稳定表达HIV-1 GagPol基因真核表达载体的构建与鉴定  

Stable expression and identification of GagPol gene from HIV-1 in HEK293

作　　者：张一折[1] 王鹏[2] 张守涛[1] 刘伟[1] 刘国祥[1] 

机构地区：[1]郑州大学生物工程系,郑州450001 [2]吉林省四平市疾病预防控制中心,四平136000

出　　处：《郑州大学学报（医学版）》2009年第1期145-148,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：郑州大学人才引进基金资助项目

摘　　要：目的:构建能稳定表达HIV-1 GagPol基因的真核表达载体。方法:用PCR方法扩增HIV-1 GagPol基因,克隆入pMD18-T,然后亚克隆入真核表达质粒pcDNA3.1(-)中,将构建的重组质粒pcDNA3.1(-)/GagPol分别瞬时和稳定转染HEK293细胞,经G418筛选,建立稳定转染GagPol基因的细胞系,并应用Western Blot方法鉴定表达产物。结果:酶切鉴定和Western Blot检测证实重组质粒pcDNA3.1(-)/GagPol构建正确,并能稳定表达HIV-1 GagPol蛋白。结论:成功构建携带HIV-1 GagPol基因的真核表达载体pcDNA3.1(-)/GagPol,并在HEK293细胞中获得稳定表达。Aim ： To construct a recombinant eukaryotic expression plasmid for stable expression of HIV-1 GagPol protein and to detect its expression in HEK293 in order to lay the foundation for further development HIV-1 VLP vaccine. MoIhods：HIV-1 GagPol gene was amplified by PCR, then subcloned into eukaryotic expression vector pcDNA3. 1 （ - ）. The recombinant plasmid pcDNA3.1 （ - ）/GagPol was transfected into HEK293 cell line , and the expression of the GagPol gene was analyzed by Western Blot. Rosults： The successful construction of peDNA3. 1 （ - ）/GagPol was verified by restrictive endonuclease assay, and the expression of GagPol gene was detected in the lysate of transfected HEK293 cells by Western Blot. Conclusion： The recombinant plasmid pcDNA3.1 （ - ）/GagPol was successfully constructed and its stable expression in HEK293 cell line was also confirmed in the present study.

关 键 词：HIV-1 GagPol基因 稳定表达 病毒样颗粒疫苗 

分 类 号：R183[医药卫生—流行病学]