ACE BioSciences A/S, Roskildevej 12 C, 3400 Hillerød, Denmark.
J Microbiol Methods. 2010 Oct;83(1):13-9. doi: 10.1016/j.mimet.2010.07.011. Epub 2010 Jul 17.
Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of non-pathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cell-surface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination.
肠产毒性大肠杆菌（ETEC）感染的发病机制涉及细胞表面菌毛（CS）或定植菌毛抗原（CFA）介导的小肠定植。然而，针对 ETEC 的再感染的保护也是由体细胞抗原而不是毒力因子提供的。为了发现 ETEC 特异性的体细胞抗原，我们将 ETEC H10406 株的表面蛋白质组与非致病性大肠杆菌 K12 株进行了比较。在这项研究中，我们使用稳定同位素标记的氨基酸在细胞培养物（SILAC）技术对表面蛋白进行标记和相对定量，以鉴定特定存在于 ETEC 株上的多肽。分离出外膜蛋白，通过凝胶电泳分离，并通过质谱鉴定。鉴定出 23 种差异表达的 ETEC 细胞表面多肽，并通过生物信息学评估其作为蛋白疫苗候选物的潜力。这些多肽作为抗体的靶标具有高度的适用性，因为它们既暴露于表面，又存在差异，因此可用于被动或主动免疫/接种。
