Zhao Ningning, Jiang Yue, Ming Shuzhen, Liu Sidang, Zhao Xiaomin, Wang Fangkun
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Tai'an, China.
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Tai'an, China.
Front Microbiol. 2019 Aug 13;10:1878. doi: 10.3389/fmicb.2019.01878. eCollection 2019.
Elongation factor thermo-unstable (EF-Tu), an abundant multifunctional protein, is pivotal during protein synthesis and is an important antigen. Few studies have addressed the role of this protein in species, and the epitopes of this protein have not been reported. Here, we describe a monoclonal antibody (McAb), BD, for EF-Tu in . Using western blotting involving a series of partially overlapping recombinant EF-Tu truncation peptides, a novel linear B-cell epitope, QTREHIL (EF), was identified. Alanine-scanning mutagenesis revealed that residues Q, T, R, I, and L were core residues involved in recognition. Sequence alignment suggested that the epitope peptide was conserved among bacterial species but differed by one amino acid residue (I) from the host sequence. The epitope peptide was recognized by sera from -infected mice, and while recombinant epitope peptide induced a strong humoral immune response, the corresponding mouse peptide, QTREHLL, did not. These results suggested that I may be the key residue for the host immune system to distinguish between bacterial and self epitope EF sequences. Indirect immunofluorescence and western blotting assays showed that epitope peptide could be used in , human embryonic kidney cell (HEK-293), and chicken fibroblast cell (DF1) expression systems and immunoprecipitation assay. Together, our results suggested that the McAb BD is a useful tool for further investigation of the potential functions of the EF-Tu protein in pathogen-host interactions, and that the epitope tag may be useful for application as a novel affinity tag to identify other bacterial pathogens, especially convenient for the identification of intracellular bacteria.
热不稳定延伸因子（EF-Tu）是一种丰富的多功能蛋白质，在蛋白质合成过程中起关键作用，并且是一种重要的抗原。很少有研究探讨这种蛋白质在物种中的作用，其表位也尚未见报道。在此，我们描述了一种针对EF-Tu的单克隆抗体（McAb）BD。通过使用涉及一系列部分重叠的重组EF-Tu截短肽的蛋白质印迹法，鉴定出一种新的线性B细胞表位QTREHIL（EF）。丙氨酸扫描诱变显示，残基Q、T、R、I和L是参与识别的核心残基。序列比对表明，该表位肽在细菌物种中保守，但与宿主序列相差一个氨基酸残基（I）。该表位肽可被感染小鼠的血清识别，虽然重组表位肽诱导了强烈的体液免疫反应，但相应的小鼠肽QTREHLL则不能。这些结果表明，I可能是宿主免疫系统区分细菌和自身表位EF序列的关键残基。间接免疫荧光和蛋白质印迹分析表明，表位肽可用于表达系统和免疫沉淀分析，包括、人胚肾细胞（HEK-293）和鸡成纤维细胞（DF1）。总之，我们的结果表明，单克隆抗体BD是进一步研究EF-Tu蛋白在病原体-宿主相互作用中潜在功能的有用工具，并且该表位标签可能作为一种新型亲和标签用于鉴定其他细菌病原体，特别是便于鉴定细胞内细菌。
