欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (20): 98-101.doi: 10.11924/j.issn.1000-6850.2012-1656

所属专题： 生物技术； 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

猪圆环病毒Ⅱ型Cap、Rep基因原核表达质粒的构建与表达

陈福旺 于飞飞 曹宇航 杨鑫 任林柱   

  • 收稿日期:2012-04-28 修回日期:2012-05-07 出版日期:2012-07-15 发布日期:2012-07-15

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

  • Received:2012-04-28 Revised:2012-05-07 Online:2012-07-15 Published:2012-07-15

PDF (PC)

838

摘要：

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-T simple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。

关键词: 通径分析, 通径分析

Abstract:

In order to develop effective detection methods and prepare monoclonal antibody of porcine circovirus type Ⅱ , Cap and Rep genes of porcine circovirus 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-1-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.coli BL21 (DE3). Furthermore, plasmid for coexpression of two proteins expressed better than the other two plasmids.