抑制coronin-1基因表达的siRNA质粒载体的构建及筛选研究
发布时间：2018-11-09 18:40
【摘要】： 目的:构建特异性抑制Coronin-1基因表达的siRNA表达载体,并筛选出具有最高抑制效率的质粒载体。 方法:提取鼠巨噬细胞总RNA,RT-PCR扩增Coronin-1基因目标序列,将其连接到pSEB-HUS真核表达质粒上,重组质粒经酶切和DNA测序鉴定后命名为pSEB-HUS-C。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不同重组质粒对Coronin-1表达的影响。最后用实时定量PCR和Western blot法检测其对Coronin-1基因表达的抑制效率。 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Coronin-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了基础。
[Abstract]:Aim: to construct a siRNA expression vector that specifically inhibits the expression of Coronin-1 gene, and to screen the plasmid vector with the highest inhibition efficiency. Methods: the target sequence of Coronin-1 gene was amplified by total RNA,RT-PCR of mouse macrophages and ligated to the eukaryotic expression plasmid of pSEB-HUS. The recombinant plasmid was named pSEB-HUS-C. after restriction endonuclease digestion and DNA sequencing. The designed, synthesized siRNA and negative control were cloned into pSEB-HUS-C, respectively, and the recombinant pSEB-HUS-C1,pSEB-HUS-C2,pSEB-HUS-C3 and pSEB-HUS-CN plasmids were constructed. The interference plasmids were transiently transfected into A549 cells and the effects of different recombinant plasmids on the expression of Coronin-1 were observed by green fluorescence signal. Finally, real-time quantitative PCR and Western blot were used to detect the inhibition efficiency of Coronin-1 gene expression. Results: the target gene size and sequence of siRNA expression vector were confirmed by double enzyme digestion and sequencing. After transient transfection of A549 cells, the pSEB-HUS-C3 could significantly inhibit the expression of Coronin-1 mRNA and the synthesis of Coronin-1 protein, the inhibition rates were 75.9% and 75.1%, respectively. Conclusion: siRNA expression vector with high efficiency and specific inhibition of Coronin-1 expression was successfully constructed and screened, which laid a foundation for further study of the role of Coronin-1 in macrophages.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
本文编号：2321246
[Abstract]:Aim: to construct a siRNA expression vector that specifically inhibits the expression of Coronin-1 gene, and to screen the plasmid vector with the highest inhibition efficiency. Methods: the target sequence of Coronin-1 gene was amplified by total RNA,RT-PCR of mouse macrophages and ligated to the eukaryotic expression plasmid of pSEB-HUS. The recombinant plasmid was named pSEB-HUS-C. after restriction endonuclease digestion and DNA sequencing. The designed, synthesized siRNA and negative control were cloned into pSEB-HUS-C, respectively, and the recombinant pSEB-HUS-C1,pSEB-HUS-C2,pSEB-HUS-C3 and pSEB-HUS-CN plasmids were constructed. The interference plasmids were transiently transfected into A549 cells and the effects of different recombinant plasmids on the expression of Coronin-1 were observed by green fluorescence signal. Finally, real-time quantitative PCR and Western blot were used to detect the inhibition efficiency of Coronin-1 gene expression. Results: the target gene size and sequence of siRNA expression vector were confirmed by double enzyme digestion and sequencing. After transient transfection of A549 cells, the pSEB-HUS-C3 could significantly inhibit the expression of Coronin-1 mRNA and the synthesis of Coronin-1 protein, the inhibition rates were 75.9% and 75.1%, respectively. Conclusion: siRNA expression vector with high efficiency and specific inhibition of Coronin-1 expression was successfully constructed and screened, which laid a foundation for further study of the role of Coronin-1 in macrophages.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
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