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目的 通过筛选乙型肝炎病毒 (HBV)表面抗原基因启动子Ⅱ(SPⅡ)结合蛋白 ,为HBV复制机制的研究探索新的途径。方法 应用噬菌体展示技术 ,以HBV的表面抗原基因启动子Ⅱ的聚合酶链反应 (PCR)产物DNA作为固相筛选分子 ,对噬菌体人肝细胞cDNA文库进行 5轮“吸附 洗脱 扩增”筛选过程 ,经噬斑的PCR扩增后 ,构建克隆载体 ,最后对所筛选克隆进行DNA序列测定和同源性分析。结果 噬菌体经富集后 ,从随机筛选的 2 0个克隆中得到 4个阳性克隆 ,成功构建了克隆载体。序列测定后经过同源性分析 ,确定了和HBVSPⅡ结合的肝细胞蛋白———尼克酰胺腺嘌呤二核苷酸脱氢酶 4 (NADHDH4 )。结论 用噬菌体人肝cDNA文库筛选得到了HBVSPⅡ的结合蛋白 ,分析了该蛋白的编码基因 ,为进一步研究HBV的复制、转录、调节机制开辟了新的途径。
OBJECTIVE: To explore a novel approach for the study of HBV replication mechanism by screening for the promoter of hepatitis B virus surface antigen (SP Ⅱ) binding protein. Methods Phage display technology was used to screen the cDNA library of phage human hepatocyte by 5 cycles of “adsorption elution and amplification” with polymerase chain reaction (PCR) DNA of promoter of surface antigen gene of HBV as solid screening molecule After plaque PCR amplification, the cloning vector was constructed. Finally, the DNA sequence and homology analysis of the selected clones were carried out. Results After phage enrichment, 4 positive clones were obtained from 20 random clones, and cloning vectors were successfully constructed. Sequence analysis after homologous analysis to determine and HBVSP Ⅱ hepatocyte protein --- Nick amide adenine dinucleotide dehydrogenase 4 (NADHDH4). Conclusion The HBVSPⅡ binding protein was screened from phage human liver cDNA library. The gene encoding this protein was analyzed, which opened up a new way to further study the mechanism of HBV replication, transcription and regulation.