Screening And Adhesion Of Membrane Associated Proteins In Mycoplasma Bovis

Mycoplasma bovis(M.bovis)is the pathogenic agent of many diseases including bovine pneumonia,mastitis,and arthritis.It has caused serious economic losses to the cattle industry in China.At present,bovine mycoplasma disease is still an epidemic trend,and many bovine mycoplasma pneumonia outbreaks have occurred in Guizhou Province.However,the research on the pathogenesis of Mycoplasma bovis is still lacking,and the development of its prevention and control techniques are still insufficient.The pathogenic mechanism of Mycoplasma and the development of prevention and control techniques mostly depend on the screening of membrane proteins and their biological function-associated studies.Several studies have confirmed that some membrane proteins of Mycoplasma bovis have biological characteristics such as adhesion and antigenicity,and participate in the pathogenic process of Mycoplasma bovis and the immune response of the animal body.Therefore,the membrane protein of Mycoplasma bovis is the key target for study on the pathogenicity,immune prevention and control,and diagnostic techniques of Mycoplasma bovis.Given the important functions of Mycoplasma bovis membrane protein,here we selected of Mycoplasma bovis Guizhou strain(GZ-2)for screening and bioinformatics analysis of the membrane protein-related genes.Then,the prokaryotic expression,subcellular localization,and adhesion of the screened membrane proteins were studied,thus providing basic research data for exploring the pathogenesis of bovine mycoplasma,and screening target proteins for the diagnosis of bovine mycoplasma disease and the development of new vaccines.1、Cloning and bioinformatics analysis of Mycoplasma bovis M27,M32,M498,and M663genesAccording to gene sequences of the standard strain PG45 and Guizhou isolates of Mycoplasma bovis published in Gen Bank,possible membrane protein genes were screened out,and specific primers were designed and synthesized.The M27,M32,M498,and M663 genes of Mycoplasma bovis Guizhou isolate were cloned and sequenced for bioinformatics analysis.The results showed that the length of M27,M32,M498 and M663 genes of Guizhou strain of M.bovis were 726,432,498,and 663 bp,respectively,which encode 242,143,165 and 220 amino acids,respectively.The results of homology and phylogenetic tree analysis showed that the four genes had high homology with reference strains at home and abroad.Transmembrane structure and signal peptide analysis showed that M27,M32 and M663 had transmembrane structure,M32 had signal peptide,and M498 had no transmembrane structure and signal peptide.The results of B cell epitope analysis showed that the dominant epitopes of M27,M32,M498,and M663 were distributed evenly and had good hydrophilicity,flexibility,and strong surface accessibility and antigenicity.The above results indicate that the M27,M32,M498 and M663 genes of Mycoplasma bovis Guizhou isolate were highly conserved,and these proteins possessed a good antigenicity with several dominant epitopes.2、Prokaryotic expression of M27,M32,M498,and M663 putative membrane proteins of Mycoplasma bovisBased on the sequences of Mycoplasma bovis M27,M32,M498,and M663 genes with deletion of the transmembrane structure regions,specific primers were designed for amplifying the dominant epitope region fragments,which were then cloned into p ColdⅠ vector for prokaryotic expression in Escherichia coli BL21-Coodom Plus(DE3)strain.The results showed that the recombinant expression plasmids p ColdⅠ-M27,p ColdⅠ-M32,p ColdⅠ-M498,and p ColdⅠ-M663 were successfully constructed.SDS-PAGE analysis showed that potential proteins with molecular weights of 27 k Da,16 k Da,19 k Da,and 26 k Da,respectively,were expressed in a soluble form.The M27,M32,M498,and M663 putative membrane proteins were purified according to His tag-based affinity chromatography.Western-Blot analysis showed that the purified M27,M32,M498,and M663 putative membrane proteins specifically react with the His-labeled monoclonal antibody,indicating a good reactogenicity.The above results indicate that the prokaryotic expression of M27,M32,M498,and M663 proteins was successfully,and the purified proteins were obtained for subsequent studies.3.Study on subcellular localization and adhesion of Mycoplasma bovis M27,M32,M498 and M663 proteinTo analyze the subcellular localization and adhesion of Mycoplasma bovis M27,M32,M498,and M663 proteins,the purified recombinant proteins were quantitated for preparation of polyclonal antibodies by immunizing New Zealand white rabbits at a dose of 500 μg/ rabbit.Total protein,membrane protein,and cytoplasmic protein were extracted from cultured Mycoplasma bovis.The membrane localization of M27,M32,M498,and M663 proteins were analyzed by Western blot.Primary fetal bovine alveolar(EBL)cells were prepared,and the adhesion properties of M27,M32,M498 and M663 proteins and their roles in adhesion of Mycoplasma bovis to host cells were analyzed by laser confocal microscopy and flow cytometry.The results showed that the concentration and purity of the purified proteins were high,and the titer of rabbit antiserum antibodies reached more than 1:25600.The results of protein membrane localization showed that M27 and M498 were present in both cytomembrane and cytoplasmic components of Mycoplasma bovis,and M32 protein was present in the cytomembrane components,while M663 protein was not detectable in both cytomembrane and cytoplasmic components.The results of laser confocal assays showed that all the four proteins could specifically adhere to EBL cells,and the prepared specific serum antibody could effectively inhibit their adhesion.Flow cytometry results also showed that the serum antibodies of each protein inhibited the adhesion of Mycoplasma bovis to EBL cells.The M663 protein membrane localization was undetectable in Western-blot assay,but it still inhibited the adhesion of Mycoplasma bovis in laser confocal assays and flow cytometry assays,which might be attributed to the difference in antigen-antibody reaction characteristics and sensitivity between Western-blot and immunofluorescence and flow cytometry assays.The above results showed that M27,M32 and M498 proteins were present in the cell membrane of Mycoplasma bovis,and M27,M32,M498,and M663 proteins were related to the adhesion properties of Mycoplasma bovis.In summary,highly conserved Mycoplasma bovis M27,M32,M498 and M663 genes were screened in this study.The corresponding membrane proteins with good immunogenicity were successfully expressed in Escherichia coli,and the rabbit polyclonal antibody with high titers was prepared using these immunogens.Further studies showed that M27,M32,and M498 were membrane proteins of Mycoplasma bovis,and M27,M32,M498,and M663 proteins were involved in the adhesion of Mycoplasma bovis to EBL cells.Our results would lay a foundation for the development of diagnostic reagents and new vaccines,and the elucidation of pathogenesis of bovine mycoplasma disease.