Preparation Of The Dry Powdre Vaccine Of Mycoplasma Hyopneumoniae 168 Strain And Evalution Monoclonal Antibodies Against This Vaccine

Mycoplasmal pneumoniae of swine(MPS)is a chronic respiratory disease with high morbidity and low mortality,which caused by Mycoplasma hyopneumoniae(Mhp).MPS will cause secondary infections and it is diffcult to be eliminated once porcines are infected by MPS.An attentauated vaccine of Mhp 168 strain is used widely in China to control this disease.The mucosal immune system will be actived and mucosal immunity will be motivated by the vaccine.As the continuing development of swine industry,it is meaningful and promising to change the immunization routes from individual vaccination to herd vaccinaton.In this research,the spray drying technology was applied to make the dry powder vaccine of Mhp 168 strain to achieve nebulization immunization.We optimized several parameters,including excipients and inlet temperatures.The way to produce and evaluate the dry powder vaccine of Mhp 168 strain has been prepared primarily so far.Meanwhile,two kinds of monoclonal antibodies closely related to mucosal immune were also prepared in this research,which would be used to detecte the mucosal immunity induced by the dry powder vaccine of Mhp 168 strain.This research was developed as follows:1.According to the pulished data,two excipients,MTPCD and TP,were sprayed primarily and then MTPCD was selected to further study used as the basic excipient.The excipient formulations,inlet temperatures,outlet temperatures,aspirator rates and pump rates were optimized based on the titers of Mhp 168 strain,particle sizes and particle morphologys of the dry powder vaccine.We have prepared the powder vaccine with the particle size to be 2μm to 5μm and the particle morphology to be sphere,which could located in the lower aspiratory tracts by spray immunization.Furthermore,the glass transition temperatures of them were above 40℃,indicating them could be storaged at room temperature.2.The gene sequence of porcine pIgR published in GenBank was analyzed and the gene coding porcine secretory component(SC)was obtained followed by designing the primers. The porcine SC gene was amplified successfully from the RNA samples extracted from tracheal epithelial cells of porcine and then cloned into the expression vector pCold I to construct the recombinant plasmid pCold I/SC followed by ransformed into the E.coli host strain BL21(DE3).The SC recombinant protein was expressed by adding IPTG,purified by Ni column and gel recovery and confirmed by western blot.BALB/c mice were immunized with the SC recombinant protein and then the spleen cells were fused with myeloma cells.Three hybridoma cell lines were obtained after cloning for five times.The specificity detection revealed that the monoclonal antibodies(MAbs)clould recognize both porcine SC recombinant protein and SC native protein,but not porcine IgA,porcine IgG or the whole lysate of E.coli.Isotypes of the three MAbs were identified to be IgGl/K,IgGl/K and IgG2a/κ,respectively and the ELISA titers for ascites were 1:100000 uniformly.3.BALB/c mice were immunized with porcine slgA protein.The classical hybridoma cell technology was applied to prepare MAbs against porcine slgA protein.Finally,three positive hybridoma cell lines secreting MAbs were obtained,named 1D8,1F10 and 1F11.The MAbs binded to the heavy chain of porcine IgA by western blot testing and had no cross-reaction with porcine IgG.All the isotypes were IgGl/K and the highest antibody titer for ascite was 1:1000000.In this research,we tried to produce the dry powder vaccine of Mhp 168 strain.The preparation and evaluation methods have been made primarily.But further study still need to be done.To evaluate the mucosal immunity activated by the dry powder vaccine,we also prepared two kinds of MAbs which correlated to mucosal immune.