乙肝病毒核心启动子部分缺失对其活性及X蛋白转式激活作用的影响  

Effects of HBV core promoter with partial deletion on its activity and on trans-acting function of X-protein

作　　者：彭劼[1] 骆抗先[1] 郭亚兵[1] 侯金林[1] 陈金军[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：《中华微生物学和免疫学杂志》2002年第3期266-268,共3页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 (39630 2 80 )

摘　　要：目的　研究乙型肝炎病毒 (HBV) 2 0 / 2 1bp缺失 (nt 174 8/ 174 7～nt 176 7)的核心启动子(CP)的活性及CP部分缺失对X蛋白转式激活作用的影响。方法　将HBVCPnt16 0 1～ 186 0区段克隆到氯霉素乙酰转移酶 (CAT)报告基因表达载体 (pCAT3 Basic)上 ,构建重组pCAT3质粒 (2 0d pCAT3、2 1d pCAT3、wt pCAT3)转染HepG2细胞以检测CP活性。将wt pCAT3与野毒株型或CP 2 0 / 2 1bp部分缺失的含 1.2拷贝HBV全基因重组质粒 (P3.8Ⅰ、2 0d P3.8Ⅰ、2 1d P3.8Ⅰ )共转染HepG2细胞以检测X蛋白的转式激活作用。ELISA方法检测CAT表达量。结果　PCR扩增和双酶切初步筛选的克隆株 ,经测序最终鉴定重组质粒克隆成功。重组pCAT3质粒转染HepG2细胞表明 ,CP缺失的重组表达载体 (2 0d pCAT3、2 1d pCAT3)较野毒株型CP的重组表达载体 (wt pCAT3)产生的CAT量明显下降 ,均仅为后者的14 .2 % ;共转染试验表明 ,wt pCAT3可被P3.8Ⅰ产生的完整X蛋白转式激活 ,而分别不能或仅能较弱地被共转染的 2 0d P3.8Ⅰ、2 1d P3.8Ⅰ产生的截短的X蛋白所改变。结论　HBV 2 0 / 2 1bp缺失的CP活性较野毒株型CP活性显著下降 ;CP2 0 / 2 1bp缺失株的X蛋白难以发挥转式激活作用。Objective To study the activity of core promoter (CP) of hepatitis B virus (HBV) with 20/21bp deletion spanning from nt 1 748 (1 747) to nt 1 767, and to observe the effects of the CP deletion on trans acting function of X protein. Methods The partial HBV genome containing the CP region (nt 1 601 1 860) was cloned into the chloramphenicol acetyltransferase (CAT) expressing plasmid (pCAT3 Basic) to construct the recombinant CAT plasmids (20d pCAT3, 21d pCAT3, wt pCAT3). Results The transfection results showed that the levels of the CAT polypeptide in CP deletion transcripts (20d pCAT3, 21d pCAT3) were markedly reduced compared with that in wild type transcripts(wt pCAT3). In the cotransfection experiments, wt pCAT3 was transactivated by the intact X protein produced by P3.8Ⅰ. The CAT polypeptide amount of wt pCAT3, however, had no or only slight change by cotransfection with 20d pCAT3 or 21d pCAT3, which were designed to produce truncated X protein. Conclusion Promoter activity examined by transfection of the recombinant CAT plasmids containing the mutant DNA was much lower than that of wild type, and truncated X protein produced by the mutant did not possess a sufficient transactivating. [

关 键 词：X蛋白 转式激活作用 乙型肝炎病毒 核心启动子 缺失变异 表达载体 

分 类 号：R373[医药卫生—病原生物学]