干扰素对猪伪狂犬病毒增殖的影响

Effects of Interferons on the Proliferation of Porcine Pseudorabies Virus

为了探讨干扰素(IFN)的抗病毒机制,构建pcDNA3.1-IFNα、pcDNA3.1-IFNβ、pcDNA3.1-IFNγ3个重组表达载体,分别转染PK15细胞,并以pcDNA3.1空载体作为对照(NC),24 h后以感染复数(MOI)为1接种猪伪狂犬病毒(PRV)HNJY株,然后分别在接毒后24、36、48 h收细胞。提取细胞中病毒基因组DNA,采用实时荧光定量PCR(qPCR)的方法检测gD基因在各个时间点的表达量;提取细胞RNA,反转录后采用qPCR检测细胞因子IFNα、IFNβ、IFNγ、ISG15、IL6、IL1β和IL10的表达量。观察细胞表型变化,IFNγ试验组的PK15细胞感染PRV后细胞表型变化与对照组相类似,IFNα和IFNβ试验组的PK15细胞感染PRV后正常形态细胞更多。qPCR检测gD基因的表达量,结果表明,IFNα和IFNβ试验组gD基因的表达量较对照组均极显著上调(P<0.05);IFNγ试验组gD基因的表达量在24、36 h均极显著低于对照组(P<0.01),在48 h差异不显著。qPCR检测细胞因子的表达量,结果显示,与对照组相比,试验组IFNα、IFNβ、IFNγ的表达量均极显著上调(P<0.01);IFNα和IFNβ试验组能显著诱导ISG15的表达,但IFNγ试验组不能显著诱导ISG15的表达;IFNα、IFNβ和IFNγ试验组均能诱导IL6、IL1β的上调表达;IFNβ和IFNγ试验组能诱导IL10的上调表达,但IFNα试验组不能显著诱导IL10的表达。综上,不同干扰素通过调节机体不同细胞因子的表达影响PRV的增殖。

In order to explore the antiviral mechanism of interferon(IFN),three recombinant expression vectors pcDNA3.1-IFNα,pcDNA3.1-IFNβ,pcDNA3.1-IFNγwere constructed and transfected into PK15 cells respectively,and pcDNA3.1 empty vector was used as negative control(NC).After 24 hours,the porcine pseudorabies virus(PRV)HNJY strain was inoculated with multiple infection(MOI)of 1,and then the cells were harvested at 24,36 and 48 h after inoculation.Viral genomic DNA was extracted from the cells,and the expression of gD at each time point was detected by real time fluorescence quantitative PCR(qPCR).Cell RNA was extracted,and qPCR was used to detect the expression levels of cytokines IFNα,IFNβ,IFNγ,ISG15,IL6,IL1βand IL10 after reverse transcription.The morphological changes of PK15 cells were observed.Compared with the control group,the morphological changes of PK15 cells overexpressing IFNγwere similar after PRV infection.PK15 cells overexpressing IFNα/IFNβhad more normal cells after infection with PRV.The transcription of gD in the cells transfected with IFNα,IFNβand IFNγwas detected by qPCR.The results showed that the transcription of gD in the IFNαand IFNβgroups was significantly up-regulated compared with the control group(P<0.05);the transcription of gD in IFNγtransfection group was extremely significantly lower than that of control group at 24 and 36 h(P<0.01),but the difference was not significant at 48 h.The relative expression levels of IFNα,IFNβ,IFNγ,ISG15,IL6,IL1βand IL10 were detected by qPCR.Compared with the control group,the expression levels of IFNα,IFNβ,IFNγwere all extremely significantly up-regulated(P<0.01).After PRV challenge,IFNαand IFNβgroups could significantly induce the expression of ISG15,but the IFNγgroup could not.IFNα,IFNβand IFNγgroups could induce the up-regulated expression of IL6 and IL1β.IFNβand IFNγgroups could induce the up-regulation of IL10 expression,but the IFNαgroup could not significantly induce the expression of IL10.Therefore,different interferons affect the proliferation of PRV by regulating the expression of different cytokines in the body.