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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 investigate the effects of deletion of CP part on the transactivation of X protein by detecting the activity of the core promoter (CP) of 20 2/2 1bp deletion (nt 174 8/174 7 ~ nt 176 7) of hepatitis B virus. Methods The HBVCPnt16 0-1 ~ 186 0 fragment was cloned into the chloramphenicol acetyltransferase (CAT) reporter gene expression vector (pCAT3 Basic) to construct recombinant pCAT3 plasmid (20d pCAT3, 21d pCAT3, wt pCAT3) and transfected into HepG2 Cells were tested for CP activity. HepG2 cells were co-transfected with wt pCAT3 and the wild-type strain or CP2 0/2 1bp partially containing the full-length HBV recombinant plasmid (P3.8I, 20d P3.8I, 21d P3.8I) to detect X Transactivation of proteins. ELISA method to detect the expression of CAT. Results The clones which were screened by PCR and double enzyme digestion were successfully identified by sequencing. Recombinant pCAT3 plasmid transfected HepG2 cells showed that CP deletion recombinant expression vector (20d pCAT3,21d pCAT3) compared with wild-type CP recombinant expression vector (wt pCAT3) produced by the amount of CAT decreased significantly, are only the latter 14.2%. The co-transfection assay showed that wt pCAT3 could be transactivated by the entire X protein produced by P3.8Ⅰ, while the transcripts of 20d P3.8Ⅰ, 21d P3.8 Ⅰ truncated X protein produced by the change. Conclusions The CP activity of HBV 2 0/2 1bp deletion is significantly lower than that of the wild-type CP. The X protein of CP2 0/2 1bp deletion is difficult to transactivate.