可诱导表达小反刍兽疫病毒P蛋白细胞系的建立

Establishment a des petits ruminants virus phosphoprotein inducible expressing cell line

为研究小反刍兽疫病毒(PPRV)磷蛋白(P)的生化功能,本研究通过PCR将PPRV p全长基因扩增后克隆至真核表达载体pcDNA4/TO中,构建了重组质粒pcDNA4/TO-PPRVP。将该质粒转染至已稳定转入pcDNA6/TR质粒的T-REx293细胞中,并用博来霉素和稻瘟菌素筛选存活克隆。将强力霉素添加至存活细胞克隆中诱导P蛋白表达,Western blot及间接免疫荧光试验鉴定出可诱导表达P蛋白的细胞克隆,扩增阳性克隆获得能够稳定表达P蛋白的细胞系。Western blot显示该细胞系表达的P蛋白能够与山羊PPRV阳性血清反应,并能够有效抑制聚肌胞苷酸诱导的STAT1磷酸化,表明该细胞系表达的重组PPRV P蛋白具有与野生型P蛋白类似的生物活性。本研究利用Tet on系统建立了表达PPRV P蛋白的真核细胞系,为深入研究PPRV的致病机制奠定了基础。

In order to study the biochemical mechanism of phosphoprotein (P) of the peste des petits ruminants virus (PPRV), a Tet on cell line capable of inducing the expression of PPRV P was established in this study. First, the full-length PPRV P gene was cloned into eukaryotic expression plasmid pcDNA4/TO by PCR to construct a recombinant plasmid pcDNA4/TO-PPRVP. The T-REx293 cells were transfected with pcDNA4/TO-PPRVP and screened for the surviving clones in present of zeocin and blasticidin. After inducing by doxycycline, the expression of phosphoprotein was detected by indirect immunofluorescence assay and western blot. The positive clones that expressed phosphoprotein were proliferated and designated T-REx293-PPRV-P. Western blot detection results showed that the phosphoprotein expressed in the induced cell line reacted with PPRV positive sera and effectively inhibited the phosphorylation of STAT1 simulated by Poly I: C, indicating that the induced cell line was able to express PPRV phosphoprotein with natural biological activity, which would facilitate the pathogenesis study of PPRV.