HBV转录激活蛋白HBx、MHBs^(t78)MHBs^(t155)真核重组载体构建  

Construction of Eukaryotic Recombinant Plasmids Encoding Hepatitis B Virus Transacivator HBx,MHBs^(t78), MHBst^(155) Proteins

作　　者：杨林[1] 江元森[1] 陈幼明[1] 李志刚[1] 李刚[1] 陈文思[1] 姚集鲁[1] 

机构地区：[1]中山大学附属第三医院传染科,广州510630

出　　处：《热带医学杂志》2004年第3期237-239,252,共4页Journal of Tropical Medicine

基　　金：国家教育部留学回国人员科研启动基金;广东省自然科学基金重点项目(No.20013116)。

摘　　要：目的分别构建人乙型肝炎病毒(HBV)X蛋白、羧基端截断的中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,以便进一步研究其转录激活功能及对宿主细胞信号传导通路的影响。方法设计合成3对寡核苷酸引物,以adr亚型HBV质粒pHBVDNA为模板,采用PCR法分别扩增HBVX基因、MHBst78与MHBst155编码基因片段;用HindⅢ,KpnⅠ双酶切HBVⅩ基因;用HindⅢ和BamHⅠ双酶切MHBst78与MHBst155编码基因片段后,分别定向插入到真核表达载体pcDNA3.1相应酶切位点,转化宿主菌JM109,提取质粒,分别用上述内切酶酶切及DNA测序鉴定重组质粒。结果酶切重组体显示所切下的片段大小均与预计相符,测序结果与文献报道序列及预计结果一致。结论成功构建了HBVX基因、羧基端截断的HBV中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,为进一步研究HBV转录激活蛋白HBx、MHBst78MHBst155对宿主细胞信号转导通路的影响奠定基础。Objective To construct the eukaryotic expression recombinant plasmids encoding hepatitis B virus (HBV)X protein, C terminally truncated middle surface protein MHBst78 and MHBst155 for the further investigation of the effects of HBV transactivator proteins on cell signaling transduction pathway.Methods Three pairs of specific primers were designed and synthesized according to the sequence of adr subtype HBV DNA, using plasmid pHBV containing adr subtype HBV as template.These gene fragments encoding HBV X, MHBst78 or MHBst155 proteins were amplified by polymerase chain reaction. After being digested using restriction endonuclease, HindⅢand KpnⅠ, or HindⅢand Bam HⅠ,the purified HBV X,MHBst78,and MHBst155 gene fragments were inserted into eukaryotic expression vector pcDNA3 1. Then, recombinant plasmids were identified by restriction endonuclease analysis and DNA sequencing. Result Restriction analysis and DNA sequencing confirmed that the cloned gene fragments encoding the HBV X, MHBst78,and MHBst155. Conclusion These eukaryotic recombinant plasmids encoding HBV X, MHBst78 or MHBst155 were successfully constructed, and it is available for further study of the effect of HBV transacivators on cell signaling transduction pathway.

关 键 词：乙型肝炎病毒 HBV X蛋白 羧基端截断的中分子HBV表面蛋白 真核表达载体PCDNA3.1 

分 类 号：R512.6[医药卫生—内科学]