从噬菌体肽库中筛选并鉴定CD13特异性结合肽

来源 :南方医科大学学报 | 被引量 : 0次 | 上传用户:simyhu
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目的筛选并鉴定可和单核细胞表面CD13分子特异性结合的特异性短肽。方法以CD13为靶分子,用噬菌体展示十二肽库进行筛选,通过亲和富集法筛选表达有特异性结合肽的噬菌体,ELISA鉴定所挑选噬菌体和CD13的亲和力,根据噬菌体基因序列推导出多肽序列。对筛选到的并经比对分析认为有生物学特性的多肽进行人工合成,通过免疫荧光技术检测多肽和THP-1细胞的结合情况、位置及WM15对多肽和细胞结合的阻断作用。结果经过四轮筛选,与CD13特异性结合的噬菌体得到有效富集并最终接近饱和状态。第4轮筛选后回收率与第1轮相比,富集了30倍。挑取的20个噬菌体单克隆中,经ELISA鉴定有10个和CD13的亲和力较高,有阳性意义。经比对得到2个有生物学功能的多肽序列P9、P7,它们分别和人巨细胞病毒(HCMV)UL38、UL105基因编码的相应氨基酸序列有83%、100%相似性。免疫荧光检测可见多肽P9、P7和THP-1细胞的结合位于细胞膜表面,WM15可以不同程度的阻断多肽和细胞的结合。结论成功筛选出了2条可和CD13特异性结合的短肽P9、P7,而且P9、P7可以和THP-1细胞膜表面的CD13分子特异性结合。 Objective To screen and identify specific short peptides that bind specifically to CD13 on the surface of monocytes. Methods CD13 was used as a target and screened by phage display dideopeptide library. The phages expressing specific binding peptides were screened by affinity enrichment. The affinity of selected phage and CD13 was identified by ELISA. The peptides were deduced from phage sequences sequence. Polypeptides screened and analyzed by comparative analysis were synthesized by artificial immunocytochemistry. The binding and location of peptides and THP-1 cells were detected by immunofluorescence and the blocking effect of WM15 on the binding of peptides and cells. Results After four rounds of screening, the phage that specifically bound to CD13 were effectively enriched and finally nearly saturated. After the fourth round of screening the recovery rate compared with the first round, enriched 30 times. Of the 20 phage clones picked out, 10 were identified by ELISA and had high affinity for CD13, which was positive. Two biologically functional polypeptide sequences P9 and P7 were obtained by comparison, which were respectively 83% and 100% similar to the corresponding amino acid sequences encoded by human cytomegalovirus (HCMV) UL38 and UL105 genes. Immunofluorescence showed that the binding of P9, P7 and THP-1 cells was located on the surface of cell membrane, and WM15 could block the binding of peptides and cells to varying degrees. Conclusion Two short peptides P9 and P7 specific for CD13 were successfully screened, and P9 and P7 could specifically bind to CD13 on the surface of THP-1 cell membrane.
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