Study On The Enzymatic Activity And Adhesion Characteristic Of NADH Oxidase From Mycoplasma Synoviae

Mycoplasma synoviae?MS?mainly causes subclinical respiratory diseases of chickens and turkeys,malformations of eggshell tops,and joint exudative synovitis.Many studies have shown that enzymes involved in mycoplasma metabolism not only play an enzymatic function in the cytoplasm,but also partially exist on the cell membrane,and play important roles in assisting mycoplasma adhesion and invasion of host cells.NADH oxidase?NOX?is a kind of oxidoreductase.In this experiment,we studied the enzymatic activity and adhesion characteristics of MS NOX,which providing a molecular basis for further study on the role of NOX in the pathogenic process caused by MS.Firstly,we constructed the expression strain BL21-pET28a-MSnox?DE3?.Resulting from SDS-PAGE showed that recombinant MSNOX?rMSNOX?protein expressed in the supernatant after IPTG induced.The rMSNOX protein was purified by Beaver Beads TM His Tag magnetic beads.Then,the immunogenicity of the rMSNOX protein was identified by Western blot assay.The titer of anti-rMSNOX rabbit polyclonal antibody detected by ELISA was 1:102 400,and the complement-dependent bactericidal rate mediated by anti-rMSNOX rabbit polyclonal antibody was 86.42%.NOX can catalyze the oxidation of NADH to NAD⁺in the presence of O₂.The conversion of NADH was measured at OD₃₄₀to detect the enzymatic activity of NADH oxidase.The result showed that the specific enzyme activity of rMSNOX was 14.17 IU/mg,the optimal enzymatic temperature was 37?,and the optimal p H was 7.5.Besides,the maximum reaction rate(V_max)of rMSNOX obtained from double reciprocal method was 21.8?mol/?L·min?,and the Michaelis constant[K_m?NADH?]was 244.0?mol/L.Then,We identify the subcellular localization of rMSNOX protein by Western blot and suspension immunofluorescence assays.The results showed that rMSNOX protein mainly exists in the cytoplasm of MS,and there is also a small amount distributed on the surface of MS membrane.Indirect immunofluorescence was used to detect the adhesion of rMSNOX to DF1 cells,and the mycoplasma colony counting method was used to detect the inhibition rate of anti-rMSNOX rabbit polyclonal antibody on adhesion of MS to cells.The results showed that rMSNOX could adhere to DF1 cells,in addition,the anti-rMSNOX rabbit polyclonal antibody could significantly inhibit the adhesion of MS to DF1 cells,and the inhibition rate was 67.87%.To further analyze the mechanism of rMSNOX in the adhesion of MS to host cells,we conducted the binding assays of rMSNOX to the extracellular matrix proteins Plg and Fn by Western blot and ELISA,and the results showed that rMSNOX was able to bind to cPlg and Fn.In summary,the recombinant protein of rMSNOX was expressed in vitro,which was then identified to possess NADH oxidase activity,and distribute small amount on the cell membrane of MS.In addition,rMSNOX was confirmed to play important role in the adhesion of MS to host cells.This study may provide a molecular basis for further exploring the role of NOX in the pathogenesis of MS.