狂犬病病毒糖蛋白基因重组慢病毒载体的构建及鉴定

Construction and Identification of Recombinant Lentiviral Vector with Rabies Virus Glycoprotein Gene

目的构建狂犬病病毒SRV9株糖蛋白基因重组慢病毒表达载体,并进行鉴定。方法将狂犬病病毒SRV9株糖蛋白基因克隆至慢病毒载体pLVX-ZsGreen-IRES上,筛选阳性重组克隆。将质粒pLVX-ZsGreen-IRES-SRV9G、包膜质粒pMD2.G和包装质粒psPAX2共转染293T包装细胞系,通过荧光显微镜观察报告基因表达情况。收集上清,经浓缩后接种293T细胞,进行重组慢病毒感染细胞的鉴定;用限制性内切酶切除质粒pLVX-ZsGreen-IRES-SRV9G绿色荧光蛋白基因,采用直接免疫荧光试验检测狂犬病病毒糖蛋白的表达情况。结果重组慢病毒表达载体经酶切和测序证明构建正确。荧光显微镜下可见重组慢病毒感染的293T细胞有大量绿色荧光出现,病毒滴度为3×107 IU/ml;直接免疫荧光检测表明,糖蛋白基因在293T细胞内获得有效表达。结论成功构建了狂犬病病毒SRV9株糖蛋白基因重组慢病毒载体,为狂犬病基因工程疫苗的研制奠定了基础。

Objective To construct and identify a recombinant lentiviral vector with rabies virus glycoprotein gene.Methods The glycoprotein gene of rabies virus SRV9 strain was cloned into lentiviral vector pLVX-ZsGreen-IRES,and positive recombinants were screened.293T packaging cell line was co-transfected with plasmid pLVX-ZsGreen-IRES-SRV9G,envelope plasmid pMD2.G and packaging plasmid psPAX2,and observed for expression of report gene by fluorescent microscopy.The supernatant was collected,concentrated and inoculated to 293T cells for identification of infection with recombinant lentivirus.Plasmid pLVX-ZsGreen-IRES-SRV9G was digested with restriction endonuclease to remove green fluorescent protein gene,then determined for expression of rabies virus glycoprotein by direct IFA.Results Both restriction analysis and sequencing proved that recombinant lentiviral expression vector was constructed correctly.Green fluorescence at a large quantity was observed in the 293T cells transfected with the recombinant lentivirus by fluorescent microscopy,and the virus titer was 3 × 107 IU / ml.Direct IFA proved that rabies virus glycoprotein gene was effectively expressed in 293T cells.Conclusion The recombinant lentiviral vector with glycoprotein gene of rabies virus SRV9 strain was successfully constructed,which laid a foundation of development of recombinant rabies vaccine.