猪圆环病毒2型ORF4基因编码蛋白的体外表达  

Expression of porcine circovirus type 2 ORF4 protein in vitro

作　　者：李增魁[1] 陈婷飞[2] 李益飞[2] 周继勇[2] 

机构地区：[1]青海大学农牧学院,青海西宁810003 [2]浙江大学动物科学院,浙江杭州310029

出　　处：《中国兽医科学》2008年第10期837-841,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30370052)

摘　　要：为了探讨猪圆环病毒2型(PCV-2)ORF4基因结构及其所编码蛋白在PK15细胞中的表达特性,以PCV-2杭州株病毒DNA为模板进行PCR,结果扩增出了ORF4基因,并分别构建了pGEX-4T-1-ORF4原核表达载体和pEGFP-C2-ORF4真核表达载体。序列分析、SDS-PAGE分析和Western-blot检测表明,PCV-2 ORF4基因长180 bp,共编码60个氨基酸;其编码蛋白在大肠杆菌中以包涵体形式存在,分子质量大小约为6.685 ku。真核PK15细胞的转染试验显示,ORF4重组蛋白在PK15细胞的细胞核和细胞浆中均有表达,但对其有一定的毒性。To study the ORF4 gene structure of porcine circovirus type 2（PCV-2） and the expression of ORF4 gene in PK15 cells, specific primers were designed and synthesized according to the published sequences of ORF4 gene of PCV 2. The complete ORF4 gene was amplified by PCR from viral DNA of PCV-2 Hangzhou strain（HZ0201） and inserted into the prokaryotic expression vector pGEX-4T-1 and the eukaryotic expression vector pEGFP-C2, respectively. Sequencing, SDS-PAGE analysis and Western-blot detection showed that the complete open reading frame of ORF4 gene was 180 bp in size,which encoded 60 amino acids,the ORF4 protein was expressed in Escherichia coli, mainly existing in the form of inclusion body,and the ORF4 protein was 6. 685 ku in molecular mass. In addition, the enkaryotic recombinant plasmid was transfected into PK15 cells, and the ORF4 protein could be detected and possessed certain toxicity.

关 键 词：猪圆环病毒2型 ORF4基因 原核表达 真核表达 纯化 

分 类 号：S852.659.2[农业科学—基础兽医学] Q786[农业科学—兽医学]