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利用噬菌体肽库确定IL |
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刘北一 李一 王莉 于春雷 杨贵贞 富宁 周长城 齐杰 周慧 李惟
摘 要 目的：确定IL-2Rα表位，为研制高效特异性强的小分子肽类免疫抑制剂奠定基础。方法：用抗IL-2Rα单克隆抗体5G1对噬菌体六肽库进行筛选，选择出与5G1结合较强的克隆，经DNA序列分析，获得保守序列。对含保守序列的克隆进行生物学活性鉴定。结果：选择出31个与5G1结合较强的克隆。获得Ser-Ser-Phe和Ser-Ser-Arg两种保守序列。生物学活性鉴定表明：含Ser-Ser-Arg序列克隆较含Ser-Ser-Phe被抑制效果好。结论：Ser-Ser-Arg是IL-2Rα表位的关键序列。以此表位序列合成的小分子肽可作为IL-2Rα的拮抗剂而成为免疫抑制剂。
关键词 噬菌体肽库 IL-2R表位 免疫抑制剂
中国图书分类号 R392-33
Identification of IL-2Rαepitope sequences by phage peptide library
LIU Bei-Yi, LI Yi, WANG Li et al.
Department of Immunology, Norman Bethune University of Medical Sciences, Changchun 130021
Abstract Objective: To determine the amino acid sequences of IL-2R epitope that would be useful in making small peptide drugs as immunosuppressant. Methods: A mouse monoclonal antibody that is specific for the human IL-2Rα 5G1(anti Tac antibody) was used to screen a random phage peptide library which have 6 amino-acid residues displayed. Phage clones which could highly react with 5G1 was selected. After DNA sequencing, conservative sequences were acquired. The biological activities of phage clones were studied by sIL-2R blocking assay. Results: After 4 round of biopanning 31 phage clones were selected; two kinds of conservative sequence(Ser-Ser-Phe and Ser-Ser-Arg) were determined by single-strand dideoxy-sequencing by chine termination method. Ser-Ser -Arg clones were more effective than Ser-Ser-Phe clones. Conclusion:Ser-Ser -Arg sequence is the IL-2R epitope.
Key words Phage peptide library IL-2R epitope Immunosuppressant
噬菌体呈现技术（Phage displayed techniques,PDT）是利用丝状噬菌体在体外表达外源基因的一项新技术［1］。其技术要点是将外源基因插入改建过的噬菌体外壳蛋白基因PⅢ或PⅧ区以表达外源短肽。利用PDT技术制备的噬菌体表达文库主要包括肽文库（Peptide library）和抗体库（Antibody library）。噬菌体肽文库技术目前已成功地用于抗原表位分析、研究细胞因子表位、疫苗设计等［2～7］。本研究利用抗人IL-2Rα单克隆抗体5G1筛选噬菌体六肽库以确定IL-2Rα表位为研制高效特异性强的小分子肽类免疫抑制剂奠定基础。
1 材料与方法
1.1 噬菌体肽库系统 噬菌体六肽库、受体菌K91kan、野生型噬菌体FuseI由美国Miss[1] [2] 下一页
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