新城疫病毒La Sota株NP、P和L基因表达载体的构建及真核表达  

Construction and Eukaryotic Expression of Recombinant Plasmids Containg NP,P and L Genes of NDV La Sota Strain

作　　者：呙会会 刘垒[1] 周祖涛[1] 李筱雯[1] 金卉[1] 肖运才[1] 王贵强[2] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070 [2]湖北省畜禽育种中心,湖北武汉430070

出　　处：《动物医学进展》2014年第6期1-5,共5页Progress In Veterinary Medicine

基　　金：湖北省自然科学基金项目(2010CDB10101);湖北省农业创新岗位基金

摘　　要：为研究新城疫病毒NP、P和L蛋白作用机理和新城疫病毒反向遗传操作系统,构建新城疫病毒La Sota株NP、P和L基因的真核表达载体。根据GenBank中新城疫La Sota株的全长序列(JF950510.1),设计4对特异性引物,用RT-PCR法扩增出新城疫病毒La Sota株的NP、P和L基因,将其克隆到真核载体pCl-neo上。通过酶切和测序验证克隆正确,得到重组质粒pCl-NP、pCl-P和pCl-L,瞬时转染到BHK-21细胞中。经RT-PCR、Western blot、间接免疫荧光试验分别检测NP、P和L蛋白在BHK-21细胞中的表达。经上述三种方法分别检测到NP、P和L蛋白在BHK-21细胞中的表达。重组质粒pCl-NP、pCl-P和pCl-L构建成功,为后期新城疫病毒反向遗传操作系统的构建奠定了基础。To construct the eukaryotic expression vectors containing P,NP and L genes of Newcastle disease virus La Sota strain,explore the mechanism of P,NP and L proteins and the Newcastle disease virus re-verse genetic operating system,according to the sequence of La Sota strain of NDV GenBank （JF950510. 1）,4 pairs of specific primers were designed to amplified NP,P and L gene fragments by RT-PCR,and the genes were cloned into the eukaryotic expression vector pCl-neo.By enzyme digestion and sequencing, the recombinant plasmids pCl-NP,pCl-P and pCl-L were transiently transfected into BHK-21 cells.RT-PCR,Western blot and indirect immunofluorescence assay were used to respectively detects the expression of NP,P and L proteins in BHK-21 cells.The recombinant plasmids pCl-NP,pCl-P and pCl-L were con-structed successfully.The results provide a valuable foundation for the construction of Newcastle disease virus reverse genetic operating system.

关 键 词：新城疫病毒 P和L基因 辅助质粒 

分 类 号：S852.659[农业科学—基础兽医学]