凋亡相关斑点样蛋白ASC真核表达质粒的构建及表达

Construction and expression of apoptosis-associated speck-like protein containing CARD(ASC) in eukaryotic expression vectors

目的构建凋亡相关的斑点样蛋白(apoptosis-associated speck-like protein containing CARD,ASC)真核表达质粒。方法从人胚肾细胞系HEK293T细胞的总RNA中经过逆转录和PCR获得ASC的全长cDNA双链片段,经过酶切后连接到真核表达载体pcDNA3/flag中,挑选出转化生成的阳性克隆测序,将序列正确的重组质粒命名为pcDNA3/flag-ASC。脂质体转染pcDNA3/flag-ASC质粒到HEK293T细胞中,用Western印迹检测目的蛋白的表达。同时用免疫沉淀和免疫印迹方法检测ASC蛋白与前半胱天冬酶(pro-caspase)-1的相互作用,并用ELISA方法检测ASC蛋白对IL-1β分泌的影响。结果 Western印迹实验证明pcDNA3/flag-ASC可以在HEK293T细胞中表达ASC,并且可以与pro-caspase-1结合,使IL-1β分泌明显降低。结论构建了重组质粒pcDNA3/flag-ASC,在细胞中表达ASC后具有与pro-caspase-1结合的能力,并具有降低IL-1β分泌的生物活性。

Objective To construct the eukaryotic expression plasmid of apoptosis-associated speck-like protein containing CARD（ASC）.Methods The gene encoding ASC protein was amplified by RT-PCR from total RNA of HEK293T cell line.The gene fragment was ligated into pcDNA3/flag vector digested with restriction endonucleases.The transformed clones were sequenced and the right one was named pcDNA3/flag-ASC,which was transfected into HEK293T cell line to test the expression by Western blot.We also proved the interaction of ASC and pro-caspase1 by CO-IP and analyzed the IL-1β level by ELISA.Results pcDNA3/flag-ASC expressed ASC in HEK293T cell line.Furthermore,ASC could interact with pro-caspase1 and reduce the secretion of interleukin 1-β （IL-1β） significantly.Conclusion The recombinant plasmid pcDNA3/flag-ASC is constructed successfully.The expressed ASC protein can bind to pro-caspase1 and reduce IL-1β secretion.