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 pleuropneumoniae serotype 1 clinical isolate and screened with serum produced in pigs that had been vaccinated with the anionic fraction of a sodium chloride extract. One E. coli transformant was isolated that produced a large amount of a protein with an electrophoretic mobility of about 67,000 molecular mass. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by restriction enzyme digestion and nucleotide sequence analysis which showed strong homology with the cysI gene of E. coli. One open reading 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 encoding the protein was labeled with radio-isotope and the homologous gene was isolated from an A. pleuropneumoniae serotype 5a library. The serotype 5a gene 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. Expression plasmids containing cysI from either serotype of A. pleuropneumoniae complemented an E. coli cysI mutant. Pigs vaccinated with the recombinant cysI were protected from challenge with A. pleuropneumoniae of the homologous serotype.
Willson
P J
PJ
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon. willson@sask.usask.ca
Gerlach
G F
GF
Klashinsky
S
S
Potter
A A
AA
D000189:Actinobacillus Infections / Q000517:prevention & control / Q000662:veterinary*; D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins; D001428:Bacterial Vaccines / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D005260:Female; D015723:Gene Library; D005798:Genes, Bacterial; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D015183:Restriction Mapping; D016415:Sequence Alignment / Q000662:veterinary; D012689:Sequence Homology, Nucleic Acid; D012703:Serotyping; D050889:Sulfite Reductase (Ferredoxin); D050864:Sulfite Reductase (NADPH); D013552:Swine; D014614:Vaccines, Synthetic / Q000276:immunology
D029681:Arabidopsis Proteins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D014614:Vaccines, Synthetic; D050862:Oxidoreductases Acting on Sulfur Group Donors; D050864:Sulfite Reductase (NADPH); C488954:SIR protein, Arabidopsis; D050889:Sulfite Reductase (Ferredoxin)