丙型肝炎病毒丝氨酸蛋白酶特异性结合肽的筛选和鉴定  

Selection and Characterization of Peptides That Specifically Binding to Hepatitis C Virus Serine Protease

作　　者：杜桂鑫[1] 侯利华[1] 陈万荣[1] 张永国[1] 王海涛[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071

出　　处：《微生物学报》2002年第3期298-304,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金重点项目资助 ( 396 30 0 2 0 )~~

摘　　要：丙型肝炎病毒丝氨酸蛋白酶在病毒复制和包装中的重要作用使其成为特异性抗病毒药物研究的首选靶标。根据丝氨酸蛋白酶晶体结构特点 ,用柔性连接子连接NS3丝氨酸蛋白酶结构域和NS4A的核心序列 ,构建成单链丝氨酸蛋白酶基因并且在大肠杆菌中获得高水平的可溶性表达 ,纯化后的目的蛋白能够切割重组蛋白底物NS5ab。随后 ,以单链丝氨酸蛋白酶为靶分子对噬菌体展示的随机十二肽库进行了三轮淘筛 ,挑选的 44个克隆中有 3 7个克隆能够特异性地结合丝氨酸蛋白酶 ,并且这种结合作用为竞争性ELISA试验结果所支持。对 1 3个克隆进行序列测定 ,得到 6种序列 ,它们在氨基酸组成上存在明显偏性 ,富含组氨酸和色氨酸 ,缺乏酸性氨基酸 ;The HCV NS3 serine protease that plays important role in the processing of polyprotein and the replication of virus is a prime target for antiviral drugs and therapy research. Based on the crystallographic structure of HCV sreine protease, a single-chain protease was contstructed in which the central sequence of NS4A was fused to the N-terminus of NS3 serine protease domain via a flexible linker and it was expressed at high level in soluble form in E.coli. The purified protease could cleave the recombinant protein NS5ab into two parts. The purified protease was used as target to screen binding peptides from phage displayed peptide library. After three rounds of affinity screening, 37 out of 44 randomly selected phages could bind specifically with the single-chain serine protease and their specificity were verified by competitive ELISA. The 13 sequenced clones represents 6 kinds of sequences of which the amino acids composition is in bias and there is a consensus sequence:[H/F/W]-H-W-X-X-W.

关 键 词：丙型肝炎病毒 丝氨酸蛋白酶 特异性结合肽 筛选 鉴定 噬菌体展示 肽库 

分 类 号：R346[医药卫生—基础医学]