猪ASC及Ub基因的克隆及真核表达重组质粒的构建

Cloning and Eukaryotic Expression Plasmid Construction of Porcine ASC and Ub Genes

炎症小体不仅参与机体的天然免疫反应,同时还参与机体抗病毒等多种宿主防御反应。在多种疾病相关信号通路反应中都伴随着蛋白质的泛素化修饰。NOD样受体(NLRs)介导的炎症小体信号通路中,凋亡相关点状样蛋白(ASC)是关键的接头蛋白。为了解猪瘟病毒(CSFV)感染对ASC泛素化水平的影响,通过RT-PCR从猪血管内皮细胞(ECs)中扩增获得ASC和泛素(Ub)的全长cDNA双链片段,连接至克隆载体pMD-18T,获得重组克隆质粒pMD-ASC和pMD-Ub。双酶切pMD-ASC获得ASC目的序列,连接至pcDNA3.1-Flag,双酶切pMD-Ub获得Ub目的序列,连接至pcDNA3.0-HA,获得重组表达质粒pcDNA3.1-Flag-ASC和pcDNA3.0-HA-Ub。将pcDNA3.1-Flag-ASC和pcDNA3.0-HA-Ub转染ECs,Western blot在蛋白水平检测ASC和Ub的表达。结果表明,成功构建了带有Flag标签的ASC真核表达质粒和带有HA标签的Ub真核表达质粒,且在ECs中获得高效表达,为进一步体外研究CSFV调控猪血管内皮细胞ASC泛素化的机制提供了实验基础。

Inflammasome plays a key role in the body's anti-innate immunity,and also participates in many host defense responses such as antivirus. Ubiquitination of proteins is accompanied by a variety of disease-related signaling pathways.ASC is a linker protein in NLRPs mediated inflammasome signaling pathway.To find the effect of CSFV infection on ubiquitination of ASC,the full-length double-stranded cDNA fragments of ASC and Ub were amplified from porcine vascular endothelial cells (ECs) by RT-PCR and ligated with the cloning vector pMD-18T to obtain recombinant plasmids pMD-ASC and pMD-Ub.Plasmid pMD-ASC was identified by enzyme digestion to obtain ASC target sequence,which was connected to pcDNA3.1-Flag.Plasmid pMD-Ub was identified by enzyme digestion to obtain Ub target sequence,which was connected to pcDNA3.0-HA.The recombinant expression plasmids pcDNA3.1-Flag-ASC and pcDNA3.0-HA-Ub were obtained.The pcDNA3.1-Flag-ASC and pcDNA3.0-HA-Ub were transfected into ECs to detect the expressions of ASC and Ub at the protein level by Western blot.The Flag tagged ASC eukaryotic expression plasmid and the HA tagged Ub eukaryotic expression plasmid were successfully constructed,and they were highly expressed in ECs.This study provided experimental basis for the further study on the mechanism of CSFV regulating the ubiquitination of ASC in porcine vascular endothelial cells.