Fraunhofer Institute for Biomedical Engineering, Am Muehlenberg, Potsdam, Germany.
PLoS One. 2013 May 29;8(5):e65837. doi: 10.1371/journal.pone.0065837. Print 2013.
Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium's pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify the identification of structural epitopes, as this would extend the spectrum of novel epitopes to be detected.
空肠弯曲菌仍然是我们这个时代的主要肠道病原体之一。它的人畜共患病性质和在工业化国家的广泛分布要求有一种快速可靠的诊断工具。基于抗体的检测提供了一种识别致病菌的合适方法。然而，对免疫显性靶标的了解是有限的。因此，本文提出了一种方法，可以快速筛选大量 cDNA 衍生的表达克隆，以鉴定新的抗原。对免疫显性蛋白的深入了解有助于诊断工具的设计，并进一步深入了解细菌的致病性，同时揭示潜在的疫苗候选物。我们已经成功筛选了一个表达文库的 1536 个克隆，以鉴定 22 种以前未被描述为免疫显性的蛋白质。在亚克隆相应的 22 个基因和全长蛋白的表达后，我们通过微阵列和 ELISA 研究了免疫显性特征。随后，选择了 7 种蛋白质进行表位作图。对于 cj0669 和 cj0920c，鉴定了线性表位。对于 cj0669，特异性测定显示了一个特定的线性表位位点。因此，通过丙氨酸扫描分析了 11 个氨基酸残基序列 TLIKELKRLGI，结果表明甘氨酸残基对抗体的结合很重要。本文提出的创新方法是在组合微阵列平台的情况下生成原核生物的 cDNA，从而省去了繁琐的纯化步骤，有助于阐明空肠弯曲菌的新免疫显性蛋白。特定线性表位的发现为大量未来的研究和在血清筛选等诊断应用中的潜在用途铺平了道路。此外，目前的方法很容易适应其他高度相关的细菌，使其成为未来发现抗原和潜在生物标志物的有力工具。因此，简化结构表位的鉴定是可取的，因为这将扩大要检测的新表位的范围。
