重组质粒pEGFP-C3-HCVc的构建及在RBE细胞中的表达

doi:10.3969/j.issn.1000-8535.2016.05.003

广州医药 ›› 2016, Vol. 47 ›› Issue (5): 7-10.DOI: 10.3969/j.issn.1000-8535.2016.05.003

重组质粒pEGFP-C3-HCVc的构建及在RBE细胞中的表达

曾兵^1,2, 蔡灿锋¹, 辛海洋¹, 周泉波², 周佳佳², 程帝², 曾军¹, 唐超明¹

1 广州医科大学附属第六医院普外科(清远 511518)
2 中山大学孙逸仙纪念医院胆胰外科(广州 510120)

收稿日期:2016-06-30 出版日期:2016-09-20 发布日期:2021-12-02
通讯作者: 曾兵,E-mail:zengbing2007@163.com
基金资助:
国家自然科学基金(81401996);广东省自然科学基金(2015A030310099)

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

Zeng Bing, Cai Canfeng, Xin Haiyang, Zeng Bing, Zhou Quanbo, Zhou Jiajia, et al

Zeng Bing, Cai Canfeng, Xin Haiyang, et al. Department of General Surgery, The sixth affiliated hospital of Guangzhou Medical University, Qinyuan 511518, China
Zeng Bing, Zhou Quanbo, Zhou Jiajia, et al. Department of pancreatobiliary Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-senUniversity, Guangzhou 510120, China

Received:2016-06-30 Online:2016-09-20 Published:2021-12-02

摘要/Abstract

摘要： 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。

关键词: HCVc, 肝内胆管癌, RBE

Abstract: 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.

Key words: Hepatitis C Virus core protein, Intrahepatic cholangiocarcinoma, RBE

曾兵, 蔡灿锋, 辛海洋, 周泉波, 周佳佳, 程帝, 曾军, 唐超明. 重组质粒pEGFP-C3-HCVc的构建及在RBE细胞中的表达[J]. 广州医药, 2016, 47(5): 7-10.

Zeng Bing, Cai Canfeng, Xin Haiyang, Zeng Bing, Zhou Quanbo, Zhou Jiajia, et al. Construction of recombinant plasmid of pEGFP-C3-HCVc and its expression in RBE cells[J]. Guangzhou Medical Journal, 2016, 47(5): 7-10.

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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

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