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.
出处
《中华风湿病学杂志》
CAS
CSCD
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）
关键词
HLA—B27抗原
肽库
拮抗肽
HLA-B27 antigen
Peptide library
Antagonistic peptide
