Epitopes Analysis and Immunological Studies of the
ApfA Protein of Actinobcacillus pleuropneumoniae
Advisor: Dr. Wen-Jen Yang
Institute of Biotechnology, National University of Kaohsiung Student: Wei-Fang Huang
Institute of Biotechnology, National University of Kaohsiung ABSTRACT
Actinobcacillus pleuropneumoniae, a Gram-negative bacterium, is the etiological
agent of porcine pleuropneumonia and pulmonary haemorrhagic necrosis causing swine severe respiratory disease that leads to significant economic loss. The virulence factors of this pathogen have been identified including capsular polysaccharide, lipopolysaccharide, exotoxin, and outer membrane proteins. Generally speaking, the fimbriae are necessary for bacteria to colonize on the mucous membrane surface of the host. Therefore, the fimbriae of A. pleuropneumoniae should play an important role for infection. In this study, the structural protein of A. pleuropneumoniae fimbria, ApfA, was used to investigate the epitopes analysis and the potential for subunit vaccine development.
The apfA gene has been cloned into pQE30-UA vector, expressed in E. coli expression system and the expressed ApfA protein was purified. To analyze the epitopes of ApfA protein, we predicted the epitopes using an epitope analysis program. The results show that the most possible epitopes should locate at aa55-62, aa68-75, aa77-85, and aa138, respectively. The most hydrophilic region (aa 131-138) of ApfA was synthesized an eight-mer peptide which conjugated with carrier protein multiple antigen peptides (MAPs) for further immunization analysis. Besides, we also created a serial of deleted apfA clones using ExoIII deletion method and expressed a serial of truncated proteins. The truncated ApfA proteins were detected with A. pleuropneumoniae serotype 1 polyclonal antiserum using Western blot analysis to predict the epitopes of ApfA. However, the data shown that there are cross-reactions between the antiserum and E. coli to interfere the epitopes analysis. These truncated proteins could be analyzed using monoclonal antibody in the future.
To understand the potential of ApfA as a subunit vaccine candidate, we used 50μg, 10μg recombinant ApfA protein, MAP-conjugated synthetic peptide as antigens to immunize mice. A commercial inactivated A. pleuropneumoniae vaccine and PBS also used as positive and negative controls. After second immunization, the titers of
antisera increased significantly except PBS group. These mice were challenged with 5x108 colony-forming unit (cfu) of serotype 1 of A. pleuropneumoniae after 28 days of first immunization. The survival rate of 10μg ApfA and inactivated vaccine groups were 25% and other groups were 0% after 72 hours of virulence strain challenge. The growth inhibition test was performed using the antiserum of survival mice to incubate with A. pleuropneumoniae. The results shown that antiserum can inhibit the growth of this pathogen. In summary, the data reveal that ApfA can induce significant antibody titer which can inhibit the growth of A. pleuropneumoniae and the induced immune responses can provide protection for 10 ID50 of virulence strain challenge in mice. The recombinant ApfA protein should be used as a potential subunit vaccine candidate in the future.
Keywords: Actinobcacillus pleuropneumoniae, ApfA protein, epitope,
