| Summary: | 碩士 === 國立高雄大學 === 生物科技研究所 === 95 === Actinobacillus pleuropneumoniae is a kind of pathogen which can infect the porcine respiratory tract and induce hemorrhagic, necrotic and fibrinous pleuropneumonia of swine. The pleuropneumonia is an infectious disease with high mortality rate and causes the death of swine. So far, it is still known limited about the mechanism of how does this pathogen adhere to the porcine respiratory tract. Fimbriae are the medium that the bacteria usually used to adhere to the epithelia of respiratory tract. The fimbriae of A. pleuropneumoniae are composed of thousands of structural protein ApfA, which molecular weight is approximately 15 kDa. The purpose of this study is the preparation and characterization of monoclonal antibody against ApfA and use the monoclonal antibody to analyze the epitopes of ApfA. The ApfA-pcDNA plasmid constructed previously in our laboratory was used as DNA vaccine to immunize BALB/c mice. The antibody titer against ApfA raised was confirmed after third immunization. The mice splenocytes were isolated and fused to myeloma cells. The positive hybridoma clones were confirmed by screening with ELISA and Western blot analysis. Furthermore, the positive clones were used to conduct the limiting dilution to make sure the positive reaction is produced by monoclonal hybridoma. The results show that the hybridoma can produce monoclonal antibody against ApfA was obtained. The hybridoma cells were injected into the abdomen cavity of mice through intraperitoneal administration to produce ascite fluid. The subtypes of monoclonal antibody were analyzed and the results showed that the major subtype is IgG1. The monoclonal antibody was purified from ascite fluid and the monoclonal antibody against ApfA was obtained. In addition, the cysteine residues of ApfA were replaced by glycine through site-directed mutagenesis method. Three ApfA mutant strains, C63G, C132G, and C63&132G double mutant, were obtained to analyze the influence of cysteine in ApfA epitopes. Western blot analysis was used to analyze the monoclonal antibody against these three mutant strains. The results showed that there is cross reaction between E. coli XL-1 blue which interfere with the epitope analysis. It is necessary to purify these mutated proteins for further analysis. The growth inhibition assay revealed that the monoclonal antibody could inhibit the growth of A. pleuropneumoniae. In summary, the monoclonal antibody produced in this study could provide a crucial research tool and establish the good groundwork for A. pleuropneumoniae infection mechanism research and vaccine development.
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