猪繁殖与呼吸综合征病毒M蛋白的ITIM基序对IFN-β产生的正调节作用

Positive Regulation of ITIM of PRRSV M Protein to IFN-β Production

为了研究PRRSV M蛋白的ITIM基序对IFN-β产生的激活机制,以PRRSV Hn-1/06毒株c DNA为模板扩增ORF6基因片段,分别构建了重组质粒pcDNA3. 0-GP6、pcDNA3. 0-GP6△ITIM(缺失ITIM基序)。将重组质粒分别转染Marc-145细胞,用荧光定量PCR方法检测TRIF、MyD88、TRAF6、IRF3、IRF7、NF-κB和IFN-β的mRNA转录水平;结果表明,pcDNA3. 0-GP6组与pcDNA3. 0-GP6△ITIM组相比:TRIF、IRF3、IRF7 mRNA转录水平上调(此处为总体趋势,个别时间点可能不完全相符,下同),MyD88、TRAF6 mRNA转录水平下调。分别将重组质粒和Poly(I:C)共转染Marc-145细胞,检测TRIF、IRF3、IRF7、NF-κB和IFN-βmRNA转录水平;结果表明,pcDNA3. 0-GP6组与pcDNA3. 0-GP6△ITIM组相比,TRIF、IRF3、IRF7 mRNA转录水平上调,NF-κB、IFN-βmRNA转录水平在36 h上调。将SHP-1-siRNA-781、SHP-2-siRNA-1335分别与pcDNA3. 0-GP6、pcDNA3. 0-GP6△ITIM重组质粒共转染Marc-145细胞,通过荧光定量PCR方法检测TRIF、IRF3、IRF7、NF-κB和IFN-βmRNA转录水平;结果表明,沉默SHP-1后,pcDNA3. 0-GP6组与pcDNA3. 0-GP6△ITIM组相比,TRIF、IRF3、IRF7和IFN-βmRNA转录水平12 h后显著下调;而沉默SHP-2后,TRIF、IRF3、NF-κB、IFN-βmRNA转录水平均为先升高再降低,后趋于稳定,而IRF7 mRNA转录水平被持续上调到48 h。M蛋白的ITIM基序可能通过调节TLR3-TRIF依赖型信号通路激活IFN-β的产生; SHP-1在M蛋白的ITIM基序激活Ⅰ型干扰素的信号传导途径中可能起关键调节作用,而ITIM基序与SHP-2的关系还需要进一步探索。本试验结果为进一步明确ITIM基序的作用机制提供参考。

To study the activation of ITIM motif of PRRSV M protein to IFN-βproduction,the full-length ORF6gene fragment and the full-length ORF6gene fragment without ITIM motif were amplified using the PRRSV Hn-1/06cDNA as template by PCR method,respectively.The two gene fragments were respectively inserted into the eukaryotic expression vector pcDNA3.0to yield recombinant plasmids,designated by pcDNA3.0-GP6and pcDNA3.0-GP6ΔITIM(lacking of ITIM motif).Then,the two recombinant plasmids were used to transfected the Marc-145cells,respectively.The TRIF,MyD88,TRAF6,IRF3,IRF7,NF-κB and IFN-βmRNA levels of the transfected Marc-145cells were detected by qRT-PCR.The results showed that the TRIF,IRF3and IRF7mRNA levels of the Marc-145cells transfected with pcDNA3.0-GP6recombinant plasmid were up-regulated significantly(This is the general trend,which may not be completely consistent with individual time points.The same as below),while the MyD88and TRAF6mRNA levels of the Marc-145cells transfected with pcDNA3.0-GP6recombinant plasmid were down-regulated significantly,when compared with the Marc-145cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid.The two recombinant plasmids were used to transfected the Marc-145cells transfected with Poly(I:C),respectively.The TRIF,IRF3,IRF7,NF-κB and IFN-βmRNA levels of the transfected Marc-145cells were detected by qRT-PCR.The results showed that the TRIF,IRF3and IRF7mRNA levels of the Marc-145cells transfected with pcDNA3.0-GP6recombinant plasmid were up-regulated significantly,while the NF-κB and IFN-βmRNA levels of the Marc-145cells transfected with pcDNA3.0-GP6recombinant plasmid were up-regulated significantly at36h,when compared with the Marc-145cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid.The recombinant plasmids pcDNA3.0-GP6and pcDNA3.0-GP6△ITIM were respectively transfected into the Marc-145cells silenced with SHP-1-siRNA-781or SHP-2-siRNA-1335,then the TRIF,IRF3,IRF7,NF-κB and IFN-βmRNA levels of the transfected Marc-145cells were detected by qRT-PCR.The TRIF,IRF3,IRF7,NF-κB and IFN-βmRNA levels of the SHP-1silenced Marc-145cells transfected with pcDNA3.0-GP6recombinant plasmid were down-regulated significantly at any timepoints,compared with the SHP-1silenced Marc-145cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid,the TRIF,IRF3,IRF7,NF-κB and IFN-βmRNA levels of the SHP-2silenced Marc-145cells transfected with pcDNA3.0-GP6recombinant plasmid were down-regulated significantly at early phase,then recovery to normal levels at later phase,when compared with the SHP-2silenced Marc-145cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid.In summary,the ITIM motif of PRRSV M protein may induce the IFN-βproduction by interacting with SHP-1in TLR3-TRIF-dependent signaling pathway,however the relationship between ITIM motif and SHP-2needs to further study.This study provides an important reference for understandin the role of ITIM motif.