中国流行的C基因型HBV稳定复制表达细胞系的建立  

Establishment of a stable expression and replication cell line transfected with HBV genotype C prevailed in China

作　　者：王琳[1,2] 刘文[2] 刘妍[2] 纪冬[2] 思兰兰[2] 钟彦伟[2] 徐东平[2] 

机构地区：[1]军事医学科学院,北京100850 [2]解放军302医院传染病研究所病毒性肝炎研究室

出　　处：《解放军医学杂志》2010年第12期1425-1428,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家“十一五”传染病重大专项子课题(2008ZX10002-011);北京市自然科学基金重点课题(7091006)

摘　　要：目的建立中国流行的C基因型HBV的稳定复制表达细胞系。方法从1例慢性乙肝患者血清中分离克隆得到1株C基因型病毒DNA全序列,以此为模板扩增产生1.3倍体HBV DNA,包含完整基因组(3.2kb)、从1825位点(polyA)至2599位点大小为775bp的3′端冗余序列,以及至1400位点大小为425bp的5′端冗余序列,将其连接改建重组后的pcDNA3.1(+)载体,构建pN31-51-6-1表达质粒,转染肝癌细胞系HepG2。采用ELISA检测HBsAg、HBeAg的瞬时表达情况,选择双高表达细胞孔进一步行G418筛选稳定表达细胞系。通过ELISA、免疫组化、直接免疫荧光流式细胞术以及HBV复制中间体核心颗粒DNA、细胞内cccD-NA、分泌上清中病毒DNA的RT-PCR检测等方法评估稳定细胞克隆的复制表达情况。结果获得1株具有高抗原表达和高复制能力的细胞克隆HepG2-51-6-1,已稳定传代26代。检测HBsAg、HBeAg吸光度(A)值分别为>3.5和1.29;直接免疫荧光流式细胞术证实细胞表面存在s抗原,免疫组化染色可见细胞内病毒表达。分泌到培养上清中的HBV DNA和细胞内的HBV复制中间体水平分别为2.0×104和5.5×105拷贝/ml,HBVccc DNA为6～8拷贝/细胞。结论成功获得HBV-1.3倍体转染的C基因型HBV稳定复制表达细胞系,为研究我国流行HBV的生物特性和抗HBV新药筛选奠定了基础。Objective To construct a cell line stably replicating and expressing HBV with HBV genotype C prevailed in China. Methods A full-length genotype C HBV genome was isolated from the serum sample of a patient with chronic hepatitis B.An 1.3-unit HBV DNA was amplified which contained the full-length HBV genome（3.2kb）,a 3’-end redundant nucleotide sequence from nt1825 （polyA）to nt2599（775bp）and a 5’-end redundant nucleotide sequence from nt1825to nt1400（425bp）.The 1.3-unit HBV DNA was ligated to a modified pCNDA3.1（＋）to construct a expression plasmid pN31-51-6-1.HepG2cells were then transfected with pN31-51-6-1.The secreted HBsAg and HBeAg in the supernatant were detected by ELISA.The cells with double high expression（A〉1）were selected and screened with antibiotic G418.The viral replication capacity and the expression level of stable clones were assessed by ELISA, immunohistochemistry,FCM and the detection of HBV replication intermediates in core particle,covalently closed circular DNA（cccDNA） and secreted HBV DNA by real-time quantitative PCR.Results The HBV cell line HepG2-51-6-1with high replicating and expressing ability was acquired for stable propagation of 26generations.Average A value of HBsAg and HBeAg were higher than 3.5and 1.29, respectively.The HBsAg was verified by FACS analysis and immunohistochemistry staining.Secreted HBV DNA and intracellular replication intermediate levels were 2.0 104 copies/ml and 5.5 105copies/ml,respectively.The cccDNA was 6to 8copies/cell.Conclusions A stable replicating and expressing HBV cell strain derived from genotype C 1.3-unit HBV is successfully established,which provides a tool for investigation of HBV virological features and screening of newly-developed anti-HBV agents.

关 键 词：肝炎病毒 乙型 基因型 病毒复制 细胞系 抗原 

分 类 号：R512.62[医药卫生—内科学]