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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。
Objective To establish a serum hepatitis B virus (HBV) DNA clone and sequence analysis of patients with severe hepatitis B and analyze the relationship between HBV gene mutation and the incidence of severe hepatitis B from the whole gene level. Methods HBV DNA was extracted from serum of 10 patients with severe hepatitis B, and HBV whole genome was amplified by polymerase chain reaction (PCR). PCR products were constructed into pUCm-T vector and transformed into E. coli competent DH-5α cells. After identification by restriction enzyme digestion, the recombinant Clostridium was obtained with 3.2 Kb HBV DNA. The whole genome was sequenced and the nucleotide and Amino acid changes. Results Four HBV DNA clones were successfully constructed and the complete genome sequencing was completed. Three of them had G1896A mutation in the pre-C region, resulting in a stop codon, which resulted in the deletion of HBeAg. One case showed mutations in both the 1762 and 1764 C promoter regions. There were multiple point mutations and deletion mutations in PreS2 region and The C region is known to have cytotoxic epitopes of cytotoxic T lymphocytes, B lymphocytes and T lymphocytes. Conclusion The method can be used to study the relationship between the gene structure of HBV virus and the incidence of severe hepatitis B in clinical study and lay a foundation for further study on its HBV gene function.