靶向载脂蛋白M基因小干扰RNA表达载体构建及其功能  

Construction and Functional Analysis of Short Interfering RNA Expression Vectors of Targeting Gene Apolipoprotein M

作　　者：黄刚[1] 祝成亮[2] 潘峰[1] 刘芳[2] 

机构地区：[1]华中科技大学医院,湖北省武汉市430074 [2]武汉大学生命科学学院病毒学国家重点实验室,湖北省武汉市430072

出　　处：《中国动脉硬化杂志》2007年第5期345-348,共4页Chinese Journal of Arteriosclerosis

基　　金：湖北省自然科学基金(2004ABA166)

摘　　要：目的设计和构建载脂蛋白M基因特异性的小干扰RNA体内表达载体,筛选抑制载脂蛋白M表达的有效小干扰RNA,并初步探讨载脂蛋白M的功能。方法以载脂蛋白M为目的基因,以产生小干扰RNA质粒载体pSilencerTM2.1-U6为表达模板,细胞内转录合成4条小干扰RNA,并构建携带荧光素酶报告基因的重组质粒载体plucF-载脂蛋白。将重组质粒载体plucF-载脂蛋白与产生小干扰RNA的质粒pSilencerTM2.1-U6共转染293T细胞,定量测量荧光素酶活性,初步筛选出抑制荧光素酶表达的有效小干扰RNA,然后将小干扰RNA转染L-02,逆转录聚合酶链反应检测载脂蛋白M mRNA的表达,进一步证实小干扰RNA对载脂蛋白M表达的抑制效果,酶联免疫吸附法检测有效小干扰RNA对载脂蛋白AⅠ、载脂蛋白B和载脂蛋白E合成和分泌的影响。结果合成的4条小干扰RNA中有2条抑制荧光素酶表达,抑制效率分别为86%和91%,并特异性抑制肝细胞载脂蛋白M mRNA的表达;有效小干扰RNA能够有效抑制载脂蛋白AⅠ合成和分泌(P<0.05),而载脂蛋白B和载脂蛋白E的含量无明显改变(P>0.05)。结论成功构建并筛选到针对载脂蛋白M基因表达的有效小干扰RNA质粒,为研究冠心病的发病机制奠定基础。　Aim To design and construct the expression vector that can express the short interfering RNA（siRNA） against apolipoprotein M （ApoM）gene, screen the effective target sequence of siRNA and explore the function of ApoM. Methods The four siRNA against ApoM gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilencerTM 2.1-U6, and the ApoM gene was inserted into the reporter gene in order to construct the recombinant plasmid vector plucF-ApoM. The recombinant plasmid and siRNA producing plasmid were cotransfected into 293T cells and screened out the effective siRNA that inhibiting the expression of luciferase, siRNA was transfected into L-02, the expression of ApoM mRNA was detected by reverse transcription polymerase chain reaction （RT-PCR） to further identify the inhibiting function of siRNA on ApoM expression, enzyme linked immunosorbent assay （ELISA） method was used to detect the effect of effective siRNA on synthesis and secretion of apolipoprotein AⅠ （ApoAⅠ）, apolipoprotein B （ApoB）, apolipoprotein E （ApoE）. Results Two siRNA of the four synthesized siRNA displayed inhibitory effects on the lucifermase expression with the inhibitory rate being 86% and 91% respectively, the expression of ApoM mRNA was specially inhibited, effective siRNA could inhibit synthesis and secretion of ApoAⅠ effectively （P〈0.05）, while there was no change in ApoB and ApoE secretion （P〉0.05）. Conclusion Effective siRNA plasmids against ApoM gene were constructed and screened out successfully which created a favourable condition for exploring the mechanisms of coronary heart disease.

关 键 词：生物工程学 RNA干扰 载脂蛋白M 小干扰RNA 表达载体 基因表达 冠心病 

分 类 号：Q81[生物学—生物工程]