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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。
OBJECTIVE: To construct and screen siRNA expression vector which can efficiently and specifically inhibit Coronin-1 gene expression. Methods: Total RNA was extracted from murine macrophages. The target sequence of Coronin-1 gene was amplified by RT-PCR and cloned into pSEB-HUS eukaryotic expression plasmid to construct pSEB-HUS-C recombinant plasmid. The designed and synthesized three siRNAs and the negative control were cloned into pSEB-HUS-C respectively to construct the recombinant pSEB-HUS-C1, pSEB-HUS-C2, pSEB-HUS-C3 and pSEB-HUS-CN plasmids. The interference plasmids were transiently transfected into A549 cells, and the effect of Coro-nin-1 gene expression was detected by green fluorescence signal and real-time quantitative PCR and Western blot. Results: The double-enzyme digestion and sequencing confirmed that the constructed siRNA expression vector gene size, the sequence in line with expectations. After transient transfection of A549 cells, pSEB-HUS-C3 could significantly inhibit the expression of Coronin-1 mRNA and the synthesis of Coronin-1 protein with the inhibitory rates of 75.9% and 75.1%, respectively. CONCLUSION: The siRNA expression vector that efficiently and specifically inhibits the expression of Coro-nin-1 was successfully constructed and screened, which laid the foundation for further study on the role of Coronin-1 in macrophages.