Screening Of Adhesion Proteins And Preparation Of Monoclonal Antibodies Against Mycoplasma Leachii

Mycoplasmaleachii(M. leachii)causes polyarthritis and pneumonia in calves, and mastitis in cows, which belongs to the Mycoplasma mycoides. Despite the mortality of polyarthritisin calves is not very high, the damage to the joints is irreversible. Therefore, it is an important pathogen that threatens to cattle industry because of the high elimination rate in calves. In2009, M. leachiiwas first isolated from synovial fluid of calveswith polyarthritis in China. Until now, most of the domestic and foreign researches on M. leachii focus on isolation, genetic evolution analysis, classification and pathogenicity. The research focus on the pathogenic mechanism, transmission route and prevention measureis comparatively little. Previous studies have shown that adhesion of Mycoplasma is the prerequisite for the colonization and infection to host, so selection and characterization of adhesion proteins will contribute to elucidate the pathogenic mechanism of Mycoplasma.In this study, several methods such as sequence analysis, homology comparision, recombine proteins expression and cell adhesion inhibition experiments were used to select and identify the adhesion proteins of M. leachii. Based on genome sequence analysis and homologous adhesion proteins comparison, LPPA and a-enolase were speculated to be adhesion-ralated proteins. Thus, LPPAand a-enolase were expressed in E.coli and the anti-LPPA and anti-a-enolase antibodies were prepared in mice. Western blot analysis showed that LPPA is an outer membrane lipoprotein and a-enolase is secreted in Mycoplasma membranes. The results of adhesion and adhesion inhibition experiments showed that the adhesion property of M. leachii was specifically inhibited by anti-LPPA and anti-a-enolase antibodies. Meanwhile, the image analysis by Columbas software showed that the adherence inhibition rates of1:25diluted anti-LPPA antibodies and anti-a-enolase antibodies were58.3%and55.82%, respectively, comparing with the79.26%of anti-M.leachiiantibodies. Furthermore, the sandwich ELISA detection showed that the LPPA protein possessed the ability of adhesion to joint synovial cell membrane proteins, and this adhesion was specifically inhibited by the anti-LPPA antibodies in a dose-depend manner, while a-enolase does not adhesion to joint synovial cell membrane proteins. These results demonstrated that LPPA and a-enolase were adhesion-related proteins of M. leachii.Monoclonal antibodies (MAbs) are secreted by the fusion cells of single spleen cells and hybridoma cells. The fusion cells possess the abilities of tumor cells proliferation and immune cells secreting antibodies. With high specificity, uniformity and stable, MAb has been widely used in diagnosis, treatment and experimental research. Previous studies have shown that MAbs play an important role in the diagnosis and adhesion proteins identification of Mycoplasma. In this study, M. leachii total proteins were used as inmmunogen to immune the BALB/c mice to produce MAbs against M. leachii. The positive clones were selected by indirect ELISA, Western blot and after three generations of cloning by limited dilution,10hybridized cell clones secreted MAbs against M. leachii were obtained and the ELISA titers of the ascites were1:103-1:106. According to the results of Western blot, all MAbs specifically reacted with M. leachii. Meanwhile,2MAbs possessed the ability of inhibiting M. leachii adherence to synovial cells. These results will contribute to further characterization of adhesion proteins of M. leachii.