猪圆环病毒Ⅱ型Cap、Rep基因原核表达质粒的构建与表达

Construction of Prokaryotic Expression Vector of Cap and Rep Protein of Porcine Circovirus Type Ⅱ and Expression in E.coli BL21

为进一步建立猪圆环病毒Ⅱ型(PCVⅡ)的有效检测方法,以及制备结构蛋白Cap和非结构蛋白Rep的单克隆抗体。根据GenBank中登录的PCVⅡ序列,设计2对特异性引物,PCR扩增出Cap、Rep蛋白基因,连接到pMD18-Tsimple质粒上,测序正确的Cap和Rep基因双酶切后分别插入到pACYCDuet-1上,构建成原核表达质粒pACYCDuet-1-Cap、pACYCDuet-1-Rep和pACYCDuet-1-Rep-Cap,然后将上述原核表达质粒转化BL21(DE3),并用IPTG进行诱导表达。结果表明:成功地在大肠杆菌BL21(DE3)中表达了猪圆环病毒Ⅱ型的Cap、Rep蛋白,且Rep、Cap蛋白共表达质粒pACYCDuet-1-Cap-Rep的表达效果优于其他2种质粒。

In order to develop effective detection methods and prepare monoclonal antibody of porcine circovirus type Ⅱ, Cap and Rep genes of porcine eircovirus type Ⅱ were amplified and cloned into pMD18-Tsimple vector. And the genes were sequenced. Then Cap and Rep genes were sub-cloned into pACYCDuet-1 vector to generate three prokaryotic expression plasmids named pACYCDuet-I-Cap, pACYCDuet-1-Rep, and pACYCDuet-1-Rep -Cap. The plasmids were transformed into E.coli BL21 （DE3） and expressed. The results of Western-blotting showed that the two proteins were expressed successfully in E. eoli BL21 （DE3）. Furthermore, plasmid for coexpression of two proteins expressed better than the other two plasmids.