第三代慢病毒包装系统优化及Rev表达质粒对慢病毒包装效率作用初探

发布时间：2018-07-22 16:52

【摘要】：目的:对目前最为常用的三质粒慢病毒包装系统进行优化,以期明确各质粒表达的病毒成分对提高慢病毒包装效率的重要性。方法:在限定总质粒量为10μg的情况下,将表达绿色荧光蛋白(GFP)的目的质粒、表达gag/pol、Rev、VSVG的包装质粒按不同比例混合,转染293T细胞进行病毒包装,48 h后收集上清用于感染293T及K562细胞,72 h后经流式细胞术检测GFP+阳性细胞比例,分析所获病毒的感染效率。结果:采用不同的质粒混合比例包装的病毒感染效率具有明显差异,其中携带GFP的目的质粒量影响作用最为显著,当目的质粒量从15%(1.5μg)增至35%(3.5μg)时,GFP+293T细胞比例从14.2%升至45.1%,增加了3.2倍。当固定目的质粒量为35%,同时分别将表达gag/pol或Rev的质粒量从15%提高到25%时,改变Rev组的GFP+阳性率提升最明显,为1.5倍;而改变VSVG质粒量在已测试的混合比例中作用不显著。包装病毒感染K562细胞的结果与293T细胞类似。结论:通过对比包装病毒的感染效率,优化了慢病毒包装的混合质粒条件,并成功地应用于感染白血病细胞系;首次发现提高Rev质粒量可以更有效地提高病毒的包装效率,为利用慢病毒表达体系研究多种基因在血液系统中的功能奠定了技术基础。
[Abstract]:Aim: to optimize the three plasmids lentivirus packaging system so as to clarify the importance of the viral components expressed by the three plasmids in improving the efficiency of lentivirus packaging. Methods: under the condition of limiting the total amount of plasmid to 10 渭 g, the target plasmid expressing green fluorescent protein (GFP) and the packaging plasmid expressing gag/ polo Revn VSVG were mixed in different proportion. After transfection of 293T cells for 48 h, the supernatants were collected for infection with 293T and K562 cells for 72 h. The percentage of GFP positive cells was detected by flow cytometry, and the infection efficiency was analyzed. Results: there were significant differences in the efficiency of virus infection with different plasmids mixed proportion packaging, among which the effect of the target plasmid quantity carrying GFP was the most significant. When the amount of the target plasmid increased from 15% (1.5 渭 g) to 35% (3.5 渭 g), the percentage of GFP293T cells increased from 14.2% to 45.1%, an increase of 3.2 times. When the number of fixed target plasmids was 35% and the amount of plasmids expressing gag/pol or Revs was increased from 15% to 25%, the positive rate of GFP in the modified Rev group was 1.5 times higher than that in the control group, but the change of the amount of VSVG plasmids did not play a significant role in the tested mixing ratio. The results of packaging virus infection on K562 cells were similar to 293T cells. Conclusion: by comparing the infection efficiency of packaging virus, the mixed plasmid conditions of lentivirus packaging were optimized and successfully applied to infected leukemia cell lines, and it was found for the first time that increasing the amount of Rev plasmid could improve the packaging efficiency of the virus more effectively. It lays a technical foundation for studying the function of many genes in blood system by using lentivirus expression system.
【作者单位】：中国医学科学院、北京协和医学院血液学研究所血液病医院实验血液学国家重点实验室;
【基金】：国家重点基础研究发展计划(2012CB966504) 国家自然科学基金面上项目(81170459) 协和医学院协和学者项目(2010-2013年) 天津市应用基础及前沿技术研究计划(11JCYBJC27400) 人事部留学人员择优资助项目(2012年) 实验血液学国家重点实验室自主课题(Z-12-02,Z-13-02)
【分类号】：R373;Q78

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