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目的 构建乙型肝炎病毒 (HBV)核心启动子 (CP) 2 0 /2 1bp部分缺失 (nt 1748/1747至nt 1767)及同时存在的A1896点变异真核载体 ,以进一步研究其对病毒复制与转录的影响。方法 利用线性化的、从CP上游顺序开始的、含完整转录单元的HBV基因克隆 (P3 .8Ⅰ质粒 )为工具 ,采用分子克隆、人工定点突变等技术构建突变重组载体。用脂质体法将重组载体转染HepG2细胞 ,提取细胞内DNA和RNA分别进行Southernblot、Northernblot分析。结果 经聚合酶链反应 限制性片段长度多态性 (PCR RFLP)初步鉴定和测序最终鉴定 ,突变重组载体构建成功 ;重组载体转染HepG2细胞后 ,提取细胞内DNA的Southernblot分析及细胞内RNA的Northernblot分析结果 ,均提示各变异株较野生株的HBVDNA复制水平及前C/前基因组mRNA、前S/SmRNA转录水平均显著下降。结论 HBVCP 2 0 /2 1bp部分缺失株及同时存在的A1896点变异株的病毒复制及转录水平均较野株显著下降
Objective To construct partial deletion (nt 1748/1747 to nt 1767) of 20 2 bp nucleocapsid (HBV) core promoter (CP) and simultaneous expression of A1896 point mutation eukaryotic vector in order to further study its effect on viral replication and The impact of transcription. Methods The linearized cloned HBV gene (P3.81 plasmid) containing the complete transcriptional unit from the upstream of CP was used as a tool to construct the mutant recombinant vector by molecular cloning and artificial site-directed mutagenesis. The recombinant vector was transfected into HepG2 cells by lipofectamine and the intracellular DNA and RNA were extracted and analyzed by Southern blot and Northern blot respectively. Results The recombinant plasmid was identified by PCR RFLP and identified by sequencing. The recombinant plasmid was successfully constructed. The recombinant plasmid was transfected into HepG2 cells and Southern blot analysis of intracellular DNA was performed. The result of Northern blot analysis indicated that the HBVDNA replication levels, pre-C / pre-mRNA and pre-S / SmRNA transcription levels of all mutants were significantly lower than those of wild-type strains. Conclusion The viral replication and transcription of HBVCP 2 0/2 1bp partial deletion and simultaneous A1896 point mutation were significantly lower than those of wild strain