人类白细胞抗原-B*2704/B*2705重链拮抗肽的筛选和初步鉴定

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爪随机肽库,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拈抗肽,共有5种序列,分别为HTSFCSTHLCLI(×4),QHCSPTLCQIHR(×5),ARCTITL-CYLSN(×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拈抗肽噬菌体克隆与HLA-B·2705细胞株的阳性结合率为45.69%.结论 筛选获得的HLA-B27拈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.

Screening and identification of HLA-B * 2704/B * 2705 antagonistic peptides LI Yan-bo, SUN Yu-ying,

Objective To screenand identify the B * 2704/B * 2705 H-chain antagonistic peptide from Ph.D.12TM phage displayed random peptide library. Methods Purified expression protein of B*2704/B *2705 were used as substrates for, the screening of Ph.D.12TM 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, lmmunofluorescence analysis and flow cytometry 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), ARCTTTLCYISN(×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.