HBV 1.3倍基因组HepG2稳转细胞模型优势单克隆株的筛选及鉴定

Screening and identification of dominant monoclonal HepG2 cell strain with 1.3-fold HBV genome

背景乙型肝炎病毒(hepatitis B virus,HBV)体外感染模型是开展HBV生命周期、致病机理、药物筛选等研究的基础.随着临床抗HBV治疗进入“功能性治愈”“完全治愈”新时代的趋势,对能稳定模拟共价闭合环状DNA分子(covalently closed circular DNA,cccDNA)转录机制,乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)致病机理的细胞模型的需求日益迫切.HBV1.3倍(1.3-fold HBV)全基因组包含了全部HBV生物信息,能依赖自身启动子启动转录过程,支持cccDNA形成和完整病毒复制,最接近于HBV体内感染状态下的生命周期.慢病毒转染是一种以慢病毒为载体,将外源分子如DNA,RNA等导入真核细胞的技术,可形成稳定转染.目的采用慢病毒转染技术构建HBV 1.3倍基因组HepG2稳转细胞模型,筛选并鉴定出能稳定、高效表达HBV生物标志物的优势单克隆株.方法构建含1.3-fold HBV基因组信息的慢病毒质粒并包装慢病毒;以最佳感染复数(MOI)值将1.3-fold HBV慢病毒液侵染靶细胞HepG2,以抗生素杀稻瘟菌素(blasticidin,BSD)最佳筛选剂量筛选出稳定整合1.3-fold HBV基因的HepG2细胞系(1.3-fold HBV-HepG2),继而采用PCR法鉴定该细胞模型中的HBV DNA序列.将1.3-fold HBV-HepG2稳转细胞进行培养并挑选出9株候选阳性单克隆,通过测序各单克隆细胞的侧翼序列,以确定9株候选阳性单克隆相应的基因组在HepG2细胞基因组中的插入位置,再根据各候选单克隆株表达HBsAg、HBeAg的水平,筛选出最优势单克隆株.将该优势单克隆株与HepG2.2.15细胞株分别持续培养20代,比较这两种细胞株表达HBsAg、HBeAg、HBx、cccDNA、HBV DNA等标志物的水平及稳定性.结果(1)将慢病毒pLenti-BSD-1.3-fold HBV以MOI=30的参数侵染HepG2细胞,72 h后加入BSD抗生素(终浓度1μg/mL)进行筛选,连续培养15-20 d后,获得1.3-fold HBV-HepG2稳转细胞系,PCR鉴定出HBV DNA序列;(2)在挑选出的9株候选阳性单克隆中,编号A14的细胞株(命名为HepGA14)的HBsAg、HBeAg表达水平最高,分别为24.28 IU/mL、39.62 NCU/mL,其插入HepG2基因组位置为1:166461695-166461715,确定为优势单克隆株;(3)与HepG2.2.15细胞株比较,HepGA14优势单克隆株1-20代能稳定、高表达HBsAg、HBeAg、HBx、HBV DNA、cccDNA,差异均具有统计学意义(P<0.05).结论成功构建并筛选出1.3-fold HBV基因组HepG2稳转细胞模型优势单克隆株HepGA14,这为后续开展HBV-宿主关系、发病机制及药物筛选等奠定良好的基础.

BACKGROUND Hepatitis B virus(HBV)infection model in vitro is the basis for studying HBV life cycle and pathogenesis and for drug screening.With the clinical anti-HBV therapy entering the new trend of“functional cure”and“complete cure”,there is an urgent need for cell models that can stably simulate the transcription mechanism of covalently closed circular DNA(cccDNA)and the role of hepatitis B virus X protein(HBx).The 1.3-fold HBV genome contains all the biological information of HBV.It can start the transcription process by its own promoter,support the formation of cccDNA,and complete viral replication,which is closest to the life cycle of HBV in vivo.Lentivirus transfection is a technology that takes lentivirus as vector and introduces foreign molecules such as DNA and RNA into eukaryotic cells,which can form stable transfection.AIM To construct a HepG2 cell model with 1.3-fold HBV genome by lentivirus transfection technology,and to screen and identify the dominant monoclonal strain that can stably and efficiently express HBV biomarkers.METHODS A lentiviral plasmid containing 1.3-fold HBV genome information was constructed,and the recombinant lentivirus culture was used to infect HepG2 cells at the optimal multiplicity of infection(MOI).Blasticidin(BSD)was used to select HepG2 cell strains(1.3-fold HBV-HepG2)stably integrating the 1.3-fold HBV genome,and then the HBV in the cell model was identified by PCR.HepG2 cells stably carrying the 1.3-fold HBV genome were cultured and nine candidate positive monoclones were selected.The flanking sequences of each monoclonal cell were sequenced to determine the insertion position of the corresponding HBV genome in the genome of HepG2 cells.The most dominant monoclones were selected according to the expression levels of HBsAg and HBeAg.The expression levels and stability of HBsAg,HBeAg,HBx,cccDNA,and HBV DNA in HepG2 cells stably carrying the 1.3-fold HBV genome were compared.RESULTS The lentiviral plasmid plenti-bsd-1.3-fold HBV was used to infect HepG2 cells at an MOI of 30.After 72 h,BSD(final concentration 1μg/mL)was added for screening.After 15-20 d of continuous culture,stable 1.3-fold HBV-HepG2 cell line was obtained.HBV DNA sequence was then identified by PCR.Among the nine selected candidate positive monoclones,A14,in which the 1.3-fold HBV genome was inserted into the HepG2 genome at 1:166461695-166461715(named HepGA14),had the highest expression levels of HBsAg and HBeAg at 24.28 IU/mL and 39.62 NCU/mL,respectively.HepGA14 can stably and highly express HBV biomarkers.Compared with HepG2.2.15 cell line,the expression levels of HBx and cccDNA in Hepga14 dominant monoclonal line in 1-20 passages were significantly higher(P<0.05).CONCLUSION We have successfully constructed and screened HepGA14,a dominant monoclonal HepG cell strain with HBV 1.3-fold genome,which lays a good foundation for further research of HBV-host relationship and pathogenesis as well as for drug screening.