狂犬病病毒8202株糖蛋白膜外区真核表达载体的构建及其在DK细胞中的表达

摘要

为了研究狂犬病病毒(rabies virus,RV)糖蛋白(rgp)膜外区在以糖蛋白为免疫原的免疫应答中的作用,提取RV的RNA,通过RT-PCR扩增RV糖蛋白膜外区1375 bp的序列,将其克隆入pMD18-T载体中,经测序比对正确后,通过酶切将其亚克隆人pVAX1表达载体的多克隆酶切位点处,将酶切鉴定正确的阳性重组子命名为pVAX-rgp,构建表达狂犬病病毒糖蛋白膜外区的真核表达载体,在质脂体介导下转染DK细胞观察其表达情况.研究结果表明,经免疫荧光和Western blotting 鉴定,有特异性荧光和60 ku条带产生,证明pVAX-rgp载体中的rgp可在DK细胞中表达.本研究通过成功构建狂犬病病毒8202株糖蛋白膜外区的真核表达载体,为下一步有关功能基因组的研究和基因疫苗的研制奠定了基础.%To construct the eukaryotic expression plasmid of glycoprotein ectodomain of rabies virus, the antigen cDNA fragment (1375 bp) of glycoprotein ectodomain of rabies virus was obtained by RT-PCR method, then it was cloned into pMD18-T vector and sequenced. Subcloned the foreign gene into the multi clone sites of pVAX1 plasmid, and identify the positive recombinat plasmid by restriction enzymes, generating pVAX-rgp. Transfected the DK cells with pVAX-rgp through Lipfectamine, identified the glycoprotein expression by Western blotting and indirect immunofluorescene assay. The results showed that the glycoprotein ectodomain could be expressed in eukaryotic cells in expressing vector. The eukaryotic expression vector for glycoprotein ectodomain gene of rabies virus was successfully constructed, which laid a foundation of study on functional genome and development on nucleic acid vaccine.