含临床病毒株聚合酶逆转录酶区的乙肝病毒DNA稳定复制细胞系的构建  

Establishment of a Stable Cell Line Replicating Hepatitis B Virus DNA Carrying the Reverse Transcriptase Region Derived from a Clinical Isolate

作　　者：向明确[1] 蔡雪飞[2] 张文露[2] 黄爱龙[2] 胡接力[2] 

机构地区：[1]重庆医科大学重庆市第九人民医院感染科,重庆400700 [2]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

出　　处：《中国医学科学院学报》2013年第1期13-18,共6页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(81000732);重庆市自然科学基金(cstc2011jjA10005)~~

摘　　要：目的构建含有临床病毒株聚合酶逆转录酶(RT)区的乙肝病毒(HBV)DNA稳定复制细胞系。方法采用巢式PCR从患者血清扩增HBV DNA片段,利用片段置换反应将该片段克隆到HBV DNA复制载体,并在该载体上引入新霉素抗性基因,在确认该重组DNA体外可复制后,将其转染HepG2细胞,G418筛选,采用real-time PCR结合ELISA及Southern blot检测初筛和鉴定HBV DNA稳定复制细胞系。结果从患者血清扩增出的HBV DNA片段nt55～1654被成功置换到HBV复制质粒pLL相应区域,得到质粒p11;新霉素抗性基因表达片段被克隆到p11中HBV DNA下游,获得质粒p11-neo,Southern blot检测证实p11-neo可支持体外复制;p11-neo转染HepG2后,经筛选鉴定,获得了可支持HBV DNA稳定复制的细胞系3-10。结论建立了含有临床病毒株聚合酶RT区的HBV DNA稳定复制细胞系,real-time PCR结合ELISA有助于HBV DNA稳定复制细胞系的快速初筛鉴定。Objective To establish a stable cell line that can replicate hepatitis B virus （HBV） DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Methods Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction （ FSR）, followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay （ELISA） were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis. Results Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid pl 1. The neomycin fragment was cloned into pl 1, leading to the plasmid pl 1-neo, and pl 1-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained. Conclusions A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.

关 键 词：乙型肝炎病毒 逆转录聚合酶 复制 稳定细胞系 

分 类 号：R512.62[医药卫生—内科学]