本文關(guān)鍵詞： 乙型肝炎病毒 APOBECA 抑制 脫氨基作用　出處：《中國生物工程雜志》2015年12期 　論文類型：期刊論文

【摘要】：目的:研究APOBEC3A抑制HBV復制的分子機制。方法:首先在肝癌細胞HuH7中過表達APOBEC3A,通過MTT法檢測了APOBEC3A對細胞毒性的影響;通過免疫熒光檢測了APOBEC3A在細胞中的定位,通過IP試驗進一步證實了APOBEC3A與病毒顆粒的相互作用;并通過ELISA,特異性熒光定量PCR檢測了HBV復制的參數(shù)包括上清中HBsAg,病毒核心顆粒中HBV DNA以及核內(nèi)的cccDNA的表達水平;最后通過3D PCR方法分析了核心顆粒中HBV DNA脫氨基作用。結(jié)果:過表達APOBEC3A對HuH7細胞毒性沒有顯著性差異;APOBEC3A主要位于細胞核,但APOBEC3A可以與病毒顆粒結(jié)合,在逆轉(zhuǎn)錄環(huán)節(jié)對HBV復制發(fā)生抑制作用;共轉(zhuǎn)染HBV復制質(zhì)粒和APOBEC3A表達質(zhì)粒后,細胞上清中HBsAg,核心顆粒中的HBV DNA以及核內(nèi)的cccDNA均顯著下降;最后通過3D PCR和克隆測序表明核心顆粒中的HBV DNA負鏈發(fā)生了大量的G-A突變,同時正鏈也發(fā)生了較多的C-T突變。結(jié)論:APOBEC3A可與病毒顆粒結(jié)合,在HBV復制的逆轉(zhuǎn)錄環(huán)節(jié)可作用于HBV單鏈,發(fā)生脫氨基作用,從而抑制HBV的復制。
[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.
【作者單位】： 重慶醫(yī)科大學感染性疾病分子生物學教育部重點實驗室;
【基金】：國家自然科學基金(81471945) 重慶市科委自然科學基金(cstc2014jcyjA10075)資助項目
【分類號】：R512.62
【正文快照】： 乙型肝炎病毒(Hepatitis B virus,HBV)感染是一個嚴重的公共問題,全球大約有350萬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)和原發(fā)性肝細胞癌(hepatocellulor carcinoma,HCC)等相關(guān)疾病[2],而治療慢性乙型，

本文編號：1464581