摘要：
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(dA:dT)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PCV2 ORF1/ORF2/ORF3 pcDNA3.1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 molecu-lar mechanism,a strain of PCV2 was adapted to PK-15 cell and PAMs,and relative fluorescence quantitative PCR as-say was developed to assess the impacts of PCV2 infection,cytokines,antigen processing machineries and adaptor mol-ecules were investigated.The results indicated that the IFN-β production was significantly suppressed during PCV2 in-fection in PK-15.Using luciferase reporter assays,the result showed that PCV2 infection impairs IRF3/NF-κB media-ted IFN-β production. 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-β promoter,while IFN-β promoter was impeded by ORF2,no impact was detected by ORF3.The results showed that the inhibition of innate immunity in PCV2 could be contribu-ted to PCV2 ORF2 by downregulating of IFN production by regulating transcription factor of IRF3 and NF-кB.
