Okuni Julius Boniface, Kateete David Patrick, Okee Moses, Nanteza Anna, Joloba Moses, Ojok Lonzy
College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O. Box 7062, Kampala, Uganda.
College of Health Sciences, Makerere University, P.O. Box 7072, Kampala, Uganda.
Ir Vet J. 2017 Mar 24;70:10. doi: 10.1186/s13620-017-0088-7. eCollection 2017.
Detection of (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.
MAP Heat shock protein 70 gene was amplified and cloned into an expression vector, Champion pET-SUMO, then expressed in , 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.
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.
副结核分枝杆菌（MAP）感染的检测是控制副结核病的关键。免疫组织化学是检测组织中MAP感染的方法之一。然而，缺乏能够检测该病原体的商业抗体是免疫组织化学应用的一个限制因素。本研究旨在开发一种免疫组织化学方法，使用抗MAP重组热休克蛋白70kd的抗体来诊断感染组织中的MAP。
扩增MAP热休克蛋白70基因并将其克隆到表达载体Champion pET-SUMO中，然后在大肠杆菌中表达、纯化，并用于制备抗热休克蛋白的兔多克隆抗体。用抗HSP70多克隆抗体对35个MAP感染组织进行免疫组织化学检测。所有35个MAP感染组织在巨噬细胞、上皮样细胞和巨细胞内的MAP均呈阳性，呈团块状或单个杆菌状。在3个未感染的正常组织以及用PBS或同一只兔子的免疫前血清替代一抗的MAP感染组织中未观察到阳性染色。
本研究中产生的抗HSP70为改善动物组织中MAP的诊断、筛查以及MAP发病机制的研究提供了机会。
