Frikha-Gargouri Olfa, Gdoura Radhouane, Znazen Abir, Gargouri Boutheina, Gargouri Jalel, Rebai Ahmed, Hammami Adnene
Department of Microbiology and research laboratory Microorganismes et Pathologie Humaine, Habib Bourguiba hospital of Sfax, Tunisia.
BMC Microbiol. 2008 Dec 10;8:217. doi: 10.1186/1471-2180-8-217.
The OmcB protein is one of the most immunogenic proteins in C. trachomatis and C. pneumoniae infections. This protein is highly conserved leading to serum cross reactivity between the various chlamydial species. Since previous studies based on recombinant proteins failed to identify a species specific immune response against the OmcB protein, this study evaluated an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of C. trachomatis infections.
Using the ClustalW and Antigenic programs, we have selected two predicted specific and immunogenic regions in the OmcB protein: the N-terminal (Nt) region containing three epitopes and the C-terminal (Ct) region containing two epitopes with high scores. These regions were cloned into the PinPoint Xa-1 and pGEX-6P-1 expression vectors, incorporating a biotin purification tag and a glutathione-S-transferase tag, respectively. These regions were then expressed in E. coli. Only the pGEX-6P-1 has been found suitable for serological studies as its tag showed less cross reactivity with human sera and was retained for the evaluation of the selected antigens. Only the Ct region of the protein has been found to be well expressed in E. coli and was evaluated for its ability to be recognized by human sera. 384 sera were tested for the presence of IgG antibodies to C. trachomatis by our in house microimmunofluorescence (MIF) and the developed ELISA test. Using the MIF as the reference method, the developed OmcB Ct ELISA has a high specificity (94.3%) but a low sensitivity (23.9). Our results indicate that the use of the sequence alignment tool might be useful for identifying specific regions in an immunodominant antigen. However, the two epitopes, located in the selected Ct region, of the 24 predicted in the full length OmcB protein account for approximately 25% of the serological response detected by MIF, which limits the use of the developed ELISA test when screening C. trachomatis infections.
The developed ELISA test might be used as a confirmatory test to assess the specificity of serological results found by MIF.
OmcB蛋白是沙眼衣原体和肺炎衣原体感染中最具免疫原性的蛋白之一。该蛋白高度保守，导致不同衣原体物种之间存在血清交叉反应性。由于先前基于重组蛋白的研究未能鉴定出针对OmcB蛋白的种特异性免疫反应，本研究评估了一种通过计算机预测的来自OmcB蛋白的特异性和免疫原性抗原，用于沙眼衣原体感染的血清学诊断。
使用ClustalW和抗原性程序，我们在OmcB蛋白中选择了两个预测的特异性和免疫原性区域：包含三个表位的N端（Nt）区域和包含两个高分表位的C端（Ct）区域。这些区域分别克隆到PinPoint Xa-1和pGEX-6P-1表达载体中，分别包含生物素纯化标签和谷胱甘肽-S-转移酶标签。然后在大肠杆菌中表达这些区域。仅发现pGEX-6P-1适用于血清学研究，因为其标签与人血清的交叉反应性较低，并被保留用于评估所选抗原。仅发现该蛋白的Ct区域在大肠杆菌中表达良好，并评估了其被人血清识别的能力。通过我们内部的微量免疫荧光（MIF）和开发的ELISA试验检测了384份血清中沙眼衣原体IgG抗体的存在。以MIF作为参考方法，开发的OmcB Ct ELISA具有高特异性（94.3%）但低敏感性（23.9）。我们的结果表明，使用序列比对工具可能有助于识别免疫显性抗原中的特定区域。然而，全长OmcB蛋白中预测的24个表位中位于所选Ct区域的两个表位约占MIF检测到的血清学反应的25%，这限制了开发的ELISA试验在筛查沙眼衣原体感染时的应用。
开发的ELISA试验可作为确认试验，用于评估MIF发现的血清学结果的特异性。
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