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

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。

Optimization of the HIV-1 Based Three Plasmids Lentivirural Pack-aging System and Investigation of the Importance of Rev Expression Plasmid

Objective： In order to produce a high-tier viral particles, this study has optimized the ratio of lenti？viral packaging plasmids used for the third generation HIV-1 based lentivector expression system. Methods： The HIV-1 based expression lentivector, pHEGP（contains the GFP fluorescence gene）, was mixed with the other three packaging plasmids, expressing gag/pol, Rev and VSVG respectively, in different ratios. A fixed amount of 10 μg plasmid mixture was transiently transfected into HEK 293T cell line with LipofectAMINE 2000. Supernantant con？taining virial particles was collected and used for transducing HEK 293T and K562 cell respectively. The titer of virion was assessed by the percentage of GFP positive cells via flow cytometry. Results： The GFP positive frac？tion ranges from 14% to 68%, showing that the ratio of individual plasmids in the mixture directly affect the titer of packaged virion. When increase the expression lentivector pHEGP from 15%（1.5 μg） to 35%（3.5 μg）, a 2.2 fold increase, the corresponding GFP＋ 293T cell increased from 14.2% to 45.1%,a 3.2 fold increase. When fixed the amount of target gene expression plasmid at 35% and increase the portion of gag/pol or Rev expression packag？ing plasmid from 15% to 25%, the corresponding GFP＋ cell increased 115% and 150% respectively. Meanwhile, different ratios of VSVG plasmid did not show significant effect on the percentage of GFP ＋ cell in this study. Transducing of K562 cells with packaged virion achieved results similar to those in 293T cells. Conclusion： Our＆amp;nbsp;study optimized the ratio of individual plasmids in the lentiviral packaging system. For the first time, our results suggested that increasing the ratio of Rev expressing plasmid can improve the titer of viral particles more efficient？ly than the gag/pol and VSVG plasmid. The optimized packaging system would have potential of wide applications for further studies on gene functions in many blood and stem cells with the lentiviral expression system.