APOBEC3A通过脱氨基作用抑制HBV复制

发布时间：2018-01-26 03:46

  本文关键词： 乙型肝炎病毒 APOBECA 抑制 脱氨基作用　出处：《中国生物工程杂志》2015年12期 　论文类型：期刊论文

【摘要】：目的:研究APOBEC3A抑制HBV复制的分子机制。方法:首先在肝癌细胞HuH7中过表达APOBEC3A,通过MTT法检测了APOBEC3A对细胞毒性的影响;通过免疫荧光检测了APOBEC3A在细胞中的定位,通过IP试验进一步证实了APOBEC3A与病毒颗粒的相互作用;并通过ELISA,特异性荧光定量PCR检测了HBV复制的参数包括上清中HBsAg,病毒核心颗粒中HBV DNA以及核内的cccDNA的表达水平;最后通过3D PCR方法分析了核心颗粒中HBV DNA脱氨基作用。结果:过表达APOBEC3A对HuH7细胞毒性没有显著性差异;APOBEC3A主要位于细胞核,但APOBEC3A可以与病毒颗粒结合,在逆转录环节对HBV复制发生抑制作用;共转染HBV复制质粒和APOBEC3A表达质粒后,细胞上清中HBsAg,核心颗粒中的HBV DNA以及核内的cccDNA均显著下降;最后通过3D PCR和克隆测序表明核心颗粒中的HBV DNA负链发生了大量的G-A突变,同时正链也发生了较多的C-T突变。结论:APOBEC3A可与病毒颗粒结合,在HBV复制的逆转录环节可作用于HBV单链,发生脱氨基作用,从而抑制HBV的复制。
[Abstract]:Objective: to study the molecular mechanism of APOBEC3A inhibiting HBV replication. Methods: firstly, APOBEC3A was overexpressed in HuH7 of hepatoma cells. The effects of APOBEC3A on cytotoxicity were detected by MTT method. The localization of APOBEC3A in cells was detected by immunofluorescence, and the interaction between APOBEC3A and virus particles was further confirmed by IP assay. The parameters of HBV replication including HBsAg in supernatant were detected by ELISA-specific fluorescence quantitative PCR. The expression level of HBV DNA and cccDNA in the core particles of the virus; Finally, the deamination of HBV DNA in the core particles was analyzed by 3D PCR. Results: there was no significant difference in the cytotoxicity of HuH7 cells induced by overexpression of APOBEC3A. APOBEC3A is mainly located in the nucleus, but APOBEC3A can bind to virus particles and inhibit the replication of HBV in reverse transcription. After co-transfection of HBV replication plasmid and APOBEC3A expression plasmid, HBsAg in supernatant, HBV DNA in core granules and cccDNA in nucleus decreased significantly. Finally, 3D PCR and clone sequencing showed that a large number of G-A mutations occurred in the negative strand of HBV DNA in the core particles. At the same time, more C-T mutations occurred in the positive strand. Conclusion: the HBV replication of HBV can act on the single strand of HBV and deamination. Thus, the replication of HBV was inhibited.
【作者单位】： 重庆医科大学感染性疾病分子生物学教育部重点实验室;
【基金】：国家自然科学基金(81471945) 重庆市科委自然科学基金(cstc2014jcyjA10075)资助项目
【分类号】：R512.62
【正文快照】： 乙型肝炎病毒(Hepatitis B virus,HBV)感染是一个严重的公共问题,全球大约有350万左右的慢性乙肝患者[1]。HBV慢性感染可引起慢性乙型肝炎(chronic hepatitis B,CHB)、肝硬化(liver cirrhosis,Lc)和原发性肝细胞癌(hepatocellulor carcinoma,HCC)等相关疾病[2],而治疗慢性乙型，

本文编号：1464581