Cloning and characterization of the gene coding for NADPH-sulfite reductase hemoprotein from Actinobacillus pleuropneumoniae and use of the protein product as a vaccine

An expression library was constructed from an Actinobacillus pleuropneumoni ae serotype 1 clinical isolate and screened with serum produced in pigs tha t had been vaccinated with the anionic fraction of a sodium chloride extrac t. One E. coli transformant was isolated that produced a large amount of a protein with an electrophoretic mobility of about 67 000 molecular mass. Th e A. pleuropneumoniae-derived DNA encoding the protein was localized and ch aracterized by restriction enzyme digestion and nucleotide sequence analysi s which showed strong homology with the cysI gene of E. coli. One open read ing frame of 1764 bases in length was detected which encoded a cysI protein from serotype 1, with a calculated molecular mass of 66 678. The DNA encod ing the protein was labeled with radio-isotope and the homologous gene was isolated from an A. pleuropneumoniae serotype 5a library. The serotype 5a g ene was the same length, but the cysI protein from serotype 5a was slightly larger (66 849) due to 8 substitutions in the amino acid sequence. Express ion plasmids containing cysI from either serotype of A. pleuropneumoniae co mplemented an E. coli cysI mutant. Pigs vaccinated with the recombinant cys I were protected from challenge with A. pleuropneumoniae of the homologous serotype.