Chin Chai Fung, Teh Boon Aun, Anthony Amy Amilda, Aziah Ismail, Ismail Asma, Ong Eugene Boon Beng, Lim Theam Soon
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia.
Appl Biochem Biotechnol. 2014 Nov;174(5):1897-906. doi: 10.1007/s12010-014-1173-y. Epub 2014 Aug 23.
In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.
在我们早期的研究中，使用发热患者血清进行的免疫印迹分析显示，肠炎沙门氏菌伤寒血清型外膜蛋白组分中的一条50 kDa条带仅被伤寒血清特异性识别，而不被其他发热疾病的血清识别。在此，我们研究了免疫原性50 kDa条带中所含蛋白质的身份，以确定负责其免疫原性的抗原。我们首先使用液相色谱-串联质谱（LC-MS/MS）进行蛋白质鉴定，然后使用在线工具ANTIGENpro进行抗原性预测，并制备了主要抗原的重组蛋白，用于酶联免疫吸附测定（ELISA）验证。我们发现，TolC、GlpK和SucB蛋白对伤寒血清具有特异性，但在天然和变性条件下与抗体的反应不同。这种差异表明这些蛋白质上存在线性和构象表位。
