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

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.