Screening of strain-specific Actinobacillus pleuropneumoniae genes using a combination method

We describe a three-step method designed to identify distinct antigen-coding genes between two related bacterial genomes by: (a) constructing a subtractive library using Representational Difference Analysis (RDA), (b) characterization of gene expression in vitro using a ribosome display system combined with antibody screening and (c) gene recovery and confirmation using RT-PCR and reverse Southern hybridization, respectively. To test the efficacy of this strategy we screened the antigen-coding gene profile of Actinobacillus pleuropneumoniae (APP) strains CCVC259 and CCVC263 that do not elicit cross-protective immunity. This strategy identified six different DNA fragments from CCVC259 and 10 different DNA fragments from CCVC263. Of six sequences identified from CCVC259, 2 were not significantly similar, two were 74% and 87% homologous to the sequences encoding for the Ralstonia eutropha H16 conserved membrane protein and transcriptional regulator respectively, and two were >96% homologous to the Pseudomonas alcaligenes putative transposase subunit genes IS1474 and IS1475. Among ten unique DNA fragments identified from strain CCVC263, eight were homologous to DNA fragments encoding the TBP 1 precursor, ATP-dependent helicase HepA, glycosylase, methyltransferase and GTPase in the APP L20 genome and two genes identified had no significant similarity. Our findings indicated that the three-step method could be utilized to identify unique antigen-coding genes and may be a powerful and efficient technique for serotype-specific identification of pathogens and polyvalent vaccine design.