PERK基因表达干扰对绵羊痘病毒复制影响的研究

Study of influence of expression interference of PERK gene on sheep poxvirus replication

为探究干扰Hela细胞上PERK基因表达对内质网未折叠蛋白反应(UPRER)的作用及对绵羊痘病毒(sheep pox virus,SPPV)复制的影响。本研究在HEK293T细胞进行PERK短发夹RNA(shRNA)慢病毒包装并转染Hela细胞,通过实时定量RT-PCR、细胞免疫荧光和Western-blot检测PERK在mRNA和蛋白水平的表达量,同时,用内质网应激激活剂tunicamycin处理shPERK细胞株后检测PERK通路上eIF2α,P-eIF2α,ATF5和ATF4等基因的蛋白表达情况。另外,接种SPPV2于shPERK细胞株,观察干扰PERK表达对病毒复制的影响。结果,与空白对照组和阴性对照组相比,慢病毒介导的PERK shRNA成功转染了Hela细胞;与阴性对照组比较,试验组PERK在mRNA水平和蛋白水平都明显降低,且mRNA水平两者差异极显著(P<0.001);用tunicamycin处理后,试验组eIF2α,P-eIF2α,ATF4和ATF5基因蛋白表达下调;PERK基因表达干扰后SPPV2的复制减弱,说明PERK在调控SPPV2的复制具有重要作用。以上结果为进一步深入探究UPRER对SPPV2复制的影响及阐明病毒与宿主相互作用的分子机制奠定基础。

In order to investigate the effect of lentivirus-mediated shRNA knock-down protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK) on endoplasmic reticulum unfolded protein response(UPRER) and the influence on sheep pox virus(SPPV) in Hela cells.In present study,PERK shRNA lentivirus packaging is obtained in HEK293 T cells and then stable cell lines were established through transfecting with the lentivirus in Hela cells.Real-time PCR,cell immunofluorescence and western-blot were performed to detect the change of PERK at the levels of mRNA and protein.Meanwhile,the protein expression level of eI F2α,P-eI F2α,ATF5 and ATF4 were determined in shP ERK cell lines after treating with tunicamycin,an ER stress inducer,and then the SPPV2 replication was investigated in these cells.Data showed that lentiviral with PERK-shRNA was successfully infected into Hela cells.In comparation with negative control,the expression levels of PERK mRNA and protein were obvi ously reduced in PERK shRNA experiment group with the significant difference at mRNA level(P<0.01).In addition,the protein expression level of eI F2α,P-eI F2α,ATF4 and ATF5 were declined after treatment with tunicamycin in PERK shRNA experiment group.Furthermore,the replication of SPPV2 was inhibited in shP ERK cell lines,which suggests that PERK plays an important role in regulating the replication of SPPV2.The results would make a positive contribution to improve understanding the function of PERK in UPRERand elucidate the mechanism between host and virus during virus infection.