摘要
目的:利用噬菌体展示技术筛选重组干扰素β(IFNβ)肝细胞结合蛋白,探讨IFNβ抗病毒的分子生物学机制。方法:应用噬菌体展示技术,以重组的IFNβ蛋白包被酶联免疫板,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序列测定和生物信息学分析。结果:对肝细胞cDNA文库经过5轮的生物淘洗,从最后一轮筛选的顶层琼脂中随机挑选40个克隆,构建克隆载体,序列测定后经过同源性搜索和生物信息学分析,确定了和IFNβ蛋白具有结合作用的肝细胞蛋白。结论:筛选到与IFNβ蛋白具有结合作用的多种具有不同生理、生化功能的肝细胞蛋白。
Objective： To screen binding proteins of interferon β protein by phage display technique. Methods： Recombinant interferon βprotein was coated to the plate and incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to recombinant interferon β protein were enriched. DNA fragment from positive plaque were amplified by PCR and cloned into pGEM-Teasy vector in order to perform DNA sequence analysis and subsequently computer blasting analysis. Results： Positive phage-displayed protein binding with interferon β were enriched after five rounds of biopanning. The following proteins may have binding capacity with interferon β： homo sapiens PAI-1 mRNA-binding protein; homo sapiens nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate; homo sapiens pleckstrin homology domain interacting protein, mRNA; homo sapiens chromosome 8.Conclusion： Manyproteins with different functions could bind with interferon β protein.
出处
《中西医结合肝病杂志》
CAS
2005年第6期340-342,共3页
Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases
基金
国家自然科学基金(No.C39970674
No.C03011402
No.C39900130
No.C30070689)
