猪圆环病毒3型ORF3蛋白的原核表达及纯化  

Prokaryotic expression and purification of Porcine circovirus type 3 ORF3 protein

作　　者：关现军[1,2,3] 连凯琪 时志琪 林正丹 王英杰 宋玉伟 王双山[1,2,3] GUAN Xianjun;LIAN Kaiqi;SHI Zhiqi;LIN Zhengdan;WANG Yingjie;SONG Yuwei;WANG Shuangshan(School of Biotechnology and Food Science,Anyang Institute of Technology,Anyang 455000,China;Henan Provincial Academician Workstation for Animal Disease Prevention and Control and Nutrition Immunity,Anyang 455000,China;Henan Joint Inlrniational Research Laboratory of Veterinary Biologics Research and Application,Anyang 455000,China)

机构地区：[1]安阳工学院生物与食品工程学院,河南安阳455000 [2]河南省动物疫病防控与营养免疫院士工作站,河南安阳455000 [3]河南省兽用生物制品研发与应用国际联合实验室,河南安阳455000

出　　处：《黑龙江畜牧兽医》2021年第3期96-98,102,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：安阳工学院科研培育基金项目(YPY2019017)。

摘　　要：为了制备猪圆环病毒3型(Porcine circovirus type 3, PCV-3)ORF3蛋白的抗原,试验采用PCR技术扩增ORF3基因;通过PCR和双酶切技术鉴定重组质粒pET28a(+)-ORF3;利用SDS-PAGE鉴定重组质粒pET28a(+)-ORF3的表达情况,并分析其是否为可溶性表达;采用包涵体纯化试剂盒纯化ORF3蛋白,并利用SDS-PAGE分析纯化情况。结果表明:PCR扩增得到ORF3基因,其大小为696 bp,并成功构建出重组质粒pET28a(+)-ORF3;该重组质粒可在大肠杆菌BL21(DE3)感受态细胞中成功表达,表达的ORF3融合蛋白以包涵体形式存在,纯化的蛋白条带单一。说明PCV-3 ORF3蛋白可被成功表达并纯化。In order to prepare the antigen of Porcine circovirus type 3(PCV-3) ORF3 protein,the ORF3 gene was amplified by PCR in this experiment. The recombinant plasmid pET28 a(+)-ORF3 was identified by PCR and double enzyme digestion. The expression of recombinant plasmid pET28 a(+)-ORF3 was identified by SDS-PAGE,and was analyzed whether it was soluble expression. The ORF3 protein was purified by inclusion body purification kit,and the purification was analyzed by SDS-PAGE. The results showed that the ORF3 gene was amplified by PCR,and its size was 696 bp. The recombinant plasmid pET28 a(+)-ORF3 was successfully constructed. The recombinant plasmid could be in E. coli BL21(DE3) competent cells successfully expressed. The expressed ORF3 fusion protein existed in the form of inclusion bodies,and the purified protein had a single band. The results suggested that the PCV-3 ORF3 protein can be successfully expressed and purified.

关 键 词：猪圆环病毒3型 ORF3蛋白 蛋白表达 蛋白纯化 包涵体 融合蛋白 

分 类 号：S852.65[农业科学—基础兽医学] Q51[农业科学—兽医学]