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

doi:10.11843/j.issn.0366-6964.2018.12.017

摘要/Abstract

摘要：

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cDNA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-β 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，NF-κB、IFN-β mRNA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-β mRNA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-β mRNA转录水平12 h后显著下调；而沉默SHP-2后，TRIF、IRF3、NF-κB、IFN-β mRNA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。

Abstract:

To study the activation of ITIM motif of PRRSV M protein to IFN-β production, the full-length ORF6 gene fragment and the full-length ORF6 gene fragment without ITIM motif were amplified using the PRRSV Hn-1/06 cDNA as template by PCR method, respectively. The two gene fragments were respectively inserted into the eukaryotic expression vector pcDNA3.0 to yield recombinant plasmids, designated by pcDNA3.0-GP6 and pcDNA3.0-GP6ΔITIM (lacking of ITIM motif). Then, the two recombinant plasmids were used to transfected the Marc-145 cells, respectively. The TRIF, MyD88, TRAF6, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the transfected Marc-145 cells were detected by qRT-PCR. The results showed that the TRIF, IRF3 and IRF7 mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant 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 MyD88 and TRAF6 mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were down-regulated significantly, when compared with the Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid. The two recombinant plasmids were used to transfected the Marc-145 cells transfected with Poly (I:C), respectively. The TRIF, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the transfected Marc-145 cells were detected by qRT-PCR. The results showed that the TRIF, IRF3 and IRF7 mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were up-regulated significantly, while the NF-κB and IFN-β mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were up-regulated significantly at 36 h, when compared with the Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid. The recombinant plasmids pcDNA3.0-GP6 and pcDNA3.0-GP6△ITIM were respectively transfected into the Marc-145 cells silenced with SHP-1-siRNA-781 or SHP-2-siRNA-1335, then the TRIF, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the transfected Marc-145 cells were detected by qRT-PCR. The TRIF, IRF3, IRF7,NF-κB and IFN-β mRNA levels of the SHP-1 silenced Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were down-regulated significantly at any timepoints, compared with the SHP-1 silenced Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid, the TRIF, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the SHP-2 silenced Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were down-regulated significantly at early phase, then recovery to normal levels at later phase, when compared with the SHP-2 silenced Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid. In summary, the ITIM motif of PRRSV M protein may induce the IFN-β production by interacting with SHP-1 in TLR3-TRIF-dependent signaling pathway, however the relationship between ITIM motif and SHP-2 needs to further study. This study provides an important reference for understandin the role of ITIM motif.

中图分类号:

S852.659.6

魏凤灵, 张莹莹, 许瑞勤, 李闻, 李想通, 孙杨杨, 张留君, 杨国宇, 夏平安, 张改平. 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[J]. 畜牧兽医学报, 2018, 49(12): 2680-2689.

WEI Feng-ling, ZHANG Ying-ying, XU Rui-qin, LI Wen, LI Xiang-tong, SUN Yang-yang, ZHANG Liu-jun, YANG Guo-yu, XIA Ping-an, ZHANG Gai-ping. Positive Regulation of ITIM of PRRSV M Protein to IFN-β Production[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(12): 2680-2689.

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