shRNA慢病毒载体稳定抑制LRP16表达的HepG2细胞的构建及鉴定  

Construction and Identification of Recombinant HepG2 Cells with sh RNA Lentiviral Vector Targeting LRP16

作　　者：李婷[1] 王安平[1] 汪保安[1] 哈斯[1] 母义明[1] 

机构地区：[1]中国人民解放军总医院内分泌科,北京100853

出　　处：《现代生物医学进展》2016年第8期1406-1410,1572,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(面上项目)(81471026)

摘　　要：目的:构建LRP16基因抑制表达的Hep G2稳定细胞系,鉴定其LRP16基因抑制表达的效果。方法:构建LPP-HSH008357-LVRH1GP-200短发卡RNA(sh RNA)慢病毒表达载体,用该抑制表达慢病毒感染Hep G2细胞系,通过荧光筛选及药筛,获得LRP16基因稳定抑制表达细胞系;最终运用Q-PCR和Western blot鉴定该稳定细胞系中LRP16的表达。结果:构建了LRP16基因抑制表达及抑制表达对照的Hep G2稳定细胞系,并通过Q-PCR和Western blot进行了鉴定。Q-PCR结果显示相对于对照稳转株(LPP-CSHCTR001-LVRH1GP-100),抑制表达稳转株(LPP-HSH008357-7-LVRH1GP-200)LRP16基因的抑制效率是4组抑制表达细胞中效果最理想的一组,其抑制效率高达98%;Western blot结果也显示该抑制表达稳转株(LPP-HSH008357-7-LVRH1GP-200)的LRP16蛋白表达量明显低于野生型Hep G2细胞及对照稳转株(LPP-CSHCTR001-LVRH1GP-100),其结果与Q-PCR一致。结论:构建并鉴定了人LRP16基因抑制表达Hep G2稳定细胞系及其相应的对照稳转株。To construct a recombinant lentiviral vector of short hairpin RNA（sh RNA） targeting LRP16 gene, and to identify its inhibitory effect in Hep G2 cells. Methods： The LRP16 sh RNA sequence was designed and inserted into the lentiviral vector HSH008357-LVRH1 GP. The lentiviral vector was further packaged with accessory plasmids into lentivirus in HEK293 T cells. After verification, virus infecting, cell screening, real time PCR and Western blotting, we got recombinant Hep G2 cells with sh RNA lentiviral vector targeting LRP16. Results： LRP16 stable knockdown Hep G2 cell lines were established. The efficiency of inhibitory effect were confirmed by Q-PCR and Western blot. Q-PCR results showed that the inhibition efficiency was as high as 98 % which is the most ideal of 4 groups. Western blot results showed that the LRP16 stable knockout Hep G2 cell lines was significantly lower than its control cell and the wild Hep G2 cell, and the results were consistent with Q-PCR. Conclusion： Hep G2 cell lines stable expressing LRP 16 sh RNA were successfully established with lentiviral system and its control cell.

关 键 词：LRP16 短发卡RNA 慢病毒载体 HEP G2细胞 

分 类 号：Q78[生物学—分子生物学] R-33[医药卫生]