HBV核心启动子部分缺失重组质粒的构建  

Construction of recombinant eukaryotic expression plasmid containing hepatitis B virus genome with partial deletion of the core promoter

作　　者：彭劼[1] 骆抗先[1] 林裕龙[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,广东广州510515

出　　处：《第一军医大学学报》2001年第11期806-808,818,共4页Journal of First Military Medical University

基　　金：国家自然科学基全(39630280)

摘　　要：目的　构建乙型肝炎病毒（ＨＢＶ）核心启动子（ＣＰ）部分缺失的真核表达质粒。方法　利用线性化的、从ＣＰ上游顺 序开始且含完整转录单元的ＨＢＶ 基因克隆（Ｐ３．８Ｉ质粒）为工具，采用分子克隆、人工定点突变、ＰＣＲ－ＲＦＬＰ，鉴定和测 序分析等技术构建目的载体。结果　成功构建了ＨＢＶ全基因ＣＰ ２０／２１个ｂｐ缺失（ｎｔ １７４８／１７４７－１７６７）和ＣＰ２０／２１个ｂｐ 缺失＋１８９６热点变异的重组真核质粒表达质粒，并经ＰＣＲ－ＲＦＬＰ序列分析证实。结论　此重组真核表达质粒构建可为 体外进一步研究上述变异的生物学意义奠定基础。Objective To construct an eukaryotic expression vector containing the full-length genome with partially deleted core promoter of hepatitis B virus (HBV). Methods A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units (P3.8 I plasmid) was used, which initiated from the upstream sequences of the basic core promoter. The objective eukaryotic expression vector was constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identifcation was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)followed by cloning and sequencing analysis. Results The eukaryotic expression vectors containing HBV genomes with 20/21 bp deletion (position 1 748/1 747 to 1 767) in the core promoter or with precore stop mutation at nucleotide 1896 were constructed successfully as confirmed by sequence analysis with RFLP. Conclusion The recombinant expression vector may lay the foundation for further studies into the biological significance of the above mentioned mutations in vitro.

关 键 词：乙型肝炎病毒 核心启动子 基因缺失 基因重组 CP HBV 质粒 

分 类 号：R373.21[医药卫生—病原生物学]