RESUMEN
An Escherichia coli clone carrying the recombinant plasmid pPH24 has been found to express highly immunoreactive antigens of Pasteurella haemolytica A1. Two or three antigens of approximately 30 kDa were located to both the inner and outer membranes of the E. coli clone and in P. haemolytica A1. From the insert DNA of 8.2 kbp on pPH24, a fragment of 4.6 kbp was found to code for these antigens. Nucleotide sequence analysis of the 4.6-kbp DNA identified three genes (designated plpA, -B, and -C) arranged in tandem which code for three proteins, Plp1, -2, and -3, with predicted molecular masses of 30.1, 30.3, and 29.0 kDa, respectively. Comparison of the nucleic acid sequence of plpA, -B, and -C with GenBank sequences showed extensive homology with a Haemophilus influenzae 28-kDa lipoprotein gene. [14C]palmitate labelling coupled with glybomycin inhibition experiments showed that Plp1, -2, and -3 are also lipoproteins. In addition, plpA, -B, and -C were found to be present only in A biotypes of P. haemolytica by Southern blot analysis. Since Plp1, -2, and -3 were found to be antigenic components in a culture supernatant vaccine, they could be candidates for further investigation as vaccine components.
Asunto(s)
Genes Bacterianos , Haemophilus influenzae/genética , Lipoproteínas/genética , Mannheimia haemolytica/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano/química , Lipoproteínas/química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/química , Homología de Secuencia de AminoácidoRESUMEN
A serotype-specific antigen of Pasteurella haemolytica A1 encoded on the recombinant plasmid pSSA1 is characterized. Nucleotide sequence analysis of the insert DNA in pSSA1 identified the gene ssaI, which codes for a protein of approximately 100 kDa. In vivo labeling of pSSA1-encoded protein in Escherichia coli maxicells showed the expression of a 100-kDa protein from the insert DNA on the recombinant plasmid. Northern blot and primer extension analyses were used to identify the mRNA transcript in P. haemolytica A1 and the putative promoter of ssaI. The antigen (designated Ssa1) could be localized to the outer membrane of P. haemolytica A1 and E. coli clones carrying pSSA1. A rabbit serum against Ssa1 was produced by using whole cells of E. coli expressing Ssa1 on the surface as the immunogen, demonstrating that Ssa1 is immunogenic in rabbits. The results from colony immunoblot analysis with calf serum from animals that were resistant to P. haemolytica A1-induced pneumonia suggest indirectly that Ssa1 is also immunogenic in the animals.