基于生物质谱和亲和层析策略的大肠杆菌O157: H7特异性抗体的靶蛋白及多肽抗原表位的鉴定与研究

Escherichia coli O157:H7 is one of the most prevalent foodborne pathogens in China. Currently, the specific immunoassays against this pathogen were mainly based the monoclonal antibodies (mAbs) against the whole cell of bacteria, and had a low accuracy and reliability. This was mainly because the systemic study against the antigen and epitope of the antibodies was missing, and the mechanism of the detection was unclear. Previously, characterization of cross-reactivity of the method mainly depended on the test of a certain number of bacteria. In the preliminary work, we have prepared the paired mAbs against the E. coli O157:H7, the specificity of the antibodies and the method was good. Different with the previous study, the antibodies recognize a membrane protein with molecular weight of 35-37kDa. This project intends to research and identify the target protein and sequence of the epitope, based on the antibodies and the proteome, western blot, immunoaffinity chromatography, mass spectrometry, synthetic peptide and immunoassays. Then the homology of the target protein and the epitope of peptide with other bacteria will be analyzed by sequence alignment, combined with the known structure, function and abundance of the protein. Finally, with the further confirmation by the sandwich ELISA and western blot, the mechanism and specificity of the detection will be revealed at the molecule and sequence level.

大肠杆菌O157:H7是我国常见的食源性致病菌之一，目前能特异性检测该菌的免疫学方法多基于菌体抗原制备的抗体，方法的准确性和可靠性较低。这主要是由于抗体的靶分子和抗原表位未进行系统性研究，方法的检测机理不明确，研究交叉反应只能依赖于测试有限数量的菌株。在前期工作中，申请人制备了识别大肠杆菌O157:H7菌体抗原的配对抗体，抗体和ELISA方法特异性良好，与以往研究不同的是抗体均识别分子量为35-37kDa的膜蛋白。本项目拟在该配对抗体的基础上，结合蛋白质组、免疫印迹、亲和层析、生物质谱、合成短肽以及ELISA技术，研究并鉴定大肠杆菌O157:H7的靶蛋白和多肽抗原表位。结合该蛋白已知的结构、功能、丰度，通过序列比对研究靶蛋白和多肽抗原表位与大肠杆菌等其它细菌的同源性，并结合ELISA等的验证结果，从分子和序列水平明确方法的检测机理和特异性。

项目通过蛋白质组、免疫亲和层析、生物质谱、蛋白结构、BLAST等技术手段系统性研究、鉴定了大肠杆菌O157:H7配对抗体的检测抗原和多肽抗原表位，通过全景序列同源性比对和76株细菌交叉反应测试，研究了配对抗体及夹心ELISA方法的特异性，并应用于食品样品检测。捕获抗体2G12识别丰度较高、具有β-桶状结构的渗透压调节蛋白OmpC，具体表位为膜外2条相邻的Loop结构“LGVING”和“TQTYNATRVGSLG”。2G12对8株O157均可识别，对68株非O157菌株（含big six）活菌在高浓度（8 Log/mL）下的特异性为82.35%，低浓度（6 Log/mL）下为100%。“LGVING”区域具有较好的大肠杆菌O157:H7序列特异性，是2G12的主要识别区域。检测抗体12B1识别大肠杆菌O157:H7脂多糖的O特异性侧链，对8株O157均可识别，对68株非O157菌株活菌在高浓度下的特异性为97.06%，低浓度下为100%。该夹心ELISA方法在检测76株活菌时，对大肠杆菌O157:H7的特异性为100%，人工添加样在增菌12h后可检测到牛肉中10CFU/g的大肠杆菌O157:H7，与培养法结果一致。在典型的食品环境如高盐、低酸、低营养状态下，单抗2G12识别的大肠杆菌O157:H7的OmpC表位的表达和暴露比较稳定，与12B1识别的LPS的O特异性侧链相当，有利于磁珠分离和大肠杆菌O157:H7的免疫检测。项目研究揭示了该大肠杆菌O157:H7特异性免疫检测方法的分子基础，为其今后的改进和应用提供了方向，也为其它致病微生物抗原表位的研究奠定了技术基础。

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