BACKGROUND Detection of Mycobacterium avium subspecies paratuberculosis (MAP) infection is key to the control of Johne's disease. Immunohistochemistry is one of the methods of detection of MAP infection in tissues. However, unavailability of commercial antibodies that can detect the organism is a limiting factor for the use of immunohistochemistry. This study was aimed at developing an immunohistochemistry method to diagnose MAP in infected tissues using antibodies against MAP recombinant heat shock protein 70kd. RESULTS MAP Heat shock protein 70 gene was amplified and cloned into an expression vector, Champion pET-SUMO, then expressed in E coli, purified and used to produce polyclonal rabbit antibodies against the Heat shock protein. Immunohistochemistry was performed in 35 MAP infected tissues with anti-HSP70 polyclonal antibodies. All 35 MAP infected tissues were positive for MAP within macrophages, epithelioid cells and giant cells either in clumps or singly as individual bacilli. No positive staining was seen in the three uninfected normal tissues and in MAP infected tissues where primary antibodies were substituted with PBS or pre-immune serum from the same rabbit. CONCLUSION Anti-HSP70 produced in this study offers an opportunity for improved diagnosis, screening of MAP in animal tissues and in studies on the pathogenesis of MAP.

中文翻译：

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重组热休克蛋白70抗体在自然感染牛组织中鸟分枝杆菌亚种副结核病的免疫组织化学诊断中的应用。

背景技术鸟分枝杆菌亚种副结核病（MAP）感染的检测是控制约翰氏病的关键。免疫组织化学是检测组织中MAP感染的方法之一。然而，无法获得可检测生物体的商业抗体是使用免疫组织化学的限制因素。这项研究旨在开发一种免疫组织化学方法，使用针对MAP重组热休克蛋白70kd的抗体诊断感染组织中的MAP。结果MAP热休克蛋白70基因被扩增并克隆到表达载体Champion pET-SUMO中，然后在大肠杆菌中表达，纯化并用于生产抗热休克蛋白的多克隆兔抗体。使用抗HSP70多克隆抗体在35例MAP感染的组织中进行了免疫组织化学。35个被MAP感染的组织在巨噬细胞，上皮样细胞和巨细胞中呈团块状或单独作为单个杆菌呈阳性。在三个未感染的正常组织和MAP感染的组织中均未见阳性染色，在该组织中，一抗被PBS或同一只兔子的免疫前血清取代。结论本研究中产生的抗HSP70为改善动物组织中MAP的诊断，筛选以及MAP的发病机理提供了机会。