磷脂酰肌醇3-激酶(PI3K)在ORFV感染过程中变化规律及其对ORFV病毒复制增殖的影响

The expression regulation of PI3K from ORFV infected Hela cells and the effects on ORFV replication

磷脂酰肌醇3-激酶(phosphatidylinositol kinase 3-kinase,PI3Ks)蛋白家族参与对细胞增殖、分化、凋亡等多种细胞功能的调节。课题组前期利用Illumina/Solexa高通量测序技术对羊传染性脓疱病毒(Orf virus,ORFV)感染OFTu细胞前后mRNA表达谱进行比较分析发现,PI3K在感染后宿主细胞中显著上调表达。为进一步验证,利用荧光定量PCR、Western blot方法分别从基因转录水平和蛋白表达水平对羊传染性脓疱病毒(Orf virus,ORFV)感染不同时间段(0、3、6、12、24、48、72h)Hela细胞中PI3K的表达变化规律进行分析,与未接毒细胞相比,PI3K在感染后不同时间段均呈上升趋势,且随着感染时间延长而增加,该结果与表达谱分析结果一致。为进一步明确PI3K对ORFV侵染复制的影响,利用PI3K特异性抑制剂LY294002对PI3K进行抑制处理后接种ORFV,收集感染后不同时间段(12、24、48、72h)细胞进行定量PCR检测和TCID50测定,结果显示,加入LY294002处理的Hela细胞,其病毒滴度低于未处理细胞中的病毒滴度,由此可见阻断PI3K对ORFV的入侵具有抑制作用。上述研究结果表明,PI3K对ORFV在宿主细胞中的复制增殖具有明显的促进作用。该研究结果不仅有助于对参与ORFV侵染过程相关信号通路的研究,而且将为深入揭示ORFV致病分子机制奠定基础。

Phosphatidylinositol 3-kinase（phosphatidylinositol kinase 3-kinase,PI3Ks）family proteins are involved in cell proliferation,differentiation,apoptosis and so on,which could regulate a variety of cellular functions.In our previous study,the mRNA expression profiles of the different genes of ovine fetal turbinate（OFTu）cellsinfected with ORFV had been successfully constructed using Illumina/Solexa high-throughput sequencing technologies.The PI3 Kgene of infected host cells showed significantly up-regulated expression.To further confirm,the expression regulation of PI3 Kfrom ORFV infected Hela cells in different time points（3,6,12,24,48,72h）was measured by real-time PCR and Western blot methods.Comparing to uninfected Hela cells,the expression of PI3 Kfrom ORFV infected Hela cells appeared to be obvious up-regulation in different time points,which was consistent with the result obtained from the mRNA expression profiles of the different expressed genes of OFTu cells-infected with ORFV.In order to further identify the effects of PI3 Kgene on ORFV replication,the Hela cells inoculated with ORFV after treating with specific PI3 Kinhibitor LY294002for 12,24,48 and 72hrespectively,were collected and used for real-time PCR detection and TCID50 assay.The virus titer in Hela cells treated with LY294002 was lower than that of untreated Hela cells.The results indicated that PI3 Kcould promote the replication of ORFV in host cells.The above results would not only help to the study on the related signaling pathways involved in ORFV infection,but also establish a foundation for revealing the molecular pathogenesis of ORFV infection.