CAJ | 학술논문

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.
목적 종서균체전시수궤12태고사선인류백세포항원(HLA)-B·2704급B·2705중련길항태병주초보감정.방법 용HLA-B*2704/B*2705중련포외구단백분별사선서균체전조수궤태고,매련면역흡부시험(ELISA)감정양성극륭,DNA측정학정안기산서렬,면역형광화류식세포술감 ,정서균체극륭분별여HLA-B*2704급B*2705세포주결합적특이성.결과 경3륜사선,획득12개HLA-B·2704념항태,공유5충서렬,분별위HTSFCSTHLCLI(×4),QHCSPTLCQIHR(×5),ARCTITL-CYLSN(×1),YGLCTDWYCHIT(×1),YPLCDAILCRLP(×1);10개B*2705길항태공유4충서렬,분별위:①SHCSPHWCALPF(×6);②HLCSNSLCLLPW(×2);③EPMCSWFWCTLP(×1);④WTCSPLLCTWGA(×1).비대분석표명,B*2704여B*2705길항태적서렬시기본일치적,균함유CS(T)TXXL(W)CXL표위.전시유B*2704길항태적서균체극륭가여HLA-B*2704세포주결합,양성솔위43.55%;이B*2705념항태서균체극륭여HLA-B·2705세포주적양성결합솔위45.69%.결론 사선획득적HLA-B27념항태적서균체극륭구유일정적친화력,가여표체우세포주표면적B*2704화B**2705분자특이성결합,이여정상B세포불결합,인이표현출일정적결합특이성.
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.