噬菌体表面展示技术筛选隐源性肝炎血清蛋白结合蛋白

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.

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

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 bio- panned and the positive clones were selected 5 times. After polymerase chain reaction of bac- teriophage 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 clones screened randomly. The sequence of the posi- tive clones were researched for homology, and Homo sapiens hepatitis V virus NS5A trans- regulated protein 13 was ascertained as the se- rum 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 treat- ment of CH.