人类白细胞抗原-B＊2704／B＊2705重链拮抗肽的筛选和初步鉴定  

Screening and identification of HLA-B ＊ 2704/B ＊ 2705 antagonistic peptides

作　　者：李彦博[1] 孙玉英[1] 张立新[1] 骆媛[1] 郭斯启[1] 陆浩军[1] 奚永志[1] 

机构地区：[1]军事医学科学院附属医院免疫学实验室国家生物医学分析中心免疫学实验室,北京100071

出　　处：《中华风湿病学杂志》2008年第11期739-742,I0002,共5页Chinese Journal of Rheumatology

基　　金：国家“973”重大基础研究基金资助项目（2003CB515509）;国家自然科学基金资助项目（30470751）

摘　　要：目的从噬菌体展示随机12肽库筛选人类白细胞抗原（HLA）-B＊2704及B＊2705重链拮抗肽并做初步鉴定。方法用HLA—B＊2704／B＊2705重链胞外区蛋白分别筛选噬菌体展示随机肽库，酶联免疫吸附试验（ELISA）鉴定阳性克隆，DNA测定确定氨基酸序列，免疫荧光和流式细胞术鉴定噬菌体克隆分别与HLA—B＊2704及B＊2705细胞株结合的特异性。结果经3轮筛选，获得12个HLA—B＊2704拮抗肽，共有5种序列，分别为HTSFCSTHLCLI（×4），QHCSPTLCQIHR（×5），ARCTITLCYLSN（×1），YGLCTDWYCHIT（×1），YPLCDAILCRLP（×1）；10个B＊2705拮抗肽共有4种序列，分别为：①SHCSPHWCALPF（×6）；②HLCSNSLCLLPW（×2）；③EPMCSWFWCTLP（×1）；④WTCSPLLCTWGA（×1）。比对分析表明，B＊2704与B＊2705拮抗肽的序列是基本一致的，均含有CS（T）TXXL（W）CXL表位。展示有B＊2704拮抗肽的噬菌体克隆可与HLA—B＊2704细胞株结合，阳性率为43．55％；而B＊2705拮抗肽噬菌体克隆与HLA—B＊2705细胞株的阳性结合率为45．69％。结论筛选获得的HLA—B27拮抗肽的噬菌体克隆具有一定的亲和力，可与表达于细胞株表面的B＊2704和B＊＊2705分子特异性结合，而与正常B细胞不结合，因而表现出一定的结合特异性。Objective To screen and identify the B ＊ 2704/B ＊ 2705 H-chain antagonistic peptide from Ph.D.12^TM phage displayed random peptide library. Methods Purified expression protein of B＊2704/B ＊ 2705 were used as substrates for the screening of Ph.D. 12^TM phage displayed random peptide library. Positive phage clones were identified by ELISA. The positive clones for B ＊ 2704 and B ＊ 2705 were selected for ssDNA sequencing, respectively. Immunofluorescence analysis and flow eytometry analysis were used to demonstrate the specificity of, positive phage clones that bind to HLA-B ＊ 2704/B ＊ 2705 cell lines. Results After 3 cycles of screening, 12 clones for B ＊ 2704 and 10 clones for HLA-B ＊ 2705 were selected for ssDNA sequencing. It was shown that five binding-peptide sequences for HLA-B ＊ 2704 were HTSF-CSTHLCLI （ ×4 ）, QHCSPTLCQIHR （ ×5 ）, ARCTITLCYLSN （ × 1 ）, YGLCTDWYCHIT （ × 1 ） and YPLCDAI-LCRLP （ × 1 ）. 10 B ＊ 2705 binding peptides shared 4 sequences： SHCSPHWCALPF （×6）,HLCSNSLCLL PW （×2）, EPMCSWFWCTLP （×1）, WTCSPLLCTWGA （×1）. B ＊ 2704 and B ＊ 2705 binding peptides had a consensus sequence, CS（T）TXXL（W）CXL, which indicated that these peptides had common binding epitopes between them. The bacteriophage clones of B ＊ 2704 binding peptide could bind to HLA-B ＊ 2704 cell line with a positive rate of 43.55%, while bacteriophage clone of B ＊ 2705 binding peptide could recognize HLA-B ＊ 2705 cell line with a 45.69% positive rate. Conclusion The HLA-B ＊ 2704/B ＊ 2705 binding peptides show some affinity and specificity to B ＊ 2704 and B＊2705 molecules expressed in cell surface but do not bind to normal B cells.

关 键 词：HLA—B27抗原 肽库 拮抗肽 

分 类 号：R686[医药卫生—骨科学]