The reverse transcriptase (RT) protein of hepatitis B virus (HBV) has been successfully expressed by recombinant technology in Eschericahia coli ( E. coli) . In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus (rt306P) and the polymerase gene from a mutant, with rt306P substituted by serine (rtP306S) were separately fused to the maltose binding protein (MBP) gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Western blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.
CHAO ZHAO YONG XIANG WANG ZHENG HONG YUAN YU MEI WEN
目的验证HBsAg阳性转基因小鼠肝脏中昼夜节律表达基因dbp(coding for the D site albumin promoter binding protein)转录水平的变化。方法用实时荧光定量聚合酶链式反应方法对比研究雌、雄及个体HBsAg阳性转基因小鼠及对照鼠肝脏dbp转录水平的变化。研究不同时间点HBsAg阳性转基因小鼠及对照鼠肝脏dbp转录水平的变化。结果#59及#10品系组HBsAg阳性转基因小鼠肝组织中dbp转录水平均较对照组动物上调,但个体间差别较大。初步结果显示雌性正常小鼠dbp转录水平较雄性高。而在雌性HBsAg阳性转基因小鼠中dbp上调进一步增强。与此相反,雄性转基因小鼠dbp上调则较同性对照鼠更弱。转基因小鼠dbp转录在8:00及14:00时平均水平均高于对照鼠,然而在20:00及2:00时与对照鼠相当。结论本研究首次报道HBsAg阳性转基因小鼠肝脏中dbp表达的上调,提供了乙型肝炎病毒蛋白表达与昼夜节律基因变化有所联系的证据。至于本研究在转基因鼠中的发现是否在乙型肝炎患者中存在及其意义还有待在患者中作进一步临床验证与研究。
