| 1.1倍全长HBV基因组克隆转染细胞系的建立 |
| |
| 引用本文： | 罗强,苏怀彬,梁盼盼,袁作为,张文露,黄爱龙,胡接力. 1.1倍全长HBV基因组克隆转染细胞系的建立[J]. 第三军医大学学报, 2011, 33(16): 1672-1675 |
| |
| 作者姓名： | 罗强 苏怀彬 梁盼盼 袁作为 张文露 黄爱龙 胡接力 |
| |
| 作者单位： | 重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆,400016 |
| |
| 基金项目： | 国家自然科学基金(81000732)~~ |
| |
| 摘 要： | 目的构建pneo-CH9/3091真核表达质粒,并建立其稳定表达的HepG2细胞系。方法质粒pCH9/3091包含HBV1.1倍体和CMV启动子,将PCR扩增出的带有新霉素(neo)抗性的基因片段插入pCH9/3091构建成重组质粒pneo-CH9/3091。重组质粒经酶切,测序验证正确后,通过脂质体介导转入人肝癌细胞HepG2。G418筛选后,利用酶联免疫吸附法(ELISA)检测所得单克隆细胞培养上清液的HBV表面抗原(HBsAg)和e抗原(HBeAg),PCR检测细胞上清液的病毒颗粒,Western blot检测细胞内HBV核心蛋白(HBcAg)的表达,以及Southern blot检测细胞内核心颗粒HBV DNA的复制情况。结果有3株细胞系培养上清HBsAg和HBeAg检测呈现阳性,且HBV C区片段可通过PCR扩增出来,West-ern bot结果提示只有细胞株HepG2-H7可以表达HBV核心蛋白。Southern blot结果说明此细胞株基因组中有HBV基因组的稳定整合,并且能在细胞内检测出HBV DNA复制中间体。结论成功构建1株HBV稳定整合细胞株HepG2-H7,能持续产生HBV病毒颗粒。
|
| 关 键 词： | 乙肝病毒 HepG2细胞 稳定细胞株 |
Establishment of cloned cell line transfected with 1.1-unit-length HBV genome |
| |
| Luo Qiang,Su Huaibin,Liang Panpan,Yuan Zuowei,Zhang Wenlu,Huang Ailong,Hu Jieli. Establishment of cloned cell line transfected with 1.1-unit-length HBV genome[J]. Acta Academiae Medicinae Militaris Tertiae, 2011, 33(16): 1672-1675 |
| |
| Authors: | Luo Qiang Su Huaibin Liang Panpan Yuan Zuowei Zhang Wenlu Huang Ailong Hu Jieli |
| |
| Affiliation: | Luo Qiang,Su Huaibin,Liang Panpan,Yuan Zuowei,Zhang Wenlu,Huang Ailong,Hu Jieli(Key Laboratory of Molecular Biology on Infectious Diseases of Ministry of Education,Chongqing Medical University,Chongqing,400016,China) |
| |
| Abstract: | Objective To construct eukaryotic expression plasmid pneo-CH9/3091 and establish a HepG2 cell line with stable expression of the plasmid.Methods Neomycin resistance gene(neo) obtained by PCR was inserted into plasmid pCH9/3091 containing 1.1-unit-length HBV genome and CMV promoter to construct recombinant plasmid pneo-CH9/3091.The recombinant plasmid,after being verified by enzyme digestion and sequencing,was transferred into human hepatocarcinoma cell line HepG2 by liposome-mediated transfection.The transf... |
| |
| Keywords: | Hepatitis B virus HepG2 cells stable cell line |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
