【作者】 严莎；

【导师】 陈全；

【作者基本信息】 重庆医科大学 ， 医学免疫学， 2010， 硕士

【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。更多还原

【Abstract】 Objective: To construct the plasmid vectors of siRNA which can specifically suppress the expression of coronin-1 gene and select the most effective one for further study.Methods: The cDNA of coronin-1 was amplified from the total RNA of macrophage by RT-PCR, then it was inserted into pSEB-HUS vector. After identified by restriction digestion and DNA sequencing, the constructed recombinant plasmids were named pSEB-HUS-C. 3 synthesized siRNAs were cloned into pSEB-HUS-C respectively, in order to construct the pSEB-HUS-C1, pSEB-HUS-C2 and pSEB-HUS-C3 plasmids, which were designed for Coronin-1 as target gene. After these plasmids was transiently transfected into A549, the inhibition level of Coronin-1 in A549 cells was detected by RT-PCR, Real time PCR and Western blot.Result: The recombinant plasmids were confirmed by double-enzyme digestion and DNA sequencing. The expression of Coronin-1 mRNA and Coronin-1 protein of pSEB-HUS-C3 group decreased significantly and the inhibition rate was 75.9% and 75.1% respectively.Conclusion: Specific siRNA interference plasmid vector targeted to coronin-1 gene mRNA is successfully constructed, which provides a good basis for further research on the function of Coronin-1 against tuberculosis更多还原