Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (9): 2824-2833.DOI: 10.1016/j.jia.2023.04.006

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胞内劳森菌Hsp60蛋白单克隆抗体的制备及其应用

  

  • 收稿日期:2022-11-23 接受日期:2023-02-17 出版日期:2023-09-20 发布日期:2023-09-14

Preparation and application of a novel monoclonal antibody specific for the heat shock protein 60 of Lawsonia intracellularis

XIAO Ning1*, LÜ Yun-yun1*, LI Jian-nan1, CHEN Chang-feng1, LIN Hui-xing1, FAN Hong-jie1, 2#   

  1. 1 MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P.R.China

    2 Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, P.R.China.

  • Received:2022-11-23 Accepted:2023-02-17 Online:2023-09-20 Published:2023-09-14
  • About author:XIAO Ning, E-mail: 2018207037@njau.edu.cn; #Correspondence FAN Hong-jie, Tel/Fax: +86-25-84399592, E-mail: fhj@njau.edu.cn * These authors contributed equally to this study.
  • Supported by:

    This work was supported by the National Key Research and Development Program of China (2021YFD1800400), the National Natural Science Foundation of China (31872480), the Jiangsu Agriculture Science and Technology Innovation Fund, China (CX (19)2020), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD).


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摘要： 猪增生性肠病（Porcine proliferative enteropathy, PPE）是由胞内劳森菌(Lawsonia intracellularis，L. intracellularis) 感染引起、在世界各地猪场中普遍存在的一种重要肠道疾病，以6~20周龄生长育肥猪急性出血性下痢、间歇性下痢、食欲下降和生长发育缓慢等临床症状为主要特征，给养猪业带来严重经济损失。准确的检测临床样品中胞内劳森菌方法对于预防和控制PPE尤其重要。研究表明，单克隆抗体在胞内劳森菌的病原学检测中发挥重要作用，热休克蛋白60 (Heat shock protein 60, Hsp60) 广泛存在于多种细菌中，是一种具有免疫保护作用的抗原。因此，本研究拟制备抗胞内劳森菌 Hsp60的单克隆抗体，并以其为一抗，用于感染细胞及感染组织中胞内劳森菌的检测。鉴于此，我们首先表达并纯化了Hsp60蛋白，并以纯化后的Hsp60蛋白为免疫原免疫BALB/c小鼠，采用杂交瘤细胞技术制备单克隆抗体。随后，通过间接ELISA和Western blotting检测单克隆抗体的特异性。最后，以本研究制备的单克隆抗体为一抗，分别采用免疫荧光和免疫组化法对体外单层感染细胞以及体内感染组织中胞内劳森菌进行检测。最终，我们成功筛选、鉴定出3株能够稳定分泌抗Hsp60蛋白单克隆抗体的阳性杂交瘤细胞株，分别命名为3E5、4E2和9G6。以BALB/c小鼠制备相应腹水单抗，间接ELISA效价分别为1:1024000、1:2048000和1:2048000。单克隆抗体特异性检测结果显示，3E5、4E2和9G6只能与胞内劳森菌反应，而与猪霍乱沙门氏菌、鼠伤寒沙门氏菌、大肠杆菌、猪痢短螺旋体等猪肠道中常见病原菌均无交叉反应，说明本研究制备的单克隆抗体具有良好的特异性。进一步研究发现，以上3种单克隆抗体均可与体外单层感染细胞及PPE感染猪回肠组织中的胞内劳森菌发生特异性结合。上述单克隆抗体为胞内劳森菌临床菌株的成功分离及免疫诊断方法的开发奠定了基础。


Abstract:

Porcine proliferative enteropathy (PPE), an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.  An accurate method for detecting L. intracellularis is particularly important for preventing and controlling PPE.  Heat shock protein 60 (Hsp60) is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.  Thus, the purpose of the current investigation was to produce a novel L. intracellularis Hsp60 monoclonal antibody (mAb) useful for immunodiagnostics.  Three hybridomas secreted anti-Hsp60 termed 3E5, 4E2, and 9G6 were generated, and the titers of ascitic fluids of 3E5, 4E2, 9G6 were 1:1 024 000, 1:2 048 000 and 1:2 048 000, respectively.  The Western blotting analysis demonstrated that recombinant Hsp60 (rHsp60) was recognized by mAbs 3E5, 4E2 and 9G6.  Subsequently, analyses of specificity showed all the mAbs were highly specific to L. intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs, such as Escherichia coli, Salmonella Choleraesuis, Salmonella Typhimurium, and Brachyspira hyodysenteriae.  Furthermore, the mAbs were useful for detecting L. intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.  Our research will provide a foundation for the development of immunological diagnostic tests

Key words: Porcine proliferative enteropathy ,  Lawsonia intracellularis ,  monoclonal antibody ,  immunodiagnostics ,  heat shock protein 60