Isolation And Preliminary Identification Of Secreted Proteins From Virulent And Attenuated Mycoplasma Bovis Strains

Mycoplasma bovis is a primary pathogen in cattle and always casuse chronic disease,such as bovine respiratory complex disease,polyarthritis syndrome,mastitis and so on.Secretory proteins could directly take part in the exchange of communication between host and pathogen and were one of important component that are relevant for pathogenicity and immunity.Therefore,the use of these proteins as potential targets of drug and vaccine maybe a better choice.However,M.bovis secreted proteins and its secretome have been just starting so far and their functions are incompletely understood,due to little known protein was identified.The aim of this study was to found the differential secretory proteins between high and low virulence strain of M.bovis based on the proteomic technology and bioinformatics analysis,which may help to uncover the pathogenic mechanism of M.bovis.Results were summarized as follows:1.This study was used high virulent strain HB0801 named as P1 and its attenuated strain designed P150 as research objects.We established M.bovis culture conditions in a suitable medium without serum and improved the extract method of secreted proteins in M.bovis,which allowed to identification of differetial proteins between P1 and P150 using two-dimensional gel eletrophoresis and Label-Free for quantitative proteomic analysis.Additionally,we made a preliminary biological functions analysis of 8 secretory recombinant proteins respectively.Followed by MALDI-TOF mass spectrometry of proteomic,a total of 548 secretory protein was found from P1 and P150 and 477 effective proteins were detected with molecular weight between 10 kDa-90 kDa.Compared with the predicted secretory proteins from P150,there were 66 proteins up-regulated and 48 proteins down-regulated in P1.Only 46 secretory proteins was identified in P1 and 5 proteins was found in P150.According to KEGG pathway and GO functional terms analysis,There was a significant difference in those proteins enrichment when M.bovis P1 was compared to P150: in Biological Process,35 proteins took part in ribonucleoprotein complex biogenesis and ribosome biogenesis and cellular response to DNA damage stimulus and DNA repair and cellular component biogenesis and cellular response to stress;in Cellular Component,27 proteins belonged to intracellular ribonucleoprotein complex and ribonucleoprotein complex;in Molecular Function,24 proteins took part in structural molecule activity and structural constituent of ribosome.The pathway in which 27 the secretory proteins of P150 is significantly affected were the ribosome and Phosphotransferase system.According to predictions of subcellular location,15 proteins were located in outermembrane,2 proteins as extracellular proteins.Regarding secretion pathways,65 proteins were predicted to be secreted by the non-classical pathway,while 53 proteins were predicted as classical secreted.2.In order to investigate the biological function of these proteins,8 secretory proteins including MbovP103(PDHB)?MbovP145?MbovP174?MbovP339(VspY1)?MbovP481(EF-Tu)?MbovP676(EF-G)?MbovP725?MbovP796 were cloned,expressed and purified.We also prepared their polyclonal antibody serum confirmed the immunogenicity.Additionally,one secretory protein rMbovP339 could induce proinflammtory response during infection with bovine macrophage BoMac.Following the generation of an M.bovis HB0801 mutant library with a series of transposons based on Tn4001,we found the mutant strain of rMbovP339 by direct genome sequencing.The immune response was evaluted 12 h after M.bovis challenge at MOI of 1000 by quantitative PCR.Interestingly,the IL-1? and IL-6 mRNA level of mutant strain was decreased significant compared with the widetype strain(*** and P).Meanwhile,rMbovP339 after endotoxin removal was also confirmd its proinflammatory response which could increase production of inflammatory factors(IL-1? and IL-6)in the cell mRNA level.Overall,this study was identified the differential secretory proteins between different virulent strains of M.bovis by label-free for quantitative proteomic analysis and found rMbovP339 could induce proinflammtory response to bovine macrophage.