细丝蛋白A shRNA慢病毒载体的构建及其干扰效率的检测  

Construction and knockout efficiency of shRNA lentiviral vector for filamin A

作　　者：黄文宇[1] 龙飞燕 杨臣臣 朱映柔 黄芬[2] 初佳芮 

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130062 [2]昆明理工大学医学院,云南昆明650500 [3]云南大学数统学院,云南昆明650500

出　　处：《中国生物制品学杂志》2016年第11期1146-1149,共4页Chinese Journal of Biologicals

摘　　要：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 the sh RNA lentiviral vector for filamin A and determine its knockout efficiency in HepG2 and Huh7 cells. Methods The knockout gene fragment was synthesized and inserted into the sh RNA lentiviral vector GV298 with a cherry protein reporter. The constructed recombinant plasmids sh RNA-Filamin1 and sh RNA-Filamin2 were transfected into HepG2 and Huh7 cells respectively. The expression of cherry protein was observed by fluorescent microscopy, and the knockout efficiency of filamin A was determined by Western blot. Results Sequencing results proved that the FLNa sh RNA lentiviral vectors for filamin A were constructed correctly. Strong cherry fluorescence was observed in HepG2 and Huh7 cells 24 h after transfection. However, the knockout efficiencies of sh RNA-Filamin1 and sh RNAFilamin2 were 45. 4% and 63. 7% in HepG2 cells, and 76. 5% and 78. 3% in Huh7 cells, 48 h after transfection,respectively. Conclusion The FLNa sh RNA lentiviral vectors for filamin A were successfully constructed, of which the knockout efficiencies were high in HepG2 and Huh7 cells. Filamin A was able to interact with the receptor proteins of various viruses, thus participate in the replication cycle of the virus. It provided a new approach for study on the virus replication and the pathogenic mechanism.

关 键 词：细丝蛋白A 短发夹RNA 干扰效率 

分 类 号：Q786[生物学—分子生物学]