人类免疫缺陷病毒I型Tat真核表达载体的构建及鉴定

Construction and in Vitro Expression of an Eukaryotic Vector Encoding HIV-1 Tat

目的构建人类免疫缺陷病毒Tat基因真核表达质粒,并研究该质粒在真核细胞中的有效表达。方法以含有HIV-1全长基因的质粒pNL4-3为模板,通过PCR扩增HIV-1 Tat第一外显子,应用基因工程技术将扩增的基因片段插入真核表达质粒pcDNA3.1(+),构建重组真核表达质粒pcDNA3.1-tat,经限制性内切酶酶切分析及测序鉴定正确后,应用脂质体转染技术转染入huh-7细胞。RT-PCR检测其基因转录情况,Western Blot鉴定其是否能够表达相应的目的蛋白质。结果成功扩增了Tat基因,酶切和测序证明正确构建了重组真核表达质粒pcDNA3.1-tat,RT-PCR和Western blot方法证实该质粒能在huh-7真核细胞中有效表达HIV-1 Tat目的蛋白质。结论成功构建了HIV-1 Tat基因的真核表达质粒载体,并在huh-7中表达,为在huh-7细胞模型中研究Tat的功能奠定了基础。

Objective To construct an eukaryotic plasmid expressing HIV-1 Tat and test whether the plasmid can be expressed in the eukaryotic cell in vitro.Methods The first exon of HIV-1 Tat was amplified by PCR from the plasmid pNL4-3.Then the Tat gene was inserted into the eukaryotic expression plasmid pCDNA3.1（＋）,and the resultant recombinant plasmid was confirmed by restriction endonuclease and sequencing,then was designated as pCDNA3.1-tat.The recombinant plasmid pCDNA3.1-tat was transfected into eukaryotic cell huh-7 by lipo-fectamine.RT-PCR and Western blot were used to certify whether the purpose protein was correctly expressed in huh-7 cells. Results The 240 bp DNA fragment of tat was correctly amplified.The recombinant pCDNA3.1-tat was successfully constructed,and it could be correctly expressed in the huh-7 cells. Conclusions This recombinant plasmid pCDNA3.1-tat and the resulting huh-7 cell model provide a basis for further study on the function of HIV-1 Tat.