV112基因并对其进行原核表达,根据GenBank中CEV112基因序列信息设计1对引物,以CEV基因组为模板,采用PCR扩增出1条大小为867bp的CEV112基因,将其连接到pMD20-T载体上,构建pMD20-T-CEV112重组质粒,转化到大肠埃希菌(E.coli)DH5α感受态细胞中,提取质粒进行酶切鉴定。鉴定正确后构建重组质粒pET28a-CEV112,转化到E.coli BL21(DE3)感受态细胞中。经IPTG诱导表达,表达产物用SDS-PAGE和Western blot进行分析。结果表明,成功构建了pET-28a-CEV112原核表达载体,并在E.coli BL21(DE3)中表达了CEV112基因,表达的融合蛋白大小约36ku,且主要以包涵体形式存在,为后续开展CEV112基因的功能研究奠定了基础。

Cloning and Prokaryotic Expression of CEV112 Gene of Contagious Ecthyma Virus

To clone the CEV112 gene and make prokaryotic expression in Escherichia coli,one pair of primers were designed according to the CEV112 gene sequence in GenBank, and then a target gene fragment with 867 bp was amplified by PCR.The cloning recomenbinant plasimid pMD20-T-CEV112 was constructed and transformed into the E.coli DH5α.The prokaryotic expression recombinant plasmid pET-28a-CEV112 was constructed after appraising,and transformed into the E.coli BL21(DE3).After induction with IPTG,the protein was detected by SDS-PAGE and Western blot.The results showed that the pET-28a-CEV112 prokaryotic expression vector was successfully constructed and expressed in E.coli BL21(DE3).The expression fusion protein was about 36 ku in molecular weight and it existed in the form of inclusion bodies,which would provide the research basis in the future.