| Mycoplasma gallisepticum(MG) is a primary pathogen causing chronic respiratorydisease in chicken. People pay great attention on the prevention of MG's infection inmany country, but there was not any effective method yet now. Special vaccine's usage isan effective method in prevention of contagious disease, but in practice the vaccine ofMG is not effective enough. At present there are two kinds of vaccines about toMycoplasma gallisepticum, inactivated vaccine and attenuated vaccine, but their highprice or frangibility restrict their usage. So identifying the protein with apparenteimmunogenicity in MG and preparing the subunit vaccines are stong guarantee to preventand purify the infection in fowls. It is similar with neutralizing antibody against virus thata part of antibodies against MG can inhibite its both growth and metabolism.Theoretically, Mycoplasma gallisepticum's antigen proteins specific to monoclonalantibodies with metabolic inhibition activities are major protective protein. In order toidentify antigenic and protective proteins of Mycoplasma gallisepticum, monoclonalantibodies (McAbs) against virulent M. gallisepticum S6 strain were produced in mice.MAbs were selected for its abilities to inhibit both growth and metabolism of M.gallisepticum in vitro. The isolation and expression of specific immunogens responsiblefor protective immunity may lead to the development of effective subunit vaccines. Themain contents and results of research are summarized as following:1. Preparation of monoclone antibody (McAb) against whole M.gaIlisepticum S6 strain proteinSix week BALB/c mice were immunized with whole M. gallisepticum S6 strainprotein.By using the hybridoma technique, twenty-four ELISA positive hybridoma celllines secreting monoclonal antibodies (McAbs) against MG proteins were established.The indrect ELISA titers of these hybridoma cell supernatant are among 2.00×101～2.56×103 and ascites fluids are among 1.00×103～2.05×106, The number of chromosomeof these twenty-four hybridoma cell lines were 79～95. Eleven kinds of ascites fluidscontaining MAbs with higher indirect ELISA titer was selected. In order to determinethese eleven kinds MAbs'relative affinity to M. gallisepticum S6 strain protein, three timeindirect ELISA tests were performed on them. The results suggest that the McAbsecreting by 10B6b hybridoma cell line have maximal relative affinity, then 3G4b,10B6a,10B6c,16B8a,16B8b,3G4a,16G9a,16D5,16G9band 16B4b.2. Selection of MAbs with metabolic inhibition and Analysis ofAnti-polypeptides Antigens in order to obtain MAbs with higher metabolic inhibition titer, metabolic inhibitionassay was performed on M. gaIlisepticum S6 strain of 1000CCU. Eleven kinds MAbswere selected by three time tests repeatively, and their metabolic inhibition titer are lessthan 1:320 respectively. Mycoplasma proteins were separated by SDS-PAGE and the gelwas either stained with coomassie brilliant blue or elecetrophoretically transferred to NCmembrane for immunoblotting with the McAbs selected. The result of immunoblottingsuggested that McAb 1B3 and 3G4b bound to proteins with molecular weight of 56KDprotein, McAb16D5 with molecular weight of 105KD protein, 10B6b, 16B8b all bound toproteins with molecular weight of 64KD protein, 7Gga, 7G9b, 17C5a, 16Gga bound toproteins with molecular weight of 42 KD protein. In addition, there are three stain bandsvisualized in the NC membrane treated by McAb 17C5b, they located in 105KD, 66KDand 41KD respectively, there are two stain bands visualized in the NC membrane treatedby McAb 16H6b, their molecular weight are 42KD and 29KD respectively. According tothe result of study, protein of MG-S6 with molecular weight of 105KD,66KD,64KD,56KD,42KD,41KD,29KD have good immunogenicity, and antigen against the McAbwith metabolic inhibition are not just one kind. In order to achieve the better immunedeffect we should use immunogenic protein as many as possible when to prepare thegenetically engineering vaccine. |
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