Caracterização e expressão heteróloga de proteínas de Corynebacterium pseudotuberculosis para a otimização de um teste intradérmico de hipersensibilidade para o diagnóstico da Linfadenite Caseosa

Abstract

Corynebacterium pseudotuberculosisis a Gram-positive bacterium that causes caseous lymphadenitis (CLA), a chronic infectious disease that affects small ruminants, promoting the reduction of productivity of infected animals. The disease is characterized by the formation of abscesses in the lymph nodes surface and/or internal organs. Several methods for the diagnosis of subclinical CLA been developed, but have not reached satisfactory efficacy and practicability. Thus, this study selected six of the 104 proteins targets previously described, we characterize the genics products of the Open Reading Frame(ORF) and produce recombinant proteins for the optimization of an Intradermal Hypersensitivity Test (IHT) to LC. The inserts of the six ORFs were amplified after extraction of genomic DNA from strain 1002 of C. pseudotuberculosiswith the use of specific primers which bound to vectors pAE and pET-28a were transformed into Escherichia coliDH5for molecular cloning. To verify the expression of recombinant proteins, clones weretransformed into E. coliC43 pLysS and expression kinetics performed. Two recombinant proteins were produced and subjected to Western blotwith sera from goats infected with C. pseudotuberculosis, demonstrating antigenicity in these preliminary tests. New tests for characterization of recombinant proteins will be made and hopefully they can be used in the optimization of an IHT high accuracy for control of CLA in flocks of sheep and goats.