抑制Coronin-1基因表达的siRNA载体的构建和筛选

Construction of a siRNA plasmid for knockdown of Coronin-1

目的:构建和筛选能高效、特异性抑制Coronin-1基因表达的siRNA表达载体。方法:提取鼠巨噬细胞总RNA,RT-PCR扩增Coronin-1基因目标序列,克隆入pSEB-HUS真核表达质粒,构建pSEB-HUS-C重组质粒。将设计、合成的3条siRNA及阴性对照分别克隆入pSEB-HUS-C,构建重组pSEB-HUS-C1、pSEB-HUS-C2、pSEB-HUS-C3及pSEB-HUS-CN质粒。将干涉质粒瞬时转染A549细胞,通过绿色荧光信号观察、实时定量PCR和Western blot法检测其对Coro-nin-1基因表达的影响。结果:经双酶切及测序证实,所构建siRNA表达载体目的基因大小、序列与预期相符。经瞬时转染A549细胞后,其中的pSEB-HUS-C3能明显抑制Coronin-1 mRNA的表达和Coronin-1蛋白的合成,抑制率分别是75.9%和75.1%。结论:成功构建并筛选出高效、特异性抑制Coro-nin-1表达的siRNA表达载体,为进一步研究Coronin-1在巨噬细胞中的作用奠定基础。

AIM：To construct a siRNA plasmid to knockdown Coronin-1. METHODS：The cDNA of coronin-1 was amplified by RT-PCR from the total RNA of macrophage,and then inserted into pSEB-HUS vector to generate pSEB-HUS-C plasmid. Three synthesized siRNAs targeting Coronin-1 were cloned into pSEB-HUS-C respectively,resulting in the pSEB-HUS-C1,pSEB-HUS-C2 and pSEB-HUS-C3 plasmids. These plasmids were transiently transfected into A549,and the Coronin-1 level was detected by RT-PCR,Real time PCR and Western blot. RESULTS：All plasmids were successfully constructed as confirmed by restriction enzyme digestion and DNA sequencing. The pSEB-HUS-C3 vector had the most significant knockdown effect on Coronin-1,with 75.9% inhibition at mRNA level and 75.1% inhibition at protein level. CONCLUSION：A siRNA plasmid targeting Coronin-1 was successfully constructed and validated for its knockdown effect,which will serve as a loss-of-function tool for the further mechanistic study of Coronin-1 in tuberculosis pathology.