噬菌体表面展示技术筛选隐源性肝炎血清蛋白结合蛋白

Screening of serum protein of cryptogenic hepatitis binding protein by phage display system

目的:通过筛选隐源性肝炎血清蛋白结合蛋白,为隐源性肝炎的发病机制研究探索新途径.方法:应用噬菌体表面展示技术,将隐源性肝炎患者血清作为固相筛选蛋白,用噬菌体正常人肝细胞T7cDNA文库进行5轮生物筛选,经噬斑PCR,对筛选克隆进行DNA序列分析,将推断的氨基酸序列在蛋白质库中进行搜索,自GenBank中获得结合蛋白的cDNA和基因组的全长序列,然后再探讨该蛋白在隐源性肝炎发病机制中的作用.结果:噬菌体经过5轮生物富集后,从随机筛选的60个克隆中得到24个阳性克隆,经序列测定后进行同源搜索,初步确定人类HCVNS5A转化调节蛋白13(NS5ATP13)为人体肝脏中固有的与隐源性肝炎血清蛋白结合的蛋白.结论:应用T7cDNA噬菌体表面展示技术,我们发现隐源性肝炎血清蛋白与人类肝细胞HCVNS5A转化调节蛋白13相互作用,在隐源性肝炎发生及抗病毒治疗有重要意义.

AIM： To investigate the pathogenesis of cryptogenic hepatitis （CH） by screening its binding protein of serum protein. METHODS： Using serum protein of cryptogenic hepatitis as the selective molecule, T7 select phage from human liver cDNA library was biopanned and the positive clones were selected 5 times. After polymerase chain reaction of bacteriophage plaque, the DNA fragments of the screened clones were sequenced, and the amino acid sequence of target protein was compared with protein sequence database from BLAST in GenBank. RESULTS： After bioamplification for 5 times, 24 positive clones were obtained from 60 dones screened randomly. The sequence of the positive clones were researched for homology, and Homo sapiens hepatitis V virus NS5A transregulated protein 13 was ascertained as the serum protein of CH-binding protein, which was located at liver. CONCLUSION： Serum CH protein is interacted with Homo sapiens hepatitis V virus NS5A trans-regulated protein 13 in liver, which plays an important role in the pathogenesis and treatment of CH.