狂犬病病毒糖蛋白在Bac-to-bac杆状病毒系统中的表达及应用  

Expression of glycoprotein of rabies virus in Bac-to-bac baculovirus expression system

作　　者：范晓娟[1] 李刚[1] 李伟[1] 曾妮[1] 宫苗苗[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物营养学国家重点实验室,北京100094

出　　处：《中国预防兽医学报》2011年第5期366-369,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家高技术研究发展计划(863)项目(2008AA10Z411);国家十一五科技支撑计划(2006BAD6A13-4);中央级公益性科研院所基本科研业务费专项(0032007008)

摘　　要：为表达狂犬病病毒(RV)糖蛋白(G),本研究通过RT-PCR方法克隆RVFluryLEP病毒株G基因,将其克隆至质粒pFastBacHTα中,重组质粒pFastBac-RV-G转化DH10Bac感受态细胞,经同源重组获得重组杆状病毒穿梭质粒Bacmid-RV-G,将其转染昆虫细胞sf21获得含有G基因的重组杆状病毒。采用SDS-PAGE和westernblot对重组蛋白进行鉴定及抗原性分析,以重组G蛋白作为包被抗原的ELISA检测36份犬血清样品。结果表明,在Bac-to-bac杆状病毒系统中表达的RVG蛋白能与his-tag单克隆抗体及RV阳性血清发生特异性反应,其相对分子量为60ku;与以RV为包被抗原的商品化ELISA试剂盒相比,以重组RVG蛋白为抗原建立的间接ELISA的敏感性、特异性和符合率分别为80%、81.8%和80.6%,表明杆状病毒系统表达的RVG蛋白是检测RV抗体的理想抗原蛋白,作为一种亚单位疫苗具有潜在的应用前景。The glycoprotein gene of rabies virus Flury LEP strain was amplified by RT-PCR and cloned into pFastBacHTa vector. The resultant recombinant plasmid was transformed into competent cells of E. coli DH10Bac to construct recombinant bacmid. The recombinant baculovirus was generate by transfection of recombinant bacmid into sf21 insect cells. Expression of the recombinant glycoprotein （rG） was detected by SDS-PAGE with a MW of 60 ku, which was recongized with both ant-his tag monoclonal antibody and rabies virus positive serum by western blot test. A indirect ELISA was establish with rG as coating antigen, comparing with a commercial ELISA kit coating rabies virus as antigen, tested on 36 clinic serum samples,the indirect ELISA had 80.6% coincidence.

关 键 词：狂犬病病毒 糖蛋白 杆状病毒表达系统 

分 类 号：S852.65[农业科学—基础兽医学]