【摘要】 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。更多还原

【Abstract】 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 Mc Ab 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 Mc Ab were screened from 12-mers linear phage display peptide library. Among these positive clones,phage clones No. 39 shared the conserved sequence: Wx Hx …… AGL found in previous clone No. 31( ATWx Hx Lx SAGL),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 ’ Wx Hx Vx W-’ may be antigenic epitope as SP39,which contains a T cell epitope. Our results showed that both SP39 could bind to both anti-PGN Mc Ab and a polyclonal antibody against S. aureus. Moreover PGN could inhibit the binding of SP39 to the anti-PGN Mc Ab. These data indicated that SP39 mimic to epitopes on PGN. Conclusion:SP39( GRWxHxVxWAGLAGGS) probably display the mimotopes of PGN.更多还原