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目的构建细丝蛋白A(filamin A,FLNa)shRNA慢病毒载体,并检测其在肝癌细胞Hep G2和Huh7中的干扰效率。方法通过合成干扰基因片段,插入至shRNA慢病毒载体GV298中,构建FLNa shRNA慢病毒载体shRNAFilamin1和shRNA-Filamin2,转染Hep G2和Huh7细胞,荧光显微镜下观察红色荧光表达,Western blot法检测Filamin A干扰效率。结果构建的FLNa shRNA慢病毒载体经测序证明构建正确;转染24 h后,Hep G2和Huh7细胞可见红色荧光;转染48 h后,shRNA-Filamin1和shRNA-Filamin2的干扰效率在Hep G2细胞中分别约为45.4%和63.7%,在Huh7细胞中分别约为76.5%和78.3%。结论成功构建FLNa shRNA慢病毒载体,在Hep G2和Huh7细胞中干扰效率较高;可与多种病毒的受体蛋白相互作用,从而参与病毒的复制周期,为今后病毒复制及其致病机理的研究提供了新的思路。
Objective To construct filamin A (FLNa) shRNA lentivirus vector and detect its interference efficiency in hepatoma cells Hep G2 and Huh7. Methods Interfering gene fragment was inserted into GV298 lentiviral vector to construct FLNa shRNA lentiviral vector shRNAFilamin1 and shRNA-Filamin2, transfected into Hep G2 and Huh7 cells, the red fluorescent expression was observed under a fluorescence microscope, Filamin A Interference efficiency. Results The constructed FLNa shRNA lentiviral vector was verified by sequencing. After 24 h of transfection, red fluorescence was observed in Hep G2 and Huh7 cells. After 48 h of transfection, the interference efficiency of shRNA-Filamin1 and shRNA-Filamin2 in Hep G2 cells About 45.4% and 63.7% respectively, and about 76.5% and 78.3% respectively in Huh7 cells. Conclusions The FLNa shRNA lentiviral vector was successfully constructed and its interference efficiency was high in Hep G2 and Huh7 cells. It could interact with many kinds of viral receptor proteins and thus participate in the replication cycle of the virus, thus providing a theoretical basis for future viral replication and its pathogenesis Research provides a new way of thinking.