中国株HIV-1核心蛋白真核表达载体的构建与表达

Construction of a Eukaryotic Vector and Expression for HIV-1 Core Protein Gene

运用限制性内切酶Xba Ⅰ、Sal Ⅰ对pKSGAG进行双酶切,获得HIV-1 gag基因,并与真核表达载体pCI-neo连接,构建含有中国流行株HIV-1核心蛋白真核表达载体pCI-neoGAG。经Xba Ⅰ/Sal Ⅰ双酶切及测序鉴定证实,成功地构建了HIV-1核心蛋白真核表达载体pCI-neoGAG。通过脂质体将pCI-neoGAG转染入p815细胞,G418筛选4周后,使用间接免疫荧光方法检测表达产物。结果表明所构建的HIV-1核心蛋白真核表达载体能在p815细胞中高效表达,为下一步进行HIV-1 DNA疫苗研究奠定了基础。

In order to construct the eukaryotic expression vector of Human immunodeficieacy virus 1 (HIV-1) core protein gene(pCI-neoGAG), gag gene was acquired from the plasmid pKSGAG by restriction endonuclease digestion (Xba Ⅰ/Sal Ⅰ) and ligated onto the eukaryotic expression vector pCI-neo. The construction of pCI-neoGAG was confirmed by restriction endonuclease digestion (Xba Ⅰ/Sal Ⅰ) analysis and DNA sequencing. Then the pCI-neoGAG was transfected into p815 cells by lipofectine and the expressed product was detected by indirect immunofluorescence after G418 selection for 4 weeks. The results showed that the core protein was expressed in p815 cells successfully, which will be used for the further test of HIV-1 DNA vaccine.