重组质粒pEGFP-C3-HCVc的构建及在RBE细胞中的表达

Construction of recombinant plasmid of p EGFP-C3-HCVc and its expression in RBE cells

目的构建重组p EGFP-C3-HCVc真核表达载体,并建立稳定表达HCVc基因的肝内胆管癌细胞株RBE-core。方法采用PCR钓取目的基因HCVc,并克隆入p EGFP-C3的多克隆位点,构建p EGFP-C3-HCVc重组质粒。经过双酶切及测序验证后,采用脂质体将p EGFP-C3-HCVc质粒转染到RBE细胞中,经2周G418(200μg/m L)筛选后进行单克隆挑选及扩大培养,建立稳定表达HCVc的胆管癌细胞株RBE-core。采用RT-PCR和Western blot验证HCVc在RBE-core中的表达情况。结果 PCR成功钓取HCVc基因,大小约573 bp,并插入p EGFP-C3载体HindⅢ和Bam HⅠ多克隆位点;双酶切及测序证实目的基因HCVc正确连接到p EGFP-C3的多克隆位点。RT-PCR和Western blot分别在573 bp处和34 KD左右检测到相应的阳性条带。结论成功构建重组质粒p EGFP-C3-HCVc,并在胆管癌细胞RBE中获得稳定表达。

Objective To construct a recombinant plasmid of p EGFP-C3-HCVc containing hepatitis C virus core protein,and establish the HCVc-expressing cell line RBE-core. Methods The HCVc gene was amplified by PCR and cloned into HindⅢ and Bam HⅠsite of p EGFP-C3 plasmid. The recombinant plasmid of p EGFP-C3-HCVc was confirmed by sequencing.RBE cells were transfected with the recombinant plasmid by using Lipofectamine 2000,and then performed G418（ 200 μg / m L）selection after 2 weeks. The expressing of HCVc gene in RBE cells was confirmed by RT-RCR and western blot. Results The recombinant plasmid of p EGFP-C3-HCVc was successfully constructed. RT-PCR and western blot detected a 573 bp and 34 KD bland,indicating the stably expressing of HCVc in RBE cells. Conclusion The recombinant plasmid of p EGFP-C3-HCVc is stabled expressing in RBE cells,which provides support for the further study.