血清类粘蛋白2下调乙型肝炎病毒表面抗原基因启动子Ⅰ转录活性的研究

Down-regulating effect of orosomucoid 2 on preS1 promoter of hepatitis B virus

目的:探讨血清类粘蛋白2(ORM2)对乙型肝炎病毒(HBV)表面抗原基因启动子Ⅰ(HBV-SP Ⅰ)转录的激活作用. 方法:以我实验室前期得到的HBV-SP Ⅰ的酵母单杂交系统筛选结果为基础,利用生物信息学技术确定ORM2的基因编码区域,聚合酶链反应(PCR)扩增ORM2编码基因,克隆至真核表达载体pcDNA3.1(-)中,构建pcDNA3.1(-)- ORM2载体;将该质粒与SP Ⅰ的氯霉素乙酰转移酶(CAT) 报告载体pCAT3-SP Ⅰ共转染肝癌细胞系HepG2细胞系, 并以pCAT3-SP Ⅰ单独转染HepG2细胞系作为对照,酶联免疫吸附法(ELISA)检测CAT的表达活性. 结果:pCAT3-SPⅠ在HepG2细胞中能够启动CAT的表达; 共转染实验pCAT3-SP Ⅰ+pcDNA3.1(-)-ORM2组CAT 的表达活性较pCAT3-SP Ⅰ下降了81.9%. 结论:OKM2蛋白具有对HBV-SP Ⅰ的反式抑制作用.本实验验证了我室利用酵母单杂交技术筛选HBV-SP Ⅰ特异结合蛋白的结果,为进一步了解HBV-SP Ⅰ的转录调控机制及其与SP Ⅰ结合的反式作用因子提供了新的线索.

AIM: To investigate activity of orosomucoid 2 (ORM2) on preS1 promoter (SP Ⅰ) of hepatitis B virus (HBV). METHODS: Yeast one-hybrid system was employed in screening of DNA-binding proteins specifically recognizing HBV-SP Ⅰ sequence, in which ORM2 was identified in GenBank by bioinformatics. For further studying the interaction between ORM2 and HBV-SP Ⅰ, the sequence of ORM2 was amplified from HepG2 genome by polymerase chain reaction (PCR) technique, which was then cloned into pcDNA3.1(-) expression vector. The HepG2 cell line was transfected by pCAT3- SP Ⅰ, and co-transfected by pCAT3-SP Ⅰ and pcDNA3.1(-)-ORm2, respectively. The chloRamphenicol acetyltransferase (CAT) activity was detected by an enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: pCAT3-SP Ⅰ had higher activity of CAT than pCAT3-basic by ELJSA kit. The expression of CAT from pCAT3-SP Ⅰ was increased 81.9%, as compared with that in the co-transfection of pCAT3-SP Ⅰ and pcDNA3.1(-)-ORM2. CONCLUSION: Cell transfection and ELJSA technology are successfully used to prove the results from yeast one-hybrid system, which brings some new clues for studying the specific binding proteins of HBV- SP I and its transcrip-tional regulation mechanism.