摘要
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 investigate the intracellular and extracellular affinity between hepatitis B virus e antigen （HBeAg） and protein AK026018. Methods The gene AK026018 was amplified by reverse transcription polymerase chain reaction （RT PCR） technique from HepG2 cell. The expressive vector of pGADT7-AK was constructed by routine molecular biological methods. The auxotroph yeast cells were cotransfected with pGADT7-AK and pGBKT7-eAg and plated on synthetic dropout nutrient medium （SD/-Trp-Leu His-Ade） containing x-α-gal. To further prove the affinity between HBeAg and protein AK026018, translation was performed by using reticulocyte lysate, and immunoprecipitation was showed in vitro. Results The expressive vector was constructed and con firmed by DNA sequencing analysis and restriction enzyme digestion. The positive clones could grow on SD/ Trp-Leu-His-Ade/x-α-gal medium and were blue. The radioautographic band were found in immuno precipitation analysis. Conclusions HBeAg could bind protein AK026018 in vivo and in vitro.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2006年第1期12-14,共3页
Chinese Journal of Infectious Diseases
基金
国家自然科学基金资助项目(30100170)
