Study On The Biological Function Of Phosphoglycerate Kinase Of Mycoplasma Synoviae

ObjectiveMycoplasma synoviae（MS） is one of the most important pathogens among poultry pathogens.It mainly infects the joints,bursa and foot pads of chickens and can lead to reduced growth and egg production,causing huge economic losses to the chicken industry worldwide.In orderto confirm its role in the adhesion of mycoplasma to host cells and its bio-enzymatic and otherrelated protein functions,this study focused on the purification of the prokaryotic expression,identification of enzymatic activity,membrane localization and adhesion properties of MSPGK,in orderto furtherinvestigate the role of PGK in the pathogenesis of MS.The present study focused on the purification of the prokaryotic expression,identification of the enzymatic activity,membrane localization and adhesion properties of MSPGK,providing a molecularbasis forfurtherstudies on the role of PGK in MS pathogenesis.MethodsThe genomic DNA of MS WVU₁₈₅₃was used as a template to amplify the MSpgk gene by Overlap PCR,and the TGA was mutated into TGG that could be translated and expressed in E.coli,and then ligated into the expression vectorp ET28a（+）to construct a recombinant strain BL21（MSpgk-p ET28a）forprokaryotic expression.The purified rMSPGK protein was obtained by IPTG induction followed by purification.The rMSPGK protein was assayed by Western-blot using MS standard positive sera to determine its reactogenicity.The enzymatic activity was measured at different temperatures,p H and substrate concentrations using the GAPDH coupling method and spectrophotometric method.The enzymatic specific activity,Mie’s constant and maximum reaction rate of rMSPGK protein were obtained by calculation.The purified rMSPGK protein was emulsified and then immunized to experimental rabbits to obtain rabbit polyclonal antibody against rMSPGK.Using the prepared anti-rMSPGK rabbit polyclonal antibody as primary antibody,Western-blot reaction was performed on the extracted MS whole bacteria,membrane and cytoplasmic proteins to determine the localization of PGK in MS sub-cells.Simultaneous suspension immunofluorescence assays were performed to analyze the membrane localization of MSPGK protein.Indirect immunofluorescence assays were performed to determine whetherrMSPGK protein could adhere to chicken fibroblasts DF-1.The rMSPGK protein was also identified to bind to Fn and c Plg by Western-blot and ELISA assays using samples of extracellularmatrix proteins Fn and c Plg on host cells respectively.Results1.The full length of the point mutated MSpgk gene was obtained and the recombinant strain E.coli BL21（MSpgk-p ET28a）was successfully constructed to obtain purified rMSPGK protein.It was identified to have good reactogenicity with MS positive serum.2.The enzymatic activity of rMSPGK was measured,and the specific activity of rMSPGK protease was 100.70 IU/mg,and the reaction was fast,and the optimum temperature and p H of the reaction were 37°C and 7.5,respectively,because the PGK reaction is more complicated with two substrates,3-PGA and ATP,are required.Using the Michaelis equation to rewrite the formula,V_max（3-PGA）is 370.37μmol/（L·min）,The Michaelis constant K_m（3-PGA）value is 3.88 mmol/L;V_max（ATP）is 357.14μmol/（L·min）,K_m（ATP）value is 0.25mmol/L.3.A rabbit polyclonal antibody to rMSPGK protein was successfully prepared and the potency was measured at 12800.Subcellularlocalization analysis showed that PGK was mainly distributed in the cytoplasm of MS,with a small amount on the cytosolic membrane;suspension immunofluorescence analysis showed that PGK was distributed on the membrane surface of MS,both of which confirmed that PGK was a membrane protein of MS.4.rMSPGK protein significantly adhered to DF-1 cells,while no significant adhesion was seen with control rMSFBA protein.5.rMSPGK protein could bind significantly to Fn and c Plg,and the binding response was enhanced with the increase of protein up-sampling.Conclusions1.The purified rMSPGK protein was obtained and proved to have phosphoglycerate kinase activity with an enzyme specific activity of 100.70IU/mg,indicating that PGK has a strong enzyme specific activity and can provide energy forMS.2.It was confirmed that MSPGK protein is a membrane protein and cytoplasmic protein of MS,with good reactogenicity,and can adhere to chicken fibroblasts DF-1,indicating that it plays a role in MS adhesion to host cells.3.rMSPGK was shown to bind significantly to the extracellularmatrix proteins Fn and c Plg of host cells,suggesting that theirinteraction may play a role in the pathogenesis of MS.