HIV-1 Tat真核表达载体的构建及在内皮细胞的表达

Cloning of Tat Gene of HIV-1 into a Mammalian Expression Vector and in Vitro Expression Studies

目的:构建HIV-1 Tat真核表达质粒,为研究Tat在HIV-1致病机制中的作用奠定实验基础。方法:以pCV1(tat and rev)质粒为模板,用PCR方法扩增HIV-1 Tat基因,克隆至p cDNA3.1(+)表达载体,酶切及测序鉴定重组体。重组质粒转染ECV304细胞,用RT-PCR、转染HIV-1 LTR荧光报告基因的方法检测tat基因的表达及活性。结果:经酶切及测序鉴定,成功构建pcDNA3.1(+)-Tat重组质粒。重组体转染ECV304细胞,建立稳定表达细胞株,应用RT-PCR可检测到Tat基因mRNA的表达;荧光仪检测Tat蛋白可明显促进荧光素酶在ECV304细胞内的表达。结论:成功构建HIV-1Tat真核表达载体,并建立了HIV-1 Tat内皮细胞稳定表达细胞株。

Objective: To construct a eukaryotic expressing vector pcDNA 3.1(+)-Tat for further studing Tat protein function in pathgenesis of HIV-1.Methods: The full length gene fragment of Tat was amplified by PCR.Then it was cloned into pcDNA3.1(+)eukaryotic expressing vector.The recombinant Tat expressing construct was confirmed by using XhoⅠand BamHⅠdouble digestion and by sequencing.The pcDNA3.1(+)-Tat was transfected into ECV304 cells.The expression of Tat was examined using reserve transcription PCR and HIV-1 L...