南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (12): 1724-1727.

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HIV-1B亚型包膜糖蛋白gp120基因真核表达载体的构建及其在HepG2细胞中的表达

李德良； 马文丽； 师永霞； 李凌； 张宝； 郑文岭；   

  1. 南方医科大学基因工程研究所； 南方医科大学基因工程研究所 广东广州510515； 广东广州510515；
  • 出版日期:2006-12-20 发布日期:2006-12-20
  • 基金资助:
    国家博士后基金资助项目~~

Construction of a eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and its expression in HepG2 cells

LI De-liang,MA Wen-li,SHI Yong-xia,LI Ling,ZHANG Bao,ZHENG Wen-ling Institute of Genetic Engineering,Southern Medical University,Guangzhou 510515,China   

  1. 南方医科大学基因工程研究所； 南方医科大学基因工程研究所 广东广州510515； 广东广州510515；
  • Online:2006-12-20 Published:2006-12-20

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摘要： 目的克隆人类免疫缺陷病毒Ⅰ型B亚型包膜糖蛋白gp120基因,构建真核表达载体,并在真核细胞中表达,进一步为制备自行设计的以λ噬菌体作为载体的HIV核酸疫苗奠定基础。方法以克隆好的HIV-1B亚型U26942全基因质粒DNA作为模板,根据Genbank中gp120基因的核苷酸序列设计引物,并在引物的5’端分别引入BamHⅠ及XhoⅠ酶切位点,特异性地扩增gp120基因。TA克隆后经双酶切、测序等鉴定重组质粒,再经双酶切、连接构建含gp120编码基因的真核表达载体,并进行酶切鉴定分析pcDNA3.1(+)/gp120。在脂质体介导下转染HepG2细胞,经G418压力筛选建立稳定转染gp120基因的细胞系,用RT-PCR及Westernboltting检测其在HepG2细胞中的表达。结果重组质粒经BamHⅠ、XhoⅠ双酶切成5.4kb与1.44kb的片断,表明表达载体pcDNA3.1(+)中插入了gp120基因片断,测序结果表明编码框正确。RT-PCR及Westernblotting证实稳定转染gp120基因的HepG2细胞系中有该基因的表达。结论成功构建了HIV-1B亚型包膜糖蛋白gp120基因的真核表达载体pcDNA3.1(+)/gp120,并在HepG2细胞中获得稳定表达。

Abstract: Objective To construct an eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and obtain gp120 gene expression in HepG2 cells.Methods According to the published gp120 gene sequence in Genbank,a pair of primers was designed and synthesized.The PCR amplification product of gp120 gene was cloned into pMD-18T vector using TA cloning followed by BamHⅠ and XhoⅠ digestion and sequence analysis.The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid was sequenced and identified by restrictive endonuclease digestion,and transfected into HepG2 cells via liposome.The expression of gp120 gene was analyzed by RT-PCR and Western blotting,respectively.Results Restriction endonuclease digestion and sequence analysis verified successful construction of the recombinant vector pcDNA3.1(+)/gp120.The target fragment gp120 was identical with U26942 in Genbank,and the expression of gp120 gene was detected in the lysate of the transfected HepG2 cells by RT-PCR and Western blotting.Conclusion The eukaryotic expression plasmid for gp120 has been constructed successfully,which is capable of stable expression in HepG2 cells.

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