PCV2逃逸宿主天然免疫的分子机制研究

Molecular Mechanisms of PCV2 Escape from Innate Immunity

为了探究PCV2抑制β干扰素(IFN-β)的分子机制,采用相对荧光定量的方法检测了PCV2感染PK-15细胞及PAM细胞后的白细胞介素、抗原提呈分子、模式识别受体、干扰素及其转录因子、DNA信号通路关键接头分子的转录表达,结果表明,PCV2感染引起β干扰素下调为主的天然免疫抑制。采用IFN-β启动子的荧光素酶报告检测系统检测PCV2感染PK-15细胞后IFN-β的表达情况,结果显示,PCV2感染不能激活IFN-β的转录,并且能够抑制Poly(d A∶d T)诱导的IFN-β转录,呈现PCV2接种剂量依赖性;为了探究PCV2编码的主要蛋白在天然免疫中的作用,构建了PCV2ORF1/ORF2/ORF3 pc DNA3.1真核表达载体,采用IFN-β-Luc、NF-кB-Luc、AP-1-Luc和IRF3-Luc启动子的荧光素酶报告系统检测PCV2 ORF1、ORF2、ORF3对干扰素的影响作用,结果显示,PCV2感染抑制IFN-β的产生,但其编码的蛋白ORF1和ORF2在调控方面特性相反,ORF1能够通过NF-кB激活干扰素,ORF2则可以通过IRF3、NF-кB抑制干扰素,ORF3对干扰素通路的影响不明显。研究表明,PCV2 ORF2可通过抑制干扰素产生影响机体先天性免疫反应。

To reveal the relationship between PCV2 infection and innate immune response as well as its molecular mechanism,a strain of PCV2 was adapted to PK-15 cell and PAMs,and relative fluorescence quantitative PCR assay was developed to assess the impacts of PCV2 infection,cytokines,antigen processing machineries and adaptor molecules were investigated.The results indicated that the IFN-βproduction was significantly suppressed during PCV2 infection in PK-15.Using luciferase reporter assays,the result showed that PCV2 infection impairs IRF3/NF-κB mediated IFN-βproduction.The different PCV2 expression vectors(ORF1,ORF2,or ORF3)were cotransfected with the report plasmid of the IFN-β-Luc,IRF3-Luc,NF-κB-Luc or AP-1-Luc,respectively,the luciferase reporter assays showed that the PCV2 ORF1 enhanced activation of IFN-βpromoter,while IFN-βpromoter was impeded by ORF2,no impact was detected by ORF3.The results showed that the inhibition of innate immunity in PCV2 could be contributed to PCV2 ORF2 by downregulating of IFN production by regulating transcription factor of IRF3 and NF-кB.