摘要
乙型肝炎病毒e抗原 (HBeAg)被认为与乙型肝炎病毒 (HBV)引起免疫耐受 ,免疫系统功能障碍有关。为探讨HBeAg在乙型肝炎发病机制中的作用 ,寻找防治HBV感染的有效方法 ,应用酵母双杂交系统 3构建HBeAg诱饵质粒 ,转化酵母细胞AH10 9后 ,与含肝cDNA文库质粒的酵母Y187进行配合、双杂交 ,在营养缺陷培养基 (SD/ Trp Leu His Ade)上及X α 半乳糖苷酶 (X α gal)蓝白斑双重筛选与肝细胞蛋白结合的蛋白 ,结果获得真阳性菌落 39个 ,提取酵母质粒转化大肠杆菌 ,测序后进行生物信息学分析 ,发现其中含金属硫蛋白(MT)基因的菌落有 3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HBeAg间有确切的结合作用。
To investigate the biological functions of hepatitis B virus e antigen (HBeAg) on pathogenesis of HBV, and to seek for effective methods to prevent and cure hepatitis B, we performed the yeast two hybrid system 3 technique to construct HBeAg bait plasmid. The bait plasmid was transformed into yeast AH109 and expressed in it. After the expression of HBeAg was identified by SDS page and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 YPDA medium to form diploid yeast and plated on synthetic dropout nutrient medium (SD/ Trp Leu His Ade) and synthetic dropout nutrient medium (SD/ Trp Leu His Ade) containing x α gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. Thirty nine positive colonies were sequenced, among them three colonies were metallothionein. To further prove the interaction between HBeAg and metallothionein, translation was performed by using reticulocyte lysate, and immunoprecipitation was showed in vitro . The results indicated that hepatitis B virus e antigen could interact with metallothionein in vivo and in vitro.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2003年第5期451-453,共3页
Medical Journal of Chinese People's Liberation Army
