模拟肽聚糖表位的序列分析及其抗原性鉴定  

Sequence analysis and antigenic identification of mimic epitopes of peptidoglycan

作　　者：陈益国[1] 侯晓睿[1,2] 朱平[1,2] 刘北一[1,2] 

机构地区：[1]江西省人民医院检验科,南昌330008 [2]南方医科大学基础医学院免疫教研室,广州510515

出　　处：《中国免疫学杂志》2017年第8期1191-1196,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.31270980);广东省医学科研基金(A2016533)资助

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。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,Tcell 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 peptidelibrary. Among these positive clones,phage clones No . 39 shared the conserved sequence：WxHx...AGL found in previous cloneNo. 31（ ATWxHxLxSAGL）,which provoked an effective protective immunity against infection with S. aureus. To enhance the stability of the confor^nation as well as adding biotin on the N- ter-minal as a tag, the sequences 忆 39忆 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. harard . 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 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.

关 键 词：肽聚糖 模拟肽 噬菌体肽库 金黄色葡萄球菌 

分 类 号：R392.9[医药卫生—免疫学]