【摘要】 目的建立重型乙型肝炎患者血清乙型肝炎病毒(HBV)DNA克隆并测序,从全基因水平分析HBV基因变异与重型乙型肝炎发病的关系。方法10例重型乙型肝炎患者血清提取HBV DNA,聚合酶链反应(PCR)扩增HBV全基因。PCR产物构建到pUCm-T载体上,转化至大肠杆菌感受态DH-5α细胞, 经酶切鉴定,获得含3.2 Kb HBV DNA的重组克隆菌,全基因测序, 分析各读码框核苷酸和氨基酸变化。结果4例成功构建HBV DNA克隆,并完成全基因测序。其中3例在前C区发生G1896A变异,产生一个终止密码子,导致HBeAg缺失; 1例在C启动子区1762、1764双位点出现突变;有多处点突变及缺失变异分布于PreS2区及C区已知细胞毒T淋巴细胞、B淋巴细胞和T淋巴细胞的细胞表位。结论该法可用于临床研究HBV病毒基因结构与重型乙型肝炎发病的关系,并为进一步研究其HBV基因功能奠定基础。更多还原

【Abstract】 Objective To study the association between hepatitis B virus (HBV) mutants and the pathogenesis of severe hepatitis B by full-length HBV genome. Methods Serum samples from 10 severe hepatitis B patients were collected in our hospital. Serum HBV DNAs were extracted using DNA mini Kit, and amplified by LA Taq DNA polymerase to yield full-length HBV DNA. PCR products were isolated and cloned into vector pUCm-T, then transfected into DH-5 a cells. Positive clones were selected and checked by digestion, and full-length HBV DNAs were sequenced. Results 4 cases were cloned into vector pUCm-T successfully and completed the full-length sequencing. Among them, 3 cases had a G to A mutation at nucleotide 1896 in pre-C region and 1 had a double mutation of T1762-A1764 in the core promoter region. Some amino acid changes occurred within the known CTL, B or T cell epitopes of the PrS2 and C regions. Conclusions This method could serve to study the relationship between HBV genome and the pathogenesis of severe hepatitis B.更多还原