伪狂犬病病毒EP0基因的克隆及原核表达  

Cloning and Expression of EP0 Gene of Pseudorabies Virus

作　　者：周慧英[1] 程艺[1] 孙磊磊[1] 王雨[1] 吉艺宽[1] 任涛[1] 琚春梅[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《动物医学进展》2013年第4期4-8,共5页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31001074)

摘　　要：为研究伪狂犬病病毒EP0蛋白与病毒粒子中其他蛋白的相互作用,应用PCR方法从伪狂犬病病毒基因组中扩增EP0基因的编码区并克隆至pMD18-T载体,构建重组质粒pMD-EP0;用EcoRⅠ和NotⅠ双酶切pMD-EP0,回收EP0基因,并将其亚克隆至pGEX-4T-1中,构建带有GST标签的重组质粒pGEX-EP0;重组质粒转化大肠埃希菌BL21(DE3),经IPTG诱导后,通过SDS-PAGE和Western blot检测其表达情况。结果表明,扩增到EP0基因的编码区序列大小为1 227bp;重组表达菌经诱导后,EP0融合蛋白获得了表达,大小约71.4ku,且能与伪狂犬病病毒EP0单克隆抗体发生特异性反应。该蛋白的成功表达为进一步研究EP0蛋白与宿主细胞及病毒粒子中其他蛋白的相互作用奠定了基础。To study the interaction of EP0 with other proteins of PRV, the EP0 gene was amplified by PCR. The PCR products were ligated with pMD18-T vector and the recombinant plasmid was named pMD-EP0. EP0 gene was extracted from pMD-EP0 digested with EcoR I and Not I , and subcloned into pGEX- 4T-1 to construct a recombinant expression plasmid pGEX-EP0. This plasmid was transformed into E. coli BL21 （DE3） and induced with ITPG. The targeted protein was detected by SDS-PAGE and Western blot. The results showed that the CDS region of EP0, a fragment containing 1 227 bp was amplified successful- ly, and the EP0 fusion protein was expressed efficiently with the size of 71.4 ku and could be combined with monoclonal antibody against EP0 protein. The expression of EP0 and GST fusion protein plays an important role in the further study on the interaction of EP0 with other proteins of host cell and PRV.

关 键 词：伪狂犬病病毒 EP0基因 GST标签 融合表达 

分 类 号：S852.659.1[农业科学—基础兽医学]