武警医学2019，Vol.30Issue(1)：62-66,5.
三种方法浓缩慢病毒后绿色荧光蛋白转染效率的比较
A comparative study on transfection efficiency of green fluorescent protein after concentration of lentivirus by three methods
摘要
Abstract
Objective To compare the transfection efficiency of green fluorescent protein (GFP) after concentration of lentivirus by ultrafiltration tube, ultracentrifugation and virusconcentrate. Methods p MD2. G, ps PAX2 and GFP vector strains were shaken overnight in a shaker and the three plasmids were extracted with kits. pMD2. G, ps PAX2 and GFP vector plasmids were proportionally mixed with transfection reagents and transfected into 293 T cells to produce virus. The virus supernatant was collected and concentrated by ultrafiltration tube, ultracentrifugation and virus concentrate. The 293 T cells were inoculated into three groups, and the concentrated viral concentrates of 2, 4, 8, 16 μl were added into the culture medium of 293 T cells inoculated into each group, after 48 hours, the expression of GFP in 293 T cells transfected with lentivirus was detected by fluorescence microscopy and flow cytometry. Results The results of fluorescence microscopy showed that the expression rate of GFP increased with the increase of the transfection amount, and the highest expression rate of GFP was obtained when the virus was transfected into 293 T cells by ultrafiltration tube. Flow cytometry showed that the expression rates of 2, 4, 8, 16 μl GFP in virus concentrate group were (2. 4 ± 0. 1) ％, (3. 8 ± 0. 3) ％, (8. 2 ± 0. 6) ％ and (12. 2 ± 0. 3) ％respectively; the expression rates of 2, 4, 8, 16 μl GFP in ultrafiltration tube group were (5. 4 ± 0. 3) ％, (7. 4 ± 0. 4) ％, (12. 7 ±0. 1) ％ and (17. 3 ± 0. 6) ％ respectively; the expression rates of 2, 4, 8, 16 μl GFP in ultracentrifugation group were (0. 7 ± 0. 1) ％, (1. 2 ±0. 1) ％, (2. 1 ±0. 1) ％ and (0. 4 ±0. 2) ％, respectively. Compared with the other two groups, the ultrafiltration tube group had significant difference (P ＜ 0. 05). Conclusion The transfection efficiency of lentiviral GFP is different by different methods. The transfection efficiency of lentiviral GFP by ultrafiltration tube is the highest among the three lentiviral enrichment methods.关键词
质粒/慢病毒/绿色荧光蛋白Key words
plasmid/lentivirus/green fluorescent protein分类
医药卫生引用本文复制引用
毕冉冉,白睿,刘志强,刘惠亮..三种方法浓缩慢病毒后绿色荧光蛋白转染效率的比较[J].武警医学,2019,30(1):62-66,5.基金项目
国家自然科学基金(81500077,81672607) （81500077,81672607）
