Prokaryotic Expression Of The PDHc-E2 Of Mycoplasma Bovis And Study On Its Adhesion Property And Preparation Of Monoclonal Antibody

Mycoplasma bovis is considered to be one of the important pathogenic agent of bovine,which can persist in the host and cause many diseases,such as pneumonia,mastitis,arthritis and so on,causing huge economic losses to the world cattle industry.Therefore,the study on Mycoplasma bovis will lay a foundation for the effective diagnostic methods and control measures of Mycoplasma bovis.Based on this,this study completed the following work: 1.Cloning,sequence analysis and prokaryotic expression of pdhc gene of Mycoplasma bovis Wuwei isolated strainAccording to the pdhc gene(CP002058.1)of HB0801 in Gen Bank,the primers were designed to amplify pdhc gene of Mycoplasma bovis Wuwei isolated strain.Based on sequencing and sequences analysis,the pdhc gene were optimized using Overlap PCR.Then,the optimized pdhc gene was cloned and expressed.The result showed that the CDS of pdhc gene of Mycoplasma bovis Wuwei isolated strain is 735 bp and its nucleic acid sequence were highly conserved,which was consistent with domestic isolates of Mycoplasma bovis.Its homology with other species of Mycoplasma is less than 50%.The gene encodes 245 amino acids,has no transmembrane region,no signal peptide,and is successfully expressed in E.coli.The molecular weights of the recombinant protein was about 29 kD.2.Preparation polyclonal antibodies against PDHc-E2 of Mycoplasma bovis Wuwei isolated strain and its subcellular localizationThe purified recombinant protein was used to immunize New Zealand rabbits to prepare polyclonal antibodies.The distribution of PDHc-E2 in Mycoplasma bovis was analyzed by ELISA,Western blot and immunofluorescence assay.The results showed that rPDHc-E2 had high immunogenicity.The serum titer of rPDHc-E2 as high as 1: 100 000.The results of ELISA and Western blot showed that PDHc-E2 was distributed in the membrane and cytoplasm of Mycoplasma bovis.Immunofluorescence assay further confirmed the distribution of PDHc-E2 on the surface of Mycoplasma bovis membrane.3.In vitro bactericidal test of rPDHc-E2 antiserum and analysis its adhesion to EBL cellsIn this study,the in vitro bactericidal assay of rPDHc-E2 antiserum was used to further verify the distribution of PDHc-E2 on the membrane surface of Mycoplasma bovis and the specificity of the antibody.The cell adhesion of the PDHc-E2 was analyzed by cell adhesion and adhesion inhibition assay,invasion and invasion blocking assay.The results showed that the bactericidal rate of rPDHc-E2 antiserum was 45.86%;the adhesion and adhesion inhibition assay of PDHc-E2 to EBL cells showed that rPDHc-E2 antiserum could effectively inhibit the adhesion of Mycoplasma bovis to host cells,the adhesion inhibition rate is 42.85%,at the same time,the protein played a certain role in inhibiting the invasion of host cells by Mycoplasma bovis.The invasion blocking rate was 29.71%.4.Preparation and identification of monoclonal antibody against PDHc-E2 of Mycoplasma bovis Wuwei isolated strainThe purified recombinant protein was used to immunize BALB/c mice and monoclonal antibodies against PDHc-E2 were prepared by lymphocyte hybridoma monoclonal antibody technique then identified its subtypes after ELISA screening and limited dilution method.The results showed that the selected hybridoma cell lines could secrete specific antibodies against Mycoplasma bovis and its subtypes were IgG1 type and light chain is κ.In this study,the prokaryotic expression,cell adhesion characteristics and monoclonal antibody production of PDHc-E2 were studied in order to provide a theoretical basis for the further study of the biological function of PDHc-E2,and provide a biological material for the detection of Mycoplasma bovis.