HBV-C基因型复制细胞系的构建与鉴定

肝脏 ›› 2021, Vol. 26 ›› Issue (9): 998-1002.

HBV-C基因型复制细胞系的构建与鉴定

李瑞明, 杨燕, 曾宪煌, 孟忠吉

442000 湖北十堰市太和医院(湖北医药学院附属医院)(李瑞明,孟忠吉);华中科技大学同济医学院附属同济医院实验医学研究中心(杨燕);武汉大学基础医学院(曾宪煌)

收稿日期:2020-10-10 出版日期:2021-09-30 发布日期:2021-10-22
通讯作者: 孟忠吉,Email: zhongji.meng@163.com
基金资助:
湖北省科技厅创新群体项目(2018CFA031);湖北医药学院自由探索基金创新群体项目(FDFR201902);十堰市科学技术研究与开发项目(18K78);十堰市科技局引导性科研项目(19Y27)

Establishment of a cell line supporting the gene expression and replication of hepatitis B virus-genotype C

LI Rui-ming¹, YANG Yan², ZENG Xian-huang³, MENG Zhong-ji¹

1. Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China;
2. Division of Clinical Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;
3. School of Basic Medical Sciences, Wuhan University , Wuhan 430071,China

Received:2020-10-10 Online:2021-09-30 Published:2021-10-22
Contact: MENG Zhong-ji,Email: zhongji.meng@163.com

摘要/Abstract

摘要： 目的构建HBV临床分离株复制细胞系,为抗病毒药物筛选提供新的细胞模型。方法从乙型肝炎患者血清中PCR扩增乙肝病毒全长基因组,经SapI酶切,T4连接酶连接环化后作为模板,PCR扩增0.2拷贝HBV基因组(nt1402-nt1989)及1.0拷贝HBV基因组(nt1402-nt3215-nt1407);依次克隆入pcDNA3(-)-EGFP-ΔCMV载体得到p1.2×HBV-EGFP质粒;用Lipofectamine 2000 将重组质粒转染HepG2细胞,使用含G418(700 μg/mL)培养基培养2~3周,挑选阳性克隆,进一步传代培养获得HBV复制细胞系(HepG2X15)。收集细胞系培养上清ELISA检测HBsAg和HBeAg水平;Real-time PCR检测HBV病毒含量。收集培养细胞,Southern blot检测细胞内HBV复制中间体;免疫荧光检测细胞内HBsAg和HBcAg。结果筛选出1株来源于临床分离株的HBV-C基因型复制细胞系,在培养上清中检测到高水平HBsAg、HBeAg及HBV DNA,细胞内检测到HBV复制中间体,免疫荧光检测到细胞内高水平HBsAg和HBcAg。与HepG2.2.15细胞系相比,上清中HBsAg和HBV DNA水平更高。结论成功建立来源于临床分离株的HBV-C基因型复制细胞系,该细胞系稳定表达HBsAg、HBeAg和HBcAg,并支持高水平的HBV复制,可以用于抗HBV药物筛选及临床分离株的耐药机制等研究。

关键词: 乙型肝炎病毒, 临床分离株, 复制, 细胞系

Abstract: Objective To establish a novel cell line supporting the gene expression and replication of hepatitis B virus (HBV)-genotype C, and provide a new cell model for antiviral research. Methods HBV DNA was extracted from the serum of hepatitis B patients. Full-length of HBV genome was amplified by polymerase chain reaction (PCR). The circularized HBV full-length genome was generated by Sap I restriction enzyme cleavage and T4 ligase ligation, and was used as a template to amplify 0.2 copies of HBV genome (nt1402-nt1989) and one copy of HBV (nt1402-nt3215-nt1407), respectively. The two HBV DNA PCR products were cloned into pcDNA3(-)-EGFP-△CMV vector to obtain a p1.2×HBV-EGFP plasmid, followed by sequencing verification. The recombinant plasmid was transfected into HepG2 cells with lipofectamine 2000. Positive clones were selected by culturing the cells in the presence of G418 (700μg/ml) for 2-3 weeks. The levels of HBsAg, HBeAg, and HBV DNA in the cell culture supernatants were detected by ELISA and real-time PCR, respectively; The intracellular HBV replication intermediates were detected by Southern blotting, and the intracellular HBsAg and HBcAg were detected by immunofluorescence. Results A novel cell line, namely HepG2X15, was established for supporting the replication of HBV DNA -genotype C that derived from clinical isolates. It was evidenced that high levels of HBsAg, HBeAg, and HBV DNA were detectable in the culture supernatant, and intracellular HBV replication intermediates, HBsAg, and HBcAg were detectable in the cells. Compared with HepG2.2.15 cell line, HepG2X15 cells secrete higher levels of HBsAg and HBV virions into the culture supernatant. Conclusion A novel cell line HepG2X15 supporting the replication of HBV- genotype C was successfully established, with high production of HBsAg, HBeAg, and HBV virions, and intracellular HBV replication intermediates. It can be used for screening of anti-HBV drug and for investigating the mechanisms of anti-HBV drug resistance.

Key words: Hepatitis B virus, Clinical isolates, DNA replication, Cell line

李瑞明, 杨燕, 曾宪煌, 孟忠吉. HBV-C基因型复制细胞系的构建与鉴定[J]. 肝脏, 2021, 26(9): 998-1002.

LI Rui-ming, YANG Yan, ZENG Xian-huang, MENG Zhong-ji. Establishment of a cell line supporting the gene expression and replication of hepatitis B virus-genotype C[J]. Chinese Hepatolgy, 2021, 26(9): 998-1002.

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