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目的 筛选丙型肝炎病毒 (HCV)非结构蛋白NS4A结合蛋白 ,为HCV致病机制的研究探索新的途径。方法 应用噬菌体展示技术 ,以HCV非结构蛋白NS4A作为固相筛选分子 ,对T7噬菌体人肝细胞cDNA文库进行 5轮“吸附 洗脱 扩增”的筛选过程 ,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 ,从随机挑选的 12个克隆中得到 2个阳性克隆 ,成功构建了克隆载体。序列测定后经过序列同源性分析 ,确定了与HCV非结构蛋白NS4A结合的是肝细胞蛋白 丝裂原激活蛋白激酶(MAPK)激活蛋白激酶 5 (MAPKAPK5 )。结论 用噬菌体人肝cDNA文库筛选得到了HCV非结构蛋白NS4A的结合蛋白 ,为进一步研究HCV的致病机制奠定了良好的基础。
Objective To screen NS4A binding protein of hepatitis C virus (HCV) nonstructural protein and explore new pathways for the pathogenesis of HCV. Methods The phage display technique was used to screen the T7 phage human hepatocyte cDNA library with 5 cycles of “adsorption elution and amplification” using HCV nonstructural protein NS4A as the solid phase screening molecule. After plaque PCR amplification, The cloning vector was cloned. Finally, the DNA sequence and homology analysis of the selected clones were carried out. Results After phage enrichment, two positive clones were obtained from randomly selected 12 clones, and a cloning vector was successfully constructed. After sequence analysis, it was confirmed that MAPK activated protein kinase 5 (MAPKAPK5), which binds to HCV nonstructural protein NS4A, has been identified by sequence homology analysis. Conclusion The binding protein of NS4A, a non-structural HCV protein, was screened from phage human liver cDNA library, which laid a good foundation for further study on the pathogenesis of HCV.