乙型肝炎病毒X蛋白与细胞色素C氧化酶亚单位Ⅲ结合的定位研究  

Location of the binding position for HBV X protein functional interaction with cytochrome C oxidase Ⅲ

作　　者：李丹[1] 陈治新[1] 陈芸[1] 林纳[1] 王小众[1] 

机构地区：[1]福建医科大学附属协和医院消化科,福州350001

出　　处：《中华肝脏病杂志》2014年第10期725-730,共6页Chinese Journal of Hepatology

基　　金：国家自然科学基金（81300321）,福建省卫生系统中青年骨干人才培养项目（2014-ZQN-ZD-9）,福建省2012临床医学重点专科资助项目（闽卫科教[2012]（149号））

摘　　要：目的探索细胞色素C氧化酶亚单位Ⅲ（COXⅢ）与乙型肝炎病毒X蛋白（HBx）的具体结合位点。方法构建pAS2-1-X变异体重组载体，醋酸锂转化法将其转入酵母细胞，通过PCR法及测序法证实目的片段转入酵母细胞；Westem blot法明确变异体蛋白在酵母细胞中能否正确表达，滤膜转印法排除自身激活作用；固体培养基交合实验及β-半乳糖苷酶活性实验检测HBx和COXⅢ的结合区域。结果成功构建含HBx第1～72位氨基酸的变异体重组载体pAS2-1-Xl和第1～117位氨基酸的变异体重组载体pAS2-1-X2，测序及PCR均证实片段的正确性。Western blot证实变异体蛋白在酵母细胞中可正确表达，且变异体蛋白无自身激活作用。交合实验及β-半乳糖苷酶活性检测将HBx和COXⅢ的结合区域定位于72～117位氨基酸。结论通过酵母双杂合实验证实HBx和COXⅢ的结合区域定位于72～117位氨基酸，此结合区域的明确有望帮助进一步阐明X蛋白在体内的作用机制，并可能为慢性乙型肝炎、肝硬化及肝癌的防治提供新思路。Objective To identify the binding site position of the hepatitis B virus （HBV） X protein （HBx） functional interaction with the cytochrome C oxidase subunit Ⅲ（COXⅢ, a key regulator of mitochondrial function） by using a yeast two-hybrid system. Methods Two fragments of HBx mutants （X1 1-72aa and X2 1-117aa） were amplified by PCR and inserted into the bait plasmid pAS2-1. The resultant mutant plasmids were transfected into yeast cells using the lithium acetate-method. PCR and gene sequencing were used to confirm that the mutant fragments were expressed properly in yeast cells. Western blotting was used to verify that the mutant proteins were translated accurately in the yeast cells. Filter assay was used to exclude autoactivated mutants. Hybridization in solid medium and β-gal activity detection were used to determine the precise position of the binding site for HBx and COXⅢ interaction. Results The two mutant plasmids containing HBx 1-72aa and 1-117aa respectively were suceessfully constructed and the mutants were both properly expressed and translated in yeast cells; no autoactivated mutants were detected throughout the experimental process. The binding site of HBx and COXⅢ was found to be encompass the amino acids 72 through 117 of HBx. Conclusion Amino acids 72 through 117 of HBx are the key domain of the HBx functional interaction With COXⅢ; this domain may represent a useful target for molecular-based therapies to treat HBV-related diseases.

关 键 词：肝炎病毒 乙型 X蛋白 细胞色素C氧化酶亚单位Ⅲ 酵母双杂合 结合位点 

分 类 号：R512.62[医药卫生—内科学]