猪肿瘤坏死因子受体相关因子6的克

发布时间：2018-07-20 12:54

【摘要】：肿瘤坏死因子受体相关因子6(TRAF6)是天然免疫信号通路中重要的接头分子,笔者旨在探索猪TRAF6的抗病毒和免疫调控功能。首先利用RT-PCR从猪外周血单核细胞克隆该基因,并进行序列比对分析;其次利用Piggybac转座子系统建立猪TRAF6稳定表达的PK-15细胞系;最后在细胞水平检测猪TRAF6对伪狂犬病病毒复制的影响以及对猪IFN-β等启动子活性的影响。结果显示,猪TRAF6基因为1 623bp;将猪TRAF6基因连接到Piggybac转座子质粒成功构建真核表达载体PB-TRAF6,然后将重组质粒转染PK-15细胞,经药物处理和荧光筛选获得稳定表达TRAF6的细胞系;通过荧光定量PCR和Western blot确定稳定细胞系TRAF6的高效表达;体外抗病毒试验表明,TRAF6稳定细胞系能够显著抑制伪狂犬病病毒的复制;双荧光报告基因法检测发现,TRAF6可以促进poly(I:C)诱导的Ⅰ型干扰素应答。结果提示猪TRAF6具有显著的抗病毒和免疫调节功能,为研究TRAF6的免疫学功能奠定基础。
[Abstract]:Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an important junction molecule in innate immune signaling pathway. The purpose of this study is to explore the antiviral and immunomodulatory functions of porcine TRAF6. Firstly, the gene was cloned from porcine peripheral blood monocytes by RT-PCR and sequenced, then the PK-15 cell line with stable expression of porcine TRAF6 was established by Piggybac transposition subsystem. Finally, the effects of porcine TRAF6 on pseudorabies virus replication and IFN- 尾 promoter activity were detected at cell level. The results showed that the porcine TRAF6 gene was linked to Piggybac transposon plasmid to construct the eukaryotic expression vector PB-TRAF6, and the recombinant plasmid was transfected into PK-15 cells. The cell lines expressing TRAF6 stably were obtained by drug treatment and fluorescence screening. The high expression of the stable cell line TRAF6 was confirmed by fluorescence quantitative PCR and Western blot, and the antiviral test in vitro showed that the stable cell line could significantly inhibit the replication of pseudorabies virus. Double fluorescence reporter gene assay showed that TRAF6 could promote the type I interferon response induced by poly (I: C). The results suggested that porcine TRAF6 had significant antiviral and immunomodulatory functions, which laid a foundation for studying the immunological function of TRAF6.
【作者单位】：河南农业大学牧医工程学院;华中农业大学农业微生物学国家重点实验室;河南省动物卫生监督所;
【基金】：国家自然科学基金(31602033) 河南省高校科技创新团队支持计划(14IRTSTHN015)
【分类号】：S852.65

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