Objective To construct a Tat-dependent MazF expression vector pcDNA3.1-GFP-HA-MazF-U3TAR.Methods HIV-1 U3TAR and MazF gene were amplified from pCR2.1-U3TAR and pET28a-MazF,respectively.Two gene fragments were linked together to form U3TAR-MazF by an overlapping PCR,and then cloned into pMD18T.An N-terminal HA tag was added to MazF to form U3TAR-MazF-HA.After double enzyme digestion using EcoR Ⅰ and Hind Ⅲ,U3TAR-MazF-HA was reversely inserted into pcDNA 3.1 to form pcDNA3.1-HA-MazF-U3TAR.Then,a fluorescent reporter gene GFP was inserted into the downstream of U3TAR to form pcDNA3.1-GFP-HA-MazF-U3TAR.Results The cotransfection experiments with pcDNA3.1-tat-flag showed that pcDNA3.1-GFP-HA-MazF-U3TAR is Tat dependent.MazF was expressed only under the presence of Tat.In addition,compared with the cells transfected with pcDNA3.1-GFP-HA-MazF-U3TAR,less green fluorescent signal was observed in the cells co-transfected with pcDNA3.1-GFP-HA-MazF-U3TAR and pcDNA3.1-tat-flag,indicating that expressed MazF down-regulated the expression of GFP.Conclusions The expression vector pcDNA3.1-GFP-HA-MazF-U3TAR will provide an important tool for the development of MazF-based AIDS gene therapy strategies.%目的 建立HIV-1 Tat依赖的MazF表达载体pcDNA3.1-GFP-HA-MazF-U3TAR系统.方法 分别扩增HIV-1 U3TAR和MazF基因片段,通过重叠PCR将U3TAR和MazF连接起来,并TA克隆到pMD18T载体中.通过PCR为MazF引入HA标签,获得U3TAR-MazF-HA.经过双酶切和连接反应,将U3TAR-MazF-HA反向插入到pcDNA3.1,获得pcDNA3.1-HA-MazF-U3TAR.将GFP基因正向插入到MazF基因之后,为该载体引入了荧光报道基因,获得pcDNA3.1-GFP-HA-MazF-U3TAR.结果 与pcDNA3.1-tat-flag的共转染实验证明,构建的MazF表达载体pcDNA3.1-GFP-HA-MazF-U3TAR是Tat依赖的.对比pcDNA3.1-GFP-HA-MazF-U3TAR单转染的细胞,共转染的细胞具有更少的绿色荧光信号,表明表达的MazF可以下调报道基因GFP的表达.结论 pcDNA3.1-GFP-HA-MazF-U3TAR的构建,为探索基于MazF的艾滋病基因治疗方案提供了载体工具.
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