伪狂犬病毒gE、gI和US9三基因缺失株的构建

Constructing gE,gI and US9 gene deletion strain of pseudorabies virus

伪狂犬病毒(pseudorabies virus,PRV)是一种疱疹病毒,在自然界具有极广泛的宿主类群,是中国养猪业发展主要威胁之一。为了研究病毒的神经传导机制,实验采用Lipofectamine3 000转染试剂,将PP63质粒与伪狂犬病毒Fa株基因组DNA共转染BHK-21细胞,空斑筛选得到gI/gE/US9重组缺失病毒,并命名为SA215-T。采用PCR、基因测序、Western Blot、电镜检查和生长曲线测定等方法检测重组病毒PRV-SA215-T。研究结果显示,Western Blot未发现gE基因的表达,SA215-T株的电镜形态与野毒株无明显差异,SA215-T株与亲本毒株Fa株在细胞中的生长曲线差异不明显且均达到了较高的病毒滴度。

Pseudorabies vims （PRV） , a member of Herpes vims has an extremely broad range in nature and threat-ens the pig-industry development in our country. To explore the mechanism of nerve conduction of PRV, a vims mu-tant with a deletion in gE, gl and US9 genes was constructed. Plasmid of PP63 and pseudorabies vims Fa strain ge-nomic DNA were co-transfected into BHK-21 cells by using Lipofectamine 3000 transfection reagent, and a recombi-nant vims with the deletion of g I /gE/US 9 , named SA215-T, was screened and purified by plaque assay. PCR, gene sequencing. Western Blot, electron microscope and growth curve were used for identification of the deletion of genes, and features of the recombinant. The results showed that SA215-T was with effective deletion of g l and gE gene by PCR,and was absent in the expression of gE gene by Western Blot. There were no obvious differences in the morphology and growth curve of the recombinant vims as compared to its parental vims, they both achieved a high vi-ral titer in cells.