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

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] E. 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1：128 000-1：512 000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1：100 000稀释，免疫荧光试验中Mhp366-N多克隆抗体的工作浓度范围在1：1 000-1：10 000 000，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。

Abstract:

[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 E. coli BL21(DE3)-pET28a(+)-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 3D4/21 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 1:128 000 and 1:512 000. The optimal dilution of Mhp366-N polyclonal antibody in Western blot was 1:100 000, and in immunofluorescence assay was 1:1 000−1:10 000 000. 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.