HIV-1B亚型核心蛋白gag基因真核表达载体的构建及其在HepG2细胞中的表达

目的克隆人类免疫缺陷病毒Ⅰ型B亚型核心蛋白gag基因,构建真核表达载体,并在真核细胞中表达,为进一步制备自行设计的以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HIV1B亚型U26942全基因质粒DNA作为模板,根据Genbank中gag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,特异性的扩增gag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gag编码基因的真核表达载体,并进行酶切鉴定分析pcDNA3.1(+)/gag。在脂质体介导下转染HepG2细胞,经G418压力筛选建立稳定转染gag基因的细胞系,用RT PCR及Western印迹检测其在HepG2细胞中的表达。结果重组质粒经BamHⅠ、XhoⅠ双酶切成5.4kb与1.5kb的片断,表明表达载体pcDNA3.1(+)中插入了gag基因片断,测序结果表明编码框正确。RT PCR及Western印迹证实稳定转染gag基因的HepG2细胞系中有该基因的表达。结论成功构建了HIV1B亚型核心蛋白gag基因的真核表达载体pcDNA3.1(+)/gag,并在HepG2细胞中获得稳定表达。

Construction of the eukaryotic expression plasmid contaning the gag gene of HIV-I subtype B and its expression in HepG2 cells

To construct a eukaryotic expression plasmid containing the gag gene of HIV-1 subtype B and to detect its expression in HepG2 cells in order to lay the foundation for further development of DNA vaccine against HIV infection, a pair of primers were designed and synthesized according to the published sequence of gag gene in GenBank. After amplification with PCR, the amplified product was cloned into plasmid pIMD18-T using TA cloning followed by Bam HI and Xho I digestion and sequencing. The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1(+), and the recombinant plasmid was sequenced and identified by restrictive endonuclease digestion later on, the constructed recombinant plasmid was finally transfected into HepG2 cell line by using the lipid transfection reagent ,and the expression of the gag gene was analyzed by RT-PCR and Western blotting. It had been demonstrated that the successful construction of the recombinant vector pcDNA3.1(+)/gag was verified by restrictive endonuclease assay and sequence analysis with the same target fragment gag as U26942 in GenBank, and the expression of the gag gene was detectable in the lysate of transfected HepG2 cells as demonstrated by RT-PCR and Western blotting. Thus it is concluded the eukaryotic expression/plasmid for gag gene is successfully constructed and its expression in HepG2 cell line was also confirmed in the present study.