Production And Identification Of Monoclonal Antibodies Against P46 And Dnak Protein Of Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae (Mhp) is the causative agent of Mycoplasma Pneumoniae of Swine (MPS),which causes a chronic respiratory disease of pigs. The main symptoms of this disease are cough and asthma,with meat or shrimp-like consolidation in the leading edge of lobus cardiacus of lung as pathological features.The disease has high morbidity and low mortality.In this study,we immunized BALB/c with the protein of Mhp strain 168,in order to obtain hybridoma cell lines which can steadily secrete monoclonal antibody(McAb) against P46 protein and DnaK protein of Mhp. 1.Monoclonal antibodies against membrane protein P46 of MhpIn this study, P46 recombinant protein was expressed in E.coli expression system,and the protein was purified.At the same time,the supernatant of cultured Mhp strain 168 after sonication as immunogen which emulsified with the equivalent dose of Freund's complete adjuvant immunized 4-6 week old BALB/c mice according to 400?g per mouse by subcutaneous injection.Every 2 week immunized the mice with Mhp 168 isolation protein which emulsified with Freund's incomplete adjuvant to strengthen the immune response. Another immunization of the mice was performed 3d before the fusion.The stimulated splenocytes were fused with Sp2/0 myelomas to produce hybridomas.Screened the hybridoma cell lines which steadily secret monoclonal antibody against P46 protein by P46 protein,168 protein and vetor protein.After three times of subclones,one hybridoma cell line was obtained,designated 2F3.The results of western blot showed that the McAb can reacted with Mhp 168 isolation,and the P46 recombinant protein, has no reaction activity with vetor protein and the proteins of Mycoplasma hyorhinitis.The McAb also reacted with No 105 of 168 stain,J stain,XLW-1 isolation,XLW-2 isolation,XLW-4 isolation,AH isolation,XLW-4 isolation,WX isolation.The isotype of the 2F3 was IgG2b.The ELISA titers which reacted with P46 of cultural cell supernatant of 2F3 was 1:1600,the ELISA titers which reacted with Mhp168 of cultural cell supernatant of 2F3 was 1:1600.The ELISA titer which reacted with P46 of ascites of 2F3 was 1:204800,the ELISA titers which reacted with Mhp168 of ascites of 2F3 was1:51200.The ELISA titer of McAb puried by Octanoic Acid-ammonium sulfate precipitation method was 1:51200. 2.Monoclonal antibodies against hot shock protein DnaK of MhpIn this study,DnaK recombinant protein was expressed in E.coli expression system,and it was purified.The supernatant of Mhp 168 strain F350 after sonication as immunogen immunized 4-6 week old BALB/c mice according to 400?g per mouse by subcutaneous injection.With the hybridoma technology,the stimulated splenocytes were fused with Sp2/0 myelomas to produce hybridomas. Screened the hybridoma cell lines which steadily secret monoclonal antibody against DnaK protein by DnaK protein,168 protein and vetor protein.After three times of subclones,3 hybridoma cell lines was obtained,separately named 3E2?5D10?3A8. The results of western blot showed all of the McAb can reacted with Mhp 168 isolation and the DnaK recombinant protein,has no reaction activity with vetor protein and Mycoplasma hyorhinitis except 3A8.The McAb of 3E2?5D10 also reacted with of 168 strain F105,J strain,XLW-1 isolation,XLW-2 isolation,XLW-4 isolation,AH isolation,XLW-4 isolation,WX isolation.The isotype of the 3E2 and 5D10 was IgGl.The ELISA titers which reacted with DnaK protein of cultural cell supernatant of all antibodies were 1:51200-1:102400,the ELISA titers which reacted with Mhp 168 protein of cultural cell supernatant of all antibodies was 1:640-1:1280.The ELISA titer which reacted with DnaK protein of ascites of all antibodies was 1:106,the ELISA titers which reacted with Mhp168 protein of ascites of all antibodies was1:106.The ELISA titer of McAb puried by Octanoic Acid-ammonium sulfate precipitation method was 1:106.In this study,4 hybridoma cell lines was obtained in all.One of them are able to steadily secrete monoclonal antibody against P46 protein of Mhp.Three of them are able to steadily secrete monoclonal antibody against DnaK protein of Mhp.It should be very useful to the further study the antigen epitopes of P46 protein and DnaK protein,develope diagnose methods and prevent Mhp infection.