Identification of immunoreactive proteins of Mycobacterium avium subsp. Paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of a chronic enteritis of ruminants (bovine paratuberculosis-Johne disease) that is associated with enormous worldwide economic losses for the zootechnical industries. Diagnosis is based on observation of clinical signs, on the detection of antibodies in milk or serum or on evaluation of bacterial culture from feces. The limit of these methods is that they are not able to detect the disease in the subclinical stage and are applicable only when the disease is already in an advanced status. For this reason the main purpose of this study is to use the MAP proteome to detect novel immunoreactive proteins that may be helpful for paratuberculosis diagnoses. 2D electrophoresis and 2D immunoblotting of MAP proteins were performed using sera of control cattle and paratuberculosis infected cattle in order to highlight the specific immunoreactive proteins. Among the assigned identifiers to immunoreactive spots it was found that most of them correspond to surface-located proteins while three of them have never been described before as antigens. The identification of these proteins improves scientific knowledge that could be useful for paratuberculosis diagnoses. The sequence of the identified protein can be used for the synthesis of immunoreactive peptides that could be screened for their immunoreaction against bovine sera infected with MAP.

鸟分枝杆菌副结核亚种（Mycobacterium avium subsp. paratuberculosis，MAP）是引发反刍动物慢性肠炎（牛副结核病，又称约翰病）的病原菌，该疾病给全球畜牧养殖业造成了巨额经济损失。当前副结核病的诊断手段主要包括临床症状观察、牛奶或血清中抗体检测，以及粪便细菌培养鉴定。此类方法的局限性在于无法在亚临床阶段检出感染，仅能在疾病进展至晚期时才可应用。为此，本研究的核心目标是借助MAP蛋白质组筛选新型免疫反应性蛋白，以期为副结核病的诊断提供新的辅助手段。研究采用对照牛血清与副结核病感染牛血清，对MAP蛋白开展二维电泳（2D electrophoresis）与二维免疫印迹（2D immunoblotting）实验，以特异性识别免疫反应性蛋白斑点。对免疫反应性斑点的标识符进行分析后发现，绝大多数斑点对应的蛋白定位于细菌表面，其中3种蛋白此前从未被报道为疾病相关抗原。上述蛋白的鉴定丰富了副结核病相关的科学认知，可为该疾病的诊断提供参考依据。所鉴定蛋白的序列可用于合成免疫反应性肽段，后续可通过其与MAP感染牛血清的免疫反应性开展筛选验证。