【摘要】： 目的:構(gòu)建特異性抑制Coronin-1基因表達的siRNA表達載體,并篩選出具有最高抑制效率的質(zhì)粒載體。 方法:提取鼠巨噬細胞總RNA,RT-PCR擴增Coronin-1基因目標序列,將其連接到pSEB-HUS真核表達質(zhì)粒上,重組質(zhì)粒經(jīng)酶切和DNA測序鑒定后命名為pSEB-HUS-C。將設(shè)計、合成的3條siRNA及陰性對照分別克隆入pSEB-HUS-C ,構(gòu)建重組pSEB-HUS-C1、pSEB-HUS-C2、pSEB-HUS-C3及pSEB-HUS-CN質(zhì)粒。將干涉質(zhì)粒瞬時轉(zhuǎn)染A549細胞,通過綠色熒光信號觀察不同重組質(zhì)粒對Coronin-1表達的影響。最后用實時定量PCR和Western blot法檢測其對Coronin-1基因表達的抑制效率。 結(jié)果:經(jīng)雙酶切及測序證實,所構(gòu)建siRNA表達載體目的基因大小、序列與預(yù)期相符。經(jīng)瞬時轉(zhuǎn)染A549細胞后,其中的pSEB-HUS-C3能明顯抑制Coronin-1 mRNA的表達和Coronin-1蛋白的合成,抑制率分別是75.9%和75.1%。 結(jié)論:成功構(gòu)建并篩選出高效、特異性抑制Coronin-1表達的siRNA表達載體,為進一步研究Coronin-1在巨噬細胞中的作用奠定了基礎(chǔ)。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R346

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