抗伪狂犬病病毒gC抗体制备及其gC蛋白表达

Generation of Anti-PRV gC Antibodies and Analysis of PRV gC Expression

用纯化的重组蛋白His-gCN813制备抗PRV-gC抗体,并分析伪狂犬病病毒(pseudorabies virus,PRV)gC蛋白在真核细胞的表达情况。本研究以提取的病毒基因组为模板,PCR克隆PRV gC全长基因,构建gC真核表达质粒p3xFLAG-gC,在真核细胞中表达gC基因;纯化蛋白His-gCN813制备的抗体,Western blotting和间接免疫荧光(IFA)检测到p3xFLAG-gC转染真核细胞和PRV感染细胞中的gC蛋白。结果表明本试验成功构建了PRVgC基因真核表达系统,获得了特异性抗PRV gC抗体,重组gC真核表达质粒p3xFLAG-gC转染Vero细胞,表达的重组蛋白gC为72 ku,PRV感染Vero细胞,表达的gC蛋白为55、72和94 ku,主要定位在细胞浆,gC蛋白可以作为PRV感染的指示分子,为下一步研究PRV和宿主相互作用及PRV的复制奠定了基础。

The purified prokaryotic expression of PRV gC functional domain named His-gCN813 was used as immunogen to generate antibodies,and the PRV gC expressed in eukaryotic cell was analysised.The full length of PRV gC was cloned from PRV genome to construct recombinant plasmids p3xFLAG-gC for eukaryotic expression.The expression of PRV gC in p3xFLAG-gC transfected cells and PRV infected cells were analyzed with the anti-gC antibodies by Western blotting and immunofluorescence assay.The results indicated that the eukaryotic expressed system of PRV gC were constructed,gC specific poly