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  

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

作　　者：李德良[1] 马文丽[1] 师永霞[1] 李凌[1] 张宝[1] 郑文岭[1] 

机构地区：[1]南方医科大学基因工程研究所,广东广州510515

出　　处：《南方医科大学学报》2006年第12期1724-1727,共4页Journal of Southern Medical University

基　　金：国家博士后基金资助项目~~

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。Objective To construct an eukaryotic expression plasmid containing gp 120 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 ofgp 120 gene was cloned into pMD- 18T vector using TA cloning followed by BamH Ⅰ and Xho Ⅰ 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 ofgp 120 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 （＋）/gp 120. The target fragment gp 120 was identical with U26942 in Genbank, and the expression of gp 120 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.

关 键 词：人类免疫缺陷病毒 PCDNA3.1(+) GP120 基因表达 

分 类 号：Q78[生物学—分子生物学]