RESUMO
BACKGROUND: Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. RESULTS: A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. CONCLUSION: Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.
Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/veterinária , Animais , Antígenos de Bactérias/sangue , Bacteriófagos/genética , Sequência de Bases , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/patogenicidade , Bases de Dados de Ácidos Nucleicos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Genes Bacterianos/genética , Genoma Bacteriano , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Doenças das Cabras/microbiologia , Cabras , Linfadenite/imunologia , Linfadenite/microbiologia , Reação em Cadeia da Polimerase/veterinária , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Vacinas de DNA/uso terapêuticoRESUMO
The infectious prion protein PrP(Sc) is encoded by the PRNP gene. In cattle, insertion/deletion (indel) polymorphisms are among the changes that occur in this gene, the most studied of which are within intron 1 (12 bp) and the promoter region (23 bp). Sequence variants in this gene may affect the formation of PrP(Sc). In the present study, nucleotide variability in specific regions of the PRNP gene in Caracu cattle free of bovine spongiform encephalopathy was investigated to determine the genotypic profile of each animal within the group. Caracu cattle exhibited high allele frequency for the two polymorphic regions studied, 12ins (70 %) and 23ins (72.5 %), genotype frequencies of 50 % for 12ins/ins and 50 % for 23ins/del, and a high frequency of the 12ins-23ins haplotype (57.5 %). Of the 40 animals sampled, 15 had the 12ins-23ins/12ins-23ins diplotype.
