L-02-hNTCP稳定表达细胞系的建立与鉴定  

Establishment and stable of a HBV susceptible L-02-hNTCP cell line-expressing

作　　者：李瑞明 贺昱霖 程彬 魏志强 孟忠吉 LI Rui-ming;HE Yu-lin;CHENG Bin;MENG Zhong-ji(Taihe Hospital,Hubei University of Medicine,Shiyan Hubei,442000,China)

机构地区：[1]十堰市太和医院(湖北医药学院附属医院)生物医学研究所,湖北十堰442000

出　　处：《中西医结合肝病杂志》2021年第6期524-527,共4页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：湖北省科技厅创新群体项目(No.2018CFA031);湖北省教育厅项目(No.B2015480)。

摘　　要：目的:建立L-02-hNTCP稳定表达细胞系并筛选对HBV感染性颗粒敏感的克隆,为研究HBV入侵细胞的机制及药物筛选提供基础。方法:用Lipofectamine 3000转染hNTCP真核表达质粒至L-02细胞,加入含嘌呤霉素(0.5μg/ml)培养基筛选耐药克隆,进一步用免疫荧光筛选hNTCP高表达克隆,RT-PCR定量hNTCP转录水平;在L-02-hNTCP细胞培养基中加入HBV感染性病毒颗粒,ELISA检测培养上清HBV抗原水平,免疫荧光检测细胞内HBcAg,Real-time PCR检测复制中间体。结果:从92个耐药克隆中筛选获得1株hNTCP高表达克隆,命名为L-02-hNTCP。L-02-hNTCP胞内检测到高水平的hNTCP mRNA,免疫荧光检测到高水平Flag标签表达,且信号主要位于胞膜;经HBV病毒感染性培养基孵育24 h后,免疫荧光在细胞内检测到HBcAg,在培养上清中检测到HBsAg、HBeAg,Real-time PCR检测到细胞内HBV复制中间体(6.57±0.36)×10^(6)拷贝/孔。结论:成功建立稳定高表达hNTCP的L-02-NTCP细胞系,可以被HBV颗粒自然感染,为研究HBV入侵细胞的机制和抗病毒药物筛选提供新的模型。Objective:To establish the L-02-hNTCP cell line stable expressing that susceptive for HBV infection and provide a cell model for investigating the complete lifecycle of HBV,including the early steps of viral entry.Methods:The hNTCP eukaryotic expression plasmid was transfected into L-02 cells with lipofectamine 3000,and cultivated in the presence of puromycin(0.5μg/ml)for 3 days,positive clones were selected and further screened for the hNTCP high-expressing clones by immunofluorescence.After incubated in HBV infectious medium,the levels of HBsAg,HBeAg in the cell culture supernatants were detected by ELISA,the intracellular HBV replication intermediates were detected by Real-time PCR,the intracellular HBcAg were detected by immunofluorescence.Results:A total of 92 drug-resistant clones were selected,and the highest hNTCP-expressing clone(L-02-hNTCP)were obtained,with high level of hNTCP mRNA and high level of flag-tag,and the signal of hNTCP-flag was mainly located on the cell membrane.After treatment with HBV infectious medium,HBcAg was detected in the L-02-hNTCP cell,HBsAg and HBeAg were detected in the culture supernatants,and the amount of intracellular HBV replication intermediate was(6.57±0.36)×10^(6) copies/well.Conclusion:The novel L-02-hNTCP cell line is susceptive to HBV particles and may be used for studies in the mechanism of HBV invasion and antiviral drugs screening.

关 键 词：乙型肝炎病毒 hNTCP 病毒进入 

分 类 号：R575.3[医药卫生—消化系统] R459.9[医药卫生—内科学]