HBsAg缺失型乙型肝炎病毒复制细胞模型的建立及其对乙型肝炎病毒复制的影响  

Construction of HBV replication cell model with HBsAg deletion and effect of HBsAg on HBV replication

作　　者：潘万龙[1] 方岩 许舸[3] 罗强[3] 黄媛[3] 陶颖[3] 黄爱龙[3] 胡接力[3] 

机构地区：[1]川北医学院微生物与免疫学教研室,四川南充637100 [2]甘肃省第二人民医院ICU,甘肃兰州730000 [3]重庆医科大学感染病分子生物学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2013年第8期1063-1066,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81000732);四川省教育厅重点基金(13ZA0221);川北医学院科研发展计划(CBY12-A-ZP01)

摘　　要：目的体外构建HBsAg缺失型乙型肝炎病毒(HBV)复制细胞模型,并探讨HBsAg对HBV复制的影响。方法定点突变pch9质粒上HBV 1.1倍体基因S蛋白表达区,构建HBV1.1倍体HBsAg突变质粒,并进行测序鉴定;将鉴定正确的突变质粒瞬时转染HepG2细胞,设未突变pch9质粒转染细胞作为对照,采用ELISA法检测转染72 h的细胞培养上清中HBsAg的表达;Southern blot及Real-time PCR法检测转染72 h的细胞内HBV DNA的复制水平。结果定点突变质粒经测序证实构建正确;突变质粒转染72 h的细胞培养上清中HBsAg A450值为(0.06±0.003),表达呈阴性,对照细胞A450值为(1.82±0.010),表达呈阳性,转染突变质粒72 h的细胞内HBV DNA条带浓度明显高于转染未突变质粒的细胞;转染突变质粒的细胞内HBV DNA绝对拷贝数分别为9.33×106、5.26×106,约为转染未突变质粒(6.91×105)的7.6～13.5倍。结论已成功构建了HBsAg缺失型HBV复制细胞模型,为HBV DNA复制及相关研究提供了实验依据;推测HBsAg是HBV DNA形成的自限性因素,能够负调控HBVDNA复制。Objective To construct HBV replication cell model with HBsAg deletion in vitro and investigate the effect of HBsAg on HBV replication.Methods The gene of S protein region in plasmid pch9 was site-directed mutated to construct HBV1.1 plasmid with HBsAg mutant,which was identified by sequencing.HepG2 cells were transiently transfected with HBV1.1 plasmid with HBsAg mutant,using plasmid pch9 without mutation as control.The expression of HBsAg in culture supernatant of HepG2 cells 72 h after transfection was determined by ELISA,while the replication of HBV DNA by Southern blot and real-time PCR.Results Sequencing result proved that HBV1.1 plasmid with HBsAg mutant was constructed correctly.The A450 value of HBsAg in culture supernatant of HepG2 cells 72 h after transfection with HBV1.1 plasmid was（0.06 ± 0.003）,indicating a negative result.However,the A450 value in the cells in control groups was（1.82 ± 0.01）,indicating a positive result.The concentration of HBV DNA bands in cells 72 h after transfection with HBV1.1 plasmid was significantly higher than that with plasmid pch9.The absolute copy number of HBV DNA in cells transfected with HBV1.1 plasmid in two tests were 9.33 × 106 and 5.26 × 106 respectively,which were 7.6 ~ 13.5 times of that in the cells transfected with plasmid pch9（6.91 × 105）.Conclusions The HBV replication cell model with HBsAg deletion was successfully constructed,which provided an experimental basis for HBV DNA replication and the relevant studies.It indicated that HBsAg was a self-limiting factor in the formation of HBV DNA,which negatively regulated HBV DNA replication.

关 键 词：肝炎病毒 乙型 表面抗原 突变 模型 

分 类 号：R373.21[医药卫生—病原生物学] Q939.44[医药卫生—基础医学]