[乙型肝炎病毒抗原与金属硫蛋白之间的相互作用]

[Mutual interaction between hepatitis B virus antigen and metallothionein].

作者信息

Lu Yin-Ying, Liang Yao-Dong, Cheng Jun, Chen Tian-Yan, Wang Lin, Liu Yan, Li Ke, Zhang Ling-Xia, Yang Yong-Ping

机构信息

Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of People's Liberation Army, Beijing 100039, China.

出版信息

Zhonghua Gan Zang Bing Za Zhi. 2004 Sep;12(9):540-2.

PMID:15387906

Abstract

OBJECTIVE

To screen and identify the protein interacting with HBV antigen in hepatocytes. Then investigate the biological functions of hepatitis B virus antigen in the pathogenesis of hepatitis B and seek effective methods to prevent and treat it.

METHODS

The yeast two-hybrid system-3 technique was used to construct HBV PreS2, HBeAg, HBcAg, HBxAg bait plasmids. The bait plasmids transformed the yeast AH109 and expressed themselves in it. After being identified by SDS-PAGE and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium to form diploid yeast and was then plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. To further prove the interaction between HBV antigen and metallothionein, translation was performed by using reticulocyte lysate and coimmunoprecipitation was displayed in vitro.

RESULTS

Genes coding for HBV antigen binding protein were successfully cloned and metallothionein was found in that protein. The interaction between HBeAg, HBcAg and HBxAg and metallothionein were further proved by coimmunoprecipitation in vitro.

CONCLUSION

The interaction between HBV antigen and metallothionein indicates that metallothionein may participate in the pathogenesis of hepatitis B

摘要

目的

筛选并鉴定在肝细胞中与乙肝病毒（HBV）抗原相互作用的蛋白质。进而研究乙肝病毒抗原在乙型肝炎发病机制中的生物学功能，并探寻有效的防治方法。

方法

采用酵母双杂交系统-3技术构建HBV前S2抗原、e抗原、核心抗原、X抗原诱饵质粒。将诱饵质粒转化酵母AH109并使其在其中表达。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）鉴定后，将AH109酵母与含有肝脏cDNA文库质粒的酵母Y187在2×YPDA培养基中进行杂交形成二倍体酵母，然后接种于合成缺陷营养培养基（SD/-Trp-Leu-His-Ade）以及含X-α-半乳糖的合成缺陷营养培养基（SD/-Trp-Leu-His-Ade）上进行筛选。提取蓝色菌落的质粒并转化至大肠杆菌，随后通过DNA测序和生物信息学进行分析。为进一步证实HBV抗原与金属硫蛋白之间的相互作用，利用网织红细胞裂解液进行体外翻译并展示共免疫沉淀。