摘要
探讨乙型肝炎病毒核心抗原 (HBcAg)在乙型肝炎发病机制中的作用 ,寻找防治乙型肝炎病毒 (HBV)感染的有效方法。应用改进的酵母双杂交技术构建HBcAg诱饵质粒 ,转化酵母细胞AH10 9后 ,与含肝文库质粒的酵母Y187进行配合、双杂交 ,经营养缺陷培养基(SD/ Trp Leu His Ade)及X gal双重筛选 ,获得真阳性菌落 16个 ,PCR扩增出靶基因 ,测序后进行生物信息学分析 ,发现其中含金属硫蛋白基因的菌落有 2个。进一步用网织红细胞裂解物体外翻译、蛋白间免疫共沉淀实验证实 ,金属硫蛋白与HBcAg间有确切的结合作用。
To investigate the potential role of hepatitis B virus core protein (HBcAg) in the pathogenesis of hepatitis B and seek for an effective therapeatic approach to the treatment of hepatitis B, the improved yeast two hybrid technique was employed to construct HBcAg bait plasmid, and the plasmids were transformed into yeast AH109. Then the yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2×YPDA medium, diploid yeast was plated on both synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing X α gal to be selected and screened two times. Plasmids were extracted from sixteen positive colonies, and they were sequenced. Among them, two colonies contained metallothionein. The interaction between HBcAg and metallothionein was confirmed by in vitro reticulocyte lysate translocation and immunoprecipitation. The results suggested that the pathogenesis of hepatitis B metallothionein might play an important role in the episode process.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2003年第2期161-163,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金攻关项目资助课题 (编号C39970 674)
关键词
乙型肝炎核心抗原
金属硫蛋白
双杂交系统
hepatitis B core antigens
metallothionein
two hybrid system techniques
