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目的 :研究HBeAg与CD81分子在细胞内、外的相互作用。方法 :应用RT PCR技术 ,从HepG2细胞中扩增CD81全基因 ,并构建重组真核表达载体。将其与pGBKT7 eAg共转染营养缺陷型酵母细胞 ,观察生长情况。应用网织红细胞裂解体外翻译及免疫共沉淀试验 ,进一步验证CD81分子与HBeAg的结合。结果 :经EcoRI和BamHI酶切和DNA序列测定鉴定表明 ,构建的CD81基因的重组表达载体正确。共转染后的酵母细胞在营养缺陷的培养基中生长良好。体外免疫共沉淀试验证实 ,CD81与HBeAg出现沉淀带。结论 :HBeAg与CD81分子在细胞内、外均可结合 ,推测CD81在HBV的致病过程中起着重要的作用
Objective: To study the intracellular and extracellular interactions of HBeAg and CD81. Methods: The whole CD81 gene was amplified from HepG2 cells by RT-PCR and the recombinant eukaryotic expression vector was constructed. The pGBKT7 eAg co-transfection with auxotrophic yeast cells observed growth. In vitro reticulocyte lysate translation and co-immunoprecipitation assays were used to further confirm the binding of CD81 molecule to HBeAg. Results: EcoRI and BamHI digestion and DNA sequence identification showed that the constructed recombinant expression vector of CD81 gene was correct. Yeast cells co-transfected well grow in auxotrophic medium. Co-immunoprecipitation in vitro confirmed that CD81 and HBeAg appeared to precipitate. Conclusions: Both HBeAg and CD81 molecules can be bound both inside and outside the cell, suggesting that CD81 plays an important role in the pathogenesis of HBV