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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。
In order to clone and express prokinetic herpesvirus CEV112, a pair of primers was designed according to the sequence of CEV112 in GenBank. One CEV112 gene of 867bp was amplified by PCR using CEV genome as a template. It was ligated into pMD20-T vector to construct pMD20-T-CEV112 recombinant plasmid and transformed into E. coli DH5α competent cells. The plasmid was digested with restriction endonucleases. After identification, the recombinant plasmid pET28a-CEV112 was constructed and transformed into E. coli BL21 (DE3) competent cells. After induced by IPTG, the expressed product was analyzed by SDS-PAGE and Western blot. The results showed that the prokaryotic expression vector pET-28a-CEV112 was successfully constructed and the CEV112 gene was expressed in E.coli BL21 (DE3). The expressed fusion protein was about 36ku in size and mainly existed as inclusion bodies. Therefore, CEV112 Gene function study laid the foundation.