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

摘要

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 st21 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.%为表达狂犬病病毒(RV)糖蛋白(G),本研究通过RT-PCR方法克隆RV Flury LEP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western blot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RV G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60 ku;与以RV为包被抗原的商品化ELISA试剂盒相比,以重组RV G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RV G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.