HIV-1 Vif蛋白的体内表达及其生物学功能

目的分别构建带有6His和mbptag的HIV-1Vif基因真核表达载体,在体内表达融合蛋白,并检测其生物学功能。方法PCR扩增带有6His和mbptag的HIV-1Vif基因,克隆至真核表达载体VR1012上。将抗病毒因子APOBEC3G(A3G)分别与空载体VR1012、HIV-1野生型Vif/VR1012、Vif-mbp/VR1012和Vif-6His/VR1012共同转染293T细胞,检测Vif-mbp和Vif-6His的表达,并进行A3G体内降解试验。将A3G和/或可产生病毒的质粒HXB2Neo△Vif分别与HIV-1野生型Vif/VR1012、Vif-mbp/VR1012和Vif-6His/VR1012共同转染293T细胞,通过A3G包装进病毒和Magi细胞感染试验,进一步检测HIV-1Vif-mbp和Vif-6His融合蛋白的生物学功能。结果重组表达质粒Vif-mbp/VR1012和Vif-6His/VR1012经双酶切鉴定证明构建正确。转染293T细胞48h后,Vif-mbp和Vif-6His融合蛋白均有表达,但表达的Vif-mbp有不同程度的降解。Vif-6His可降解A3G,使其表达量下降至10%左右,而Vif-mbp几乎不能降解A3G。Vif-6His可阻止A3G包装进病毒颗粒中,而Vif-mbp无此功能。Vif-mbp可使HXB2Neo△Vif病毒感染能力恢复至15%,而Vif-6His可使其恢复至86%。结论已成功构建了带有6His和mbptag的HIV-1Vif基因真核表达载体,表达的Vif-mbp融合蛋白影响了Vif的生物学功能,但Vif-6His融合蛋白不影响Vif的生物学功能。

In Vivo Expression and Biological Function of HIV-1 Vif Protein

Objective To construct eukaryotic expression plasmids for HIV-1 Vif gene with 6His-tag and mbp-tag, express fusion protein in vivo and study its biological function. Methods Amplify Vif gene with 6His-tag and mbp-tag by PCR and clone into eukaryotic expression vector VR1012. Transfect 293T cells with antiviral factor APOBEC3G(A3G), together with empty vector VR1012, wild type HIV-1 Vif / VR1012, Vif-mbp / VR1012 and Vif-6His / VR1012 respectively, determine the expressions of Vif-mbp and Vif-6His, and perform in vivo degradation test on A3G. Transfect 293T cells with A3G and / or virus-producing recombinant plasmid HXB2NeoΔVif, together with wild type HIV-1 Vif / VR1012, Vif-mbp / VR1012 and Vif-6His / VR1012 respectively, and study the biological functions of HIV-1 Vif-mbp and Vif-6His by package of A3G into virus particle and Magi cell infectivity test. Results Restriction analysis proved that both recombinant plasmids Vif-mbp / VR1012 and Vif-6His / VR1012 were constructed correctly. Both Vif-mbp and Vif-6His were expressed in 293T cells 48 h after transfection though the Vif-mbp was degraded at different degrees. Vif-6His degraded A3G and decreased the expression level of the latter to about 10%. However, little A3G was degraded by Vif-mbp. Vif-6His inhibited the packaging of A3G into virus particles, while Vif-mbp showed no the inhibitory effect. The infectivity of HXB2NeoΔVif co-expressed with Vif-mbp and Vif-6His fusion proteins were 15% and 86% respectively, setting that in absence of A3G as 100%. Conclusion Vif-mbp fusion protein showed significant influence, while Vif-6His showed no influence on the biological function of Vif.