【摘要】：目的:對(duì)目前最為常用的三質(zhì)粒慢病毒包裝系統(tǒng)進(jìn)行優(yōu)化,以期明確各質(zhì)粒表達(dá)的病毒成分對(duì)提高慢病毒包裝效率的重要性。方法:在限定總質(zhì)粒量為10μg的情況下,將表達(dá)綠色熒光蛋白(GFP)的目的質(zhì)粒、表達(dá)gag/pol、Rev、VSVG的包裝質(zhì)粒按不同比例混合,轉(zhuǎn)染293T細(xì)胞進(jìn)行病毒包裝,48 h后收集上清用于感染293T及K562細(xì)胞,72 h后經(jīng)流式細(xì)胞術(shù)檢測(cè)GFP+陽(yáng)性細(xì)胞比例,分析所獲病毒的感染效率。結(jié)果:采用不同的質(zhì)�；旌媳壤b的病毒感染效率具有明顯差異,其中攜帶GFP的目的質(zhì)粒量影響作用最為顯著,當(dāng)目的質(zhì)粒量從15%(1.5μg)增至35%(3.5μg)時(shí),GFP+293T細(xì)胞比例從14.2%升至45.1%,增加了3.2倍。當(dāng)固定目的質(zhì)粒量為35%,同時(shí)分別將表達(dá)gag/pol或Rev的質(zhì)粒量從15%提高到25%時(shí),改變Rev組的GFP+陽(yáng)性率提升最明顯,為1.5倍;而改變VSVG質(zhì)粒量在已測(cè)試的混合比例中作用不顯著。包裝病毒感染K562細(xì)胞的結(jié)果與293T細(xì)胞類似。結(jié)論:通過(guò)對(duì)比包裝病毒的感染效率,優(yōu)化了慢病毒包裝的混合質(zhì)粒條件,并成功地應(yīng)用于感染白血病細(xì)胞系;首次發(fā)現(xiàn)提高Rev質(zhì)粒量可以更有效地提高病毒的包裝效率,為利用慢病毒表達(dá)體系研究多種基因在血液系統(tǒng)中的功能奠定了技術(shù)基礎(chǔ)。
[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.
【作者單位】： 中國(guó)醫(yī)學(xué)科學(xué)院、北京協(xié)和醫(yī)學(xué)院血液學(xué)研究所血液病醫(yī)院實(shí)驗(yàn)血液學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(2012CB966504) 國(guó)家自然科學(xué)基金面上項(xiàng)目(81170459) 協(xié)和醫(yī)學(xué)院協(xié)和學(xué)者項(xiàng)目(2010-2013年) 天津市應(yīng)用基礎(chǔ)及前沿技術(shù)研究計(jì)劃(11JCYBJC27400) 人事部留學(xué)人員擇優(yōu)資助項(xiàng)目(2012年) 實(shí)驗(yàn)血液學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室自主課題(Z-12-02,Z-13-02)
【分類號(hào)】：R373;Q78

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