Screening And Preliminary Identification Of Immune-related Membrane Proteins From Mycoplasma Bovis And Host Proteins Involved In Mycoplasma Bovis Infection

Mycoplasma bovis is an important pathogenic Mycoplasma that can infect cattle.Mycoplasma bovis can persist in the host and cause chronic diseases,including pneumonia,mastitis,otitis,reproductive disorders,arthritis,meningitis,and keratoconjunctivitis.It is difficult to prevent and control the disease caused by mycoplasma,due to the poor efficacy of antibiotics in clinic and the lack of commercially available sensitive diagnostic tools and effective vaccines.Mycoplasma bovis can cause long-term infection through the surface antigen is the major cause of the high frequency variation,avoid phagocyte phagocytosis,invasion of host cell,biofilm formation and regulating methods of host immune response to escape the host natural and adaptive immunity.Deep understanding of Mycoplasma bovis proteomics and interactions with the host cell protein,helps to develop a better Mycoplasma bovis diagnostic tools and vaccines,understand the pathogenic mechanism of mycoplasma,also will further promote the cow mycoplasma prevention and control technology development.1.Mycoplasma bovis strain membrane protein immunoprecipitation（IP）experimentNew Zealand white rabbits were immunized with the whole strain of Wuwei strain of M.bovis cultured in the middle and late logarithmic phase to prepare whole-cell polyclonal antibodies.The titer of the whole strain was tested using ELISA,and then the ammonium sulfate-octanoic acid method was used to purify the multi-antibody serum.For IgG,the titer of purified IgG was again determined using ELISA.Then the M.bovis membrane protein was extracted,and after sufficient dilution,a certain amount of purified IgG of the total Mycoplasma bovis was added.After the antigen and antibody were fully bound,the agarose A protein beads were added to form the agarose A protein beads-antigen-antibody complexes.The complex was identified by mass spectrometry.The ELISA test showed that the titer of multi-antibody serum of M.bovis strain was 1:12800,and the titer of purified IgG antibody was 1:51200.The results of mass spectrometry showed that 158 species of mycoplasmal membrane proteins with immunogenicity were selected.2.EBL membrane protein and bovine mycoplasma membrane protein immunoprecipitation（co-ip）experiment.Extract culture to 24 hours of EBL cell membrane protein and training to the middle and later periods of the logarithmic Mycoplasma bovis membrane protein,make both blending,join the purified Mycoplasma bovis wuwei plant molecular reactions in full,all bacteria(IgG form antigen antibody-do protein complexes,and then use agarose beads with A protein complex combination,make compounds and other miscellaneous protein extraction and the detection of fluid mass tandem mass spectrometry.Mass spectrum detection,according to the results of a total of 40 kinds of can with Mycoplasma bovis membrane proteins do EBL cell protein,which is 18kinds of membrane protein,for 11 kinds of mitochondrial membrane protein,endoplasmic reticulum for 11 kinds of retinal protein.3.Cloning,expression,enzymatic activity and subcellular localization of NOX1 gene from Mycoplasma bovisAccording to the sequence of GenBank NOX-1 gene of Mycoplasma bovis（GenBank No.NC₀15725.1）,the primers were designed and then the NOX-1 gene of M.bovis strain Lintao was amplified using Overlap PCR.Based on the analysis of complete sequencing,the prokaryotic expression vector pET-NOX-1 was constructed and was transformed into Escherichia coli BL21（DE3）.Then the expression of the recombinant proteins rMbNOX-1 was induced by isopropylβ-D-1-thiogalactopyranoside（IPTG）and its enzyme activity and immunogenicity were determined after the expressed product were purified.The results showed that the full-length of NOX-1 gene from M.bovis strain Lintao is 1365 bp.The molecular weight of the recombinant proteins was about 50 kDa.The recombinant protein could catalyze NADH oxidizatedion to NAD⁺and the optimal temperature was at 30℃,pH value was 7.5,the Michaelis constant（K_m）and the maximum of reaction velocity(V_max)were 256.41μmol/L and34.25μmol/（L·min）respectively.The results of Western blot and ELISA show that His-NOX-1 in M.bovis has distribution in membrane and cytoplasm is more than the amount of distribution in the cell membrane and cytoplasm.All the results of this research will lay a foundation for the further study of the biological function of M.bovis NOX.