摘要
目的构建靶向甲酰肽受体(FPR)基因的shRNA慢病毒表达载体,建立稳定转染的U87细胞系。方法设计并合成FPR基因特异性的shRNA序列,构建PDS019_pL/shRNA/GFP/F-FPR慢病毒载体,通过脂质体转染293T细胞包装成FPR慢病毒颗粒,感染U87细胞。荧光显微镜观察转染效果,实时荧光定量PCR及蛋白免疫印迹法(Western blot)法检测FPR干扰效率。结果成功构建FPR基因shRNA慢病毒表达载体。稳定转染U87细胞后,荧光显微镜下可观察到绿色荧光蛋白。实时荧光定量PCR及Western blot证实PDS019_pL/shRNA/GFP/F-FPR慢病毒载体具有良好的干扰效果。结论成功构建FPR shRNA稳定转染的U87细胞系。
Objective To construct shRNA lentiviral vector.for FPR gene,and establish stable transfection of U87 cell lines. Methods The shRNA sequences for FPR gene was synthesized and inserted into PDS019 lentiviral vector. Liposome was used to package PDS019 lentiviral particles into 293T cells and used to infect U87 cells. The green fluorescent protein （GFP） and the silen- cing effects of the reconibinant vectors were detected with the fluorescence microscope and Q-PCR,Western b｜ot. Results The re- combinant vectors were successfully constructed. In the U87 cells transfected with the recombinant vectors, the expression of GFP were detected and the interference efficiency of the recombinant vectors were confirmed. Conclusion The constructed FPR shRNA lentiviral vectors could interfere the expression of FPR in U87 cells,
出处
《重庆医学》
CAS
北大核心
2016年第26期3616-3618,3621,共4页
Chongqing medicine
基金
国家自然科学基金资助项目(81301499
81460324)
