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目的验证乙型肝炎病毒HBeAg与AK026018基因表达蛋白在细胞内及细胞外的结合作用。方法应用逆转录聚合酶链反应(RT-PCR)技术,从HepG2细胞中扩增AK026018基因序列,构建真核表达载体,与HBeAg真核表达载体共转染营养缺陷酵母细胞,观察生长情况。同时进一步应用网织红细胞裂解体外翻译及免疫共沉淀系统,验证AK026018蛋白与HBeAg间的确切结合作用。结果经限制性内切酶分析和DNA序列测定构建的AK026018重组表达载体正确。共转染后的酵母细胞在营养缺陷培养基中生长良好。体外免疫共沉淀实验可见特异放射自显影带。结论HBeAg与AK026018基因编码蛋白在细胞内及细胞外均能相互结合,推测该分子在HBV的致病过程中起着重要作用。
Objective To verify the intracellular and extracellular binding of hepatitis B virus HBeAg and AK026018 gene. Methods AK026018 gene was amplified from HepG2 cells by reverse transcription-polymerase chain reaction (RT-PCR). The eukaryotic expression vector was constructed and co-transfected with the eukaryotic expression vector of HBeAg to observe the growth of yeast cells. At the same time, further application of reticulocyte lysate in vitro translation and co-immunoprecipitation system to verify the exact binding between AK026018 protein and HBeAg. Results AK026018 recombinant expression vector constructed by restriction endonuclease analysis and DNA sequencing was correct. Yeast cells co-transfected well grow in auxotrophic medium. In vitro co-immunoprecipitation experiments showed specific autoradiography. Conclusion Both HBeAg and AK026018 genes can bind to each other both intracellularly and extracellularly, suggesting that this molecule plays an important role in the pathogenesis of HBV.