Hughes Valerie, Bannantine John P, Denham Susan, Smith Stuart, Garcia-Sanchez Alfredo, Sales Jill, Paustian Michael L, Mclean Kevin, Stevenson Karen
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, United Kingdom.
Clin Vaccine Immunol. 2008 Dec;15(12):1824-33. doi: 10.1128/CVI.00099-08. Epub 2008 Oct 8.
Mycobacterium avium subsp. paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionization-time of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis.
副结核分枝杆菌亚种可导致副结核病，一种慢性肉芽肿性肠炎。检测患有副结核感染的动物很困难，因为目前可用的工具灵敏度低且缺乏特异性；这些工具容易产生由接触环境中的鸟分枝杆菌复合群生物体导致的假阳性检测结果。为了生成用于纳入副结核病特异性检测的候选抗原，通过对副结核分枝杆菌亚种和鸟分枝杆菌亚种进行蛋白质组比较来确定亚种特异性蛋白质。分析旨在揭示仅在副结核分枝杆菌亚种蛋白质谱中表达（或占主导）的蛋白质。二维凝胶电泳从每个亚种中分离出大约1000个蛋白质斑点。蛋白质组分析确定了在副结核分枝杆菌亚种中表达谱明显增加的蛋白质斑点，通过基质辅助激光解吸电离飞行时间分析其胰蛋白酶肽谱鉴定出32个。其中30种蛋白质被克隆，并表达了它们的重组蛋白。使用绵羊副结核血清通过酶联免疫吸附测定（ELISA）、蛋白质印迹和斑点印迹分析来评估它们的免疫反应性。在至少一种免疫测定中检测到17种蛋白质，在六份检测血清中的四份中，通过ELISA检测到11种蛋白质，其光密度超过0.1的临界值。这些蛋白质的免疫反应性表明它们作为独特诊断抗原用于开发副结核病特异性血清学检测的潜力。
