核心启动子部分缺失的乙型肝炎病毒基因的真核克隆及其意义  

Cloning and implication of eukaryotic expression vectors containing hepatitis B virus genomes with partial deletion in the core promoter

作　　者：彭劼[1] 骆抗先[1] 侯金林[1] 郭亚兵[1] 王战会[1] 

机构地区：[1]第一军医大学南方医院感染内科,广州510515

出　　处：《中华传染病杂志》2002年第3期140-143,共4页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目 ( 396 30 2 80 )

摘　　要：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 /2Objective In order to further study the influence of a mutant on viral replication and transfection, a eukaryotic vector with mutation of 20/21 bp deletion (1748/ 1747 to nt 1767) in core promoter region and precore stop mutation (nt.1896) was constructed. Methods A linearized genome containing the entire HBV 3.5kb mRNA transcriptional units (P3.8Ⅰ vector) and initiating from the basic core promoter upstream sequences was used as a tool, the objective eukaryotic vectors were constructed by the molecular cloning and PCR based site directed mutagenesis in vitro. The capability of progeny virus production and transcription were examined with Southern blot and Northern blot analysis respectively, after transfection of the recombinant HBV plasmids into HepG2 cells by using liposome. Results The eukaryotic vectors were constructed successfully and their sequences were confirmed by clone sequencing. Both Southern and Northern blotting of DNA and RNA extracted from the transfected cells showed markedly reduced mutant activity to produce progeny virus, to transcript both 3.5kb precore/pregenome mRNA and 2.1kb preS/S mRNA. Conclusions The levels of replication and transcription are markedly reduced in the mutant compared with those in wild type HBV.

关 键 词：乙型肝炎病毒 基因缺失 病毒复制 核心启动子 肢质体法 分子克隆 

分 类 号：R346[医药卫生—基础医学]