乙型肝炎病毒X蛋白与细胞色素c氧化酶Ⅲ的特异性结合

Study on the interaction between hepatitis B virus X protein and cytochrome c oxidase Ⅲ

目的探讨HBVX蛋白与细胞色素c氧化酶Ⅲ（COXⅢ）之间的结合作用。方法采用交合实验验证HBVX蛋白和COXⅢ在酵母细胞内的结合作用，利用离体结合实验证实两者在体外的结合作用，采用免疫共沉淀实验证实两者在哺乳动物细胞中的结合作用。结果携带CoXⅢ基因的酵母与携带X基因的酵母交合后发生反应，β-半乳糖苷酶（LacZ）活性检测菌落呈现蓝色，离体结合实验Western印迹中出现特异性结合活性反应条带，提示X蛋白和COXⅢ在体外存在直接相互作用，免疫共沉淀实验在相对分子质量为17000处出现蛋白条带，提示在哺乳动物细胞水平仍能检测到X蛋白和COXⅢ的特异结合。结论COXⅢ与X蛋白在原核细胞和哺乳动物细胞中均存在特异性结合，HBV可能通过此反应干扰线粒体呼吸链功能及能量代谢过程。

Objective To explore the interaction between hepatitis B virus （HBV） X protein and cytochrome c oxidase Ⅲ （COX Ⅲ ）. Methods Mating experiment and in vitro binding assays were utilized to verify the interaction between HBV X protein and COX Ⅲ in vivo and in vitro respectively in yeast cells. Coimmunoprecipitation was employed to demonstrated the binding between these two proteins in mammalian cells. Results In mating experiment, the resulting diploids containing both pAS2-1-X and pACT2-COX Ⅲ plasmids turned blue in the β-gal assay, the result of Western blot displayed a positive reactive strap which indicated X protein could interact directly with COX Ⅲ in vitro binding assays. Interaction between these two proteins in mammalian cells was further confirmed by coirnmunoprecipitation, as a protein strap about 17 000 was detected in Western blot. Conclusions It is speculated the specific interaction may exist between HBV X protein and COX Ⅲ in both prokaryotic and mammalian cells. X protein may impair the mitochondrial respiration chain and energy metabolism through its association with COX Ⅲ.