目的 比较超滤管法、超速离心法及病毒浓缩液法浓缩慢病毒后绿色荧光蛋白(green fluorescent protein,GFP)的转染效率。方法 用摇床过夜摇pMD2.G、psPAX2及GFP载体菌种并用试剂盒提取这三种质粒;将pMD2.G、psPAX2及GFP载体质粒与转染试剂按比例混合后转染293T细胞生产病毒;收集病毒上清液并用超滤管法、超速离心法及病毒浓缩液法浓缩慢病毒;将接种的293T细胞分为3组,并向每组接种有293T细胞的培养液中加入2、4、8、16 μl三种方法浓缩的病毒浓缩液,48 h后用荧光显微镜观察和流式细胞仪检测三种方法浓缩慢病毒后转染293T细胞的GFP表达情况。结果 荧光显微镜观察结果显示随着转染量的增加,GFP表达率增加,超滤管法浓缩病毒转染293T细胞后GFP的表达效率最高;流式检测显示病毒浓缩液法组2、4、8、16 μl GFP的表达率分别为(2.4±0.1)%、(3.8±0.3)%、(8.2±0.6)%、(12.2±0.3)%;超滤管法组2、4、8、16 μl GFP的表达率分别为(5.4±0.3)%、(7.4±0.4)%、(12.7±0.1)%、(17.3±0.6)%;超速离心法组2、4、8、16 μl GFP的表达率分别为(0.7±0.1)%、(1.2±0.1)%、(2.1±0.1)%、(0.4±0.2)%。超滤管法组与其他两组比较,P<0.05,差异有统计学意义。结论 不同方法浓缩慢病毒后的GFP转染效率不同,三种慢病毒浓缩方法中超滤管法浓缩慢病毒后的GFP转染效率最高。
Abstract
Objective To compare the transfection efficiency of green fluorescent protein (GFP) after concentration of lentivirus by ultrafiltration tube, ultracentrifugation and virusconcentrate. Methods pMD2.G, psPAX2 and GFP vector strains were shaken overnight in a shaker and the three plasmids were extracted with kits.pMD2.G, psPAX2 and GFP vector plasmids were proportionally mixed with transfection reagents and transfected into 293T cells to produce virus.The virus supernatant was collected and concentrated by ultrafiltration tube, ultracentrifugation and virus concentrate. The 293T cells were inoculated into three groups, and the concentrated viral concentrates of 2, 4, 8,16 μl were added into the culture medium of 293T cells inoculated into each group, after 48 hours, the expression of GFP in 293T 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 293T 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
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基金
国家自然科学基金(81500077,81672607)
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