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重组质粒pEGFP-C3-HCVc的构建及在RBE细胞中的表达

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

来源期刊: 广州医药 | 7-10 发布时间:2021-12-02 收稿时间:2025/11/13 16:54:25 阅读量:7
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关键词:
HCVc肝内胆管癌RBE
Hepatitis C Virus core proteinIntrahepatic cholangiocarcinomaRBE
DOI:
10.3969/j.issn.1000-8535.2016.05.003
收稿时间:
2016-06-30 
修订日期:
 
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引用总数:
0  
目的 构建重组pEGFP-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pEGFP-C3的多克隆位点,构建pEGFP-C3-HCVc重组质粒。经过双酶切及测序验证后,采用脂质体将pEGFP-C3-HCVc质粒转染到RBE细胞中,经2周G418 (200 μg/mL) 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pEGFP-C3载体HindⅢ和BamHⅠ多克隆位点;双酶切及测序证实目的基因HCVc正确连接到pEGFP-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pEGFP-C3-HCVc,并在胆管癌细胞RBE中获得稳定表达。
Objective To construct a recombinant plasmid of pEGFP-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 BamHⅠsite of pEGFP-C3 plasmid. The recombinant plasmid of pEGFP-C3-HCVc was confirmed by sequencing. RBE cells were transfected with the recombinant plasmid by using Lipofectamine 2000, and then performed G418 (200 μg/mL) 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 pEGFP-C3-HCVc was successfully constructed. RT-PCR and western blot detected a 573bp and 34KD bland, indicating the stably expressing of HCVc in RBE cells. Conclusion The recombinant plasmid of pEGFP-C3-HCVc is stabled expressing in RBE cells,which provides support for the further study.
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