模拟肽聚糖表位的序列分析及其抗原性鉴定

摘要

Objective:To analysis the Mimotopes of the peptide mimics to PGN using online softwares.Methods: Mimotopes of PGN were screened from 12-mers linear phage display peptide library by using anti-PGN McAb and the antigenicity of selected clones was identified by ELISA.The B cell epitopes,T cell epitopes of ′GRWxHxVxWAGL′ were estimated by DNAstar and online softwares.Results: 16 phage clones that bound with anti-PGN McAb were screened from 12-mers linear phage display peptide library.Among these positive clones,phage clones No.39 shared the conserved sequence:WxHx……AGL found in previous clone No.31(ATWxHxLxSAGL),which provoked an effective protective immunity against infection with S.aureus.To enhance the stability of the conformation as well as adding biotin on the N-ter-minal as a tag,the sequences ′39′ were redesigned and synthesized by adding S(serine)A(alanine) and GG(glycine)on the C-terminal of origin sequence(named SP39).Next,we estimated or predicted antigenic epitopes,T cell epitopes and scores binding to MHC of these peptides by using DNASTAR and online softwares(http://bio.dfci.harvard.edu/Tools/antigenic.pl,www.syfpeithi.de,http://www.darrenflower.info/mhcpred),indicating that SP39 contains sites bound both mice and human MHC.The sequences ′WxHxVxW-′ may be antigenic epitope as SP39,which contains a T cell epitope.Our results showed that both SP39 could bind to both anti-PGN McAb and a polyclonal antibody against S.aureus.Moreover PGN could inhibit the binding of SP39 to the anti-PGN McAb.These data indicated that SP39 mimic to epitopes on PGN.Conclusion: SP39(GRWxHxVxWAGLAGGS) probably display the mimotopes of PGN.%目的:在线预测肽聚糖(PGN)模拟抗表原位并鉴定其抗原性.方法:利用抗PGN单克隆抗体从12线性噬菌体随机展示肽库中筛选模拟PGN表位的阳性克隆,对阳性克隆进行DNA测序和推导氨基酸序列,结合生物信息学分析阳性序列GRWxHxVxWAGL的抗原性以及T细胞表位,根据分析对阳性序列进行修饰与合成,ELISA法鉴定阳性序列的抗原性.结果:经对噬菌体12线性肽库的3轮筛选,夹心ELISA鉴定得到16个与抗PGN单克隆抗体结合的克隆,DNA 测序并推导氨基酸序列,该序列与前期具有抗金黄色葡萄球菌活性序列No.31(ATWxHxLxSAGL)含有保守序列WxHx…AGL.在线分析(http://bio.dfci.harvard.edu/Tools/antigenic.pl,www.syfpeithi.de,http://www.darrenflower.info/mhcpred)表明,含保守序列的阳性序列GRWxHxVxWAGL包含与人和小鼠MHC结合表位 (-WxHxVxW-),C端加入AGGS后具有T细胞表位(DNAstar),据此合成线性肽Biotin-GRWxHxVxWAGLAGGS(命名为SP39).ELISA结果显示,SP39能与抗PGN单抗及抗S.aureus全菌多抗结合,PGN能抑制SP39与抗PGN单克隆抗体结合.结论:经噬菌体肽库筛选获得阳性序列GRWxHxVxWAGLAGGS,该序列可能模拟PGN抗原表位.