Study On Biological Functions Of Enolase From Mycoplasma Synoviae

Mycoplasma synoviae（MS）is one of the main pathogenic mycoplasma of poultry, whichcauses chronic respiratory tract infection in chickens and turkeys. It can also lead to infectioussynovitis and tenosynovitis in chickens. Although the infection of Mycoplasma synoviaes cannot directly lead to the death of chickens, but it can retard growth in chickens and decrease thequality of the meat, laying rate or hatching rate, and bring about the immunosuppression, thuscauses serious economic losses in the worldwide poultry industry.At present, there is no relatedproducts used to provent MS infection such as vaccines in China,the documents relate to studyon MS are also rare. Therefore, the carrying out relevant research of MS are bound to lay a solidtheoretical foundation for the research of vaccine and diagnostic reagents and development oftherapeutic drugs agaist MS.In this study, the enolase gene of the WVU1853strains of MS wasamplified,expressed and then studied on it’s enzymatic activity,cellular localization andbiological charecteristics,which will lay a solid foundation for the development of vaccine anddrug to against the infection of MS.1A pair of specific primers was designed referring to the sequence of Mycoplasmasynoviaeis P53strains in GeneBank,then the enolase gene of Mycoplasma synoviaeis WVU1853was amplified and cloned into the pGEM-T Easy.after finished the sequencing and pointmutation, the gene was subcloned into pET-28a(+),the recombinant prokaryotic expressionplasmid pET-Eno was expressed in E.coli BL21(DE3) induced by IPTG,The expressedproduct(His-Eno) was used to immunize the BALB/c mouse after purified and the theanti-enolase antiserum was collected. The immunogenicity of recombinant protein His-Eno wasanalyzed by western blotting. The result show that the antiserum can specific bind to His-Enoand Mycoplasma synoviaeis.2The enzymatic activity of the expressed product was tested by using continuousmonitoring assay, it turned out that the His-Eno has the hydrolase activity with an optimum pHwas7.5and optimum temperature was50℃.Mg2+can serve as enzymes cofactors and Zn2+ofsame concentration can improve the activity of enzymes, on the contrary, Mn3+、Al2+、Ni2+、Ba2+and Li+can have different levels of inhibitory effect on the activity of enzymes. Besides,the Km and Vmax that was deduced from Lineweaver-Burk plot was1.1×10-3mol/L and0.739mmol/L/min,respectivly. 3In order to observe the distribution of enolase in MS,the membrane protein of MS WVU1853and hydrophilic protein was extracted and used to western blotting, the results showedthat enolase is present in membrane surface and the cytoplasm of MS. It was further confirmedthat the enolase is located in the membrane surface and in the cytoplasm of MS by immuneelectron microscopy method.4In order to study the role that enolase played in MS adhesion and invasion to the hostcell, the binding activity between His-Eno and plasminogen or fibronectin was tested bywestern blotting and ELISA; Besides, the bactericidal effect of the antiserum against His-Enowas tested,and the function that the antiserum against His-Eno prevent MS adhesion andinvasion to the host cell was also analysised.The ELISA method demonstrated that the His-Enocan combined of chicken blood plasminogen in a specific dose-dependent manner,accordinglythe His-Eno has the competence of binding fibronectin.Western blotting also show that theHis-Eno can specific combined with plasminogen or fibronectin and the banding was about53kDa. Serum bacterialcid assay show that the antiserum against His-Eno has excellentbactericidal effect,but the cell adhesion and adhesion blocking assay show that His-Enoadhesion blocking rate is only about5.4%.