| Mycoplasma synoviae(MS)is a member of the Mycoplasmataceae in the class of Mollicutes and mainly infects chickens and turkeys.It can cause swelling of poultry joints,inflammation of synovial sac,tendon sheath,and enlargement of parenchymal organs.The infections are very serious in domestic and foreign countries.At present,it is generally believed that it can adhere to host epithelial cells in various ways after infection.Many metabolism-related enzymes in mycoplasma have been found not only to be distributed in the cytoplasm to be biocatalytic,but.also to be distributed on the surface of cell membranes to participate in adhesion to host cells.They play pathogenic roles in psthogebesis.Dihydrolipoamide dehydrogenase(DLD)is a kind of flavin protein redox enzyme.At present,there are few studies on the DLD of MS.In this experiment,the biological function of DLD was determined to provide a molecular basis for further exploring the pathogenesis of MS.1.Prokaryotic expression and purification of dihydrolipoamide dehydrogenase of Mycoplasma synoviaeAccording to the did gene sequence of MS WVU1853 from GenBank in NCBI,specific primers for MSdld gene were designed.After amplificated by overlapping PCR,the mutated full length of MSdld gene sequence was cloned to the pLB vector and verified by sequencing.The verified MSdld gene sequence was ligated to the vector pET-28a(+).After being induced by IPTG,the recombinant MSDLD(rMSDLD)protein was confirmed to be expressed in the supernatant by SDS-PAGE electrophoresis.The supernatant rMSDLD protein was then purified by His-tag magnetic beads.2.Determination of enzymatic kinetics of dihydrolipoamide dehydrogenase of Mycoplasma synoviaeSince dihydrolipoamide dehydrogenase is a metabolic enzyme,lipoamide can be catalyzed to generate dihydrolipoamide in the presence of the substrate NADH.The amount of the substrate NADH is continuously monitored by an enzyme marker to display the enzymatic activity of rMSDLD.The results show that it has a certain enzyme activity of dihydrolipoamide dehydrogenase.Its enzymatic specific activity is 3.6 U/mg,the optimum pH is 7.0,the optimum reaction temperature is 25?,and its formula is y=4.55 x+0.1956(R2=0.9973)measured by the double inverse mapping method.The rMSDLD was obtained with a miter constant Km(NADH)of 23.2 ?mol/L and the maximum reaction rate(Vmax)of 5.1 ?mol/(L min).3.Immunogenicity and subcellular localization of dihydrolipoamide dehydrogenase of Mycoplasma SynoviaeWestern-blot assay was performed to detect the immunogenicity of the protein using MS-positive chicken serum as the primary antibody.Mouse polyclonal antibodies and rabbit polyclonal antibodies were prepared by subcutaneous immunization of Balb/c mouse and New Zealand white rabbits with rMSDLD proteins at 100 ?g and 300 ?g respectively after emulsification with adjuvant,and their titers were determined.Western-blot and suspension immunofluorescence assay were performed with the prepared mouse polyclonal antibodies to detect subcellular localization of the DLD protein in MS.Next,the immunoprotective properties of the protein were evaluated by animal experiment.At the same time,the serum mediated complement bactericidal experiment was carried out.The MS-positive chicken serum could react with 0.1 ?g of rMSDLD proteins in the immunogenicity test,and the reaction was dose dependent,which proved that the protein good immunogenicity.The mouse anti-rMSDLD serum and rabbit anti-rMSDLD serum was detected by ELISA to have an antibody titer of 2,048,000 and 102400.The subcellular localization of the DLD in MS was detected with the mouse anti-rMSDLD serum.It was found that the MSDLD mainly existed in the cytoplasmic protein of MS,and a small amount was distributed in the MS membrane components.Suspension immunofluorescence test confirmed the presence of the protein on membrane surface of MS.Then,the rMSDLD protein vaccine was prepared to immunize SPF chickens.The animal experiment showed the rMSDLD immunogen could significantly reduce the incidence of chickens when infected with MS bacteria,suggesting the MSDLD had certain protective immunity.Anti-rMSDLD serum-mediated complement bactericidal efficacy reached to 88.50%,and combining with mycoplasma can effectively activate complement,and thus show good bactericidal effect.4.Study on adhesion characteristics of dihydrolipoamide dehydrogenase in Mycoplasma synoviaeThe adhesion reaction between rMSDLD and chicken fibroblast cells(DF-1)was detected by indirect immunofluorescence and ELISA assays,and the inhibition effect of anti-rMSDLD rabbit serum on its adhesion was also detected.It was found that rMSDLD could be specifically adhered to DF-1 cells,and the adhesion could be significantly inhibited by anti-rMSDLD rabbit serum.Then,the adhesion reaction between this protein and chicken plasminogen(cPlg)of extracellular matrix protein was detected by Western-blot and ELISA,and the results showed that rMSDLD protein had obvious adhesion to extracellular matrix protein cPlg. |
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