猪肺炎支原体Mhp366-N蛋白多克隆抗体的制备及应用  

Preparation and application of polyclonal antibody against Mhp366-N protein of Mycoplasma hyopneumoniae

作　　者：文玉康 周冰倩 陈政锟 杨梅[1,2] 田亚琴 丁红雷[1,2] WEN Yukang;ZHOU Bingqian;CHEN Zhengkun;YANG Mei;TIAN Yaqin;DING Honglei(Laboratory of Veterinary Mycoplasmology,College of Veterinary Medicine,Southwest University,Chongqing 400715,China;Immunology Research Center of Medical Research Institute,Southwest University,Chongqing 400715,China)

机构地区：[1]西南大学动物医学院动物支原体学实验室,重庆400715 [2]西南大学医学研究院免疫学研究中心,重庆400715

出　　处：《微生物学通报》2021年第8期2695-2703,共9页Microbiology China

基　　金：中央高校基本科研业务费专项资金(XDJK2020B012);重庆市技术创新与应用发展专项(cstc2019jscx-msxmX0402)。

摘　　要：【背景】猪肺炎支原体是猪的一种重要的病原。该菌的研究工具较少,特别是缺少开展其致病机制研究需要的抗体。【目的】制备猪肺炎支原体Mhp366-N蛋白抗体并确定其应用范围和使用时的最佳稀释倍数。【方法】Escherichia coli BL21(DE3)-pET28a(+)-mhp366-N重组菌诱导表达Mhp366-N蛋白并纯化。纯化的蛋白免疫小鼠制备多克隆抗体。用免疫印迹和免疫荧光方法检测猪肺炎支原体AH株感染3D4/21细胞后的Mhp366蛋白,确定2种方法中Mhp366-N多克隆抗体的最佳稀释倍数;之后检测临床采集的猪肺泡巨噬细胞中的猪肺炎支原体;最后以免疫组化试验检测猪肺炎支原体感染的肺细胞。【结果】纯化的Mhp366-N蛋白纯度超过85%,免疫小鼠制备的抗血清效价在1:128000-1:512000之间。在免疫印迹试验中Mhp366-N多克隆抗体的最佳工作浓度为1:100000稀释,免疫荧光试验中Mhp366-N多克隆抗体的工作浓度范围在1:1000-1:10000000,其可用于临床采集的猪肺泡巨噬细胞和细胞系中猪肺炎支原体的检测。免疫组化试验结果显示猪肺炎支原体能够进入猪肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞。【结论】制备的Mhp366-N多克隆抗体为猪肺炎支原体致病机制研究提供了良好的研究工具。[Background]Mycoplasma hyopneumoniae is an important pathogen in pigs.The research tools of this bacterium are few,especially the antibody that is used to carry out the research on its pathogenicity.[Objective]To prepare the polyclonal antibody against Mhp366-N protein of M.hyopneumoniae and determine its application and the optimal dilution.[Methods]Expression of Mhp366-N protein was induced by recombinant bacterium Escherichia coli BL21(DE3)-pET28 a(+)-mhp366-N,and the protein was purified.The purified protein was used to immunize mice to prepare polyclonal antibody.Using Mhp366-N polyclonal antibody as the primary antibody,the Mhp366 protein in 3 D4/21 cells infected with M.hyopneumoniae AH strain was detected by Western blot and immunofluorescence,and the optimal dilution of antibody in each assay was determined.Then,M.hyopneumoniae was detected in alveolar macrophages collected clinically.Finally,the presence of M.hyopneumoniae in lung cells was detected by immunohistochemistry.[Results]The purity of purified Mhp366-N protein was more than 85%,and the titer of Mhp366-N antiserum was between 1:128000 and 1:512000.The optimal dilution of Mhp366-N polyclonal antibody in Western blot was 1:100000,and in immunofluorescence assay was 1:1000-1:10000000.In addition,the polyclonal antibody could be used to detect M.hyopneumoniae in porcine alveolar macrophages and cell lines.The results of immunohistochemistry showed that M.hyopneumoniae could enter porcine alveolar macrophages,type I and II alveolar epithelial cells.[Conclusion]Preparation of Mhp366-N polyclonal antibody could provide a good research tool for the research on the pathogenesis of M.hyopneumoniae.

关 键 词：猪肺炎支原体 多克隆抗体 免疫荧光 免疫印迹 猪肺泡巨噬细胞 

分 类 号：S858.28[农业科学—临床兽医学]