RESUMO
Streptococcus equi ssp. equi causes strangles, a highly contagious and serious disease in the upper respiratory tract of horses. Streptococcus equi ssp. zooepidemicus, another subspecies of this genus, is regarded as an opportunistic commensal in horses. The present study describes the characterization of two novel immunoglobulin G (IgG) endopeptidases of these subspecies, IdeE2 and IdeZ2. Both enzymes display sequence similarities with two previously characterized IgG endopeptidases, IdeE of S. equi ssp. equi and IdeZ of S. equi ssp. zooepidemicus. IdeE2 and IdeZ2 display high substrate-specificity in comparison with IdeE and IdeZ, as they both completely cleave horse IgG, while the activity against IgG from mouse, rabbit, cat, cow, sheep and goat is low or absent. The potential use of IdeE and IdeE2 as vaccine components was studied in a mouse infection model. In this vaccination and challenge study, both enzymes induced protection against S. equi ssp. equi infection.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Imunoglobulina G/metabolismo , Infecções Estreptocócicas/veterinária , Streptococcus equi/enzimologia , Sequência de Aminoácidos , Animais , Vacinas Bacterianas , Gatos , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , Endopeptidases/imunologia , Cabras , Cavalos , Lagomorpha , Camundongos , Dados de Sequência Molecular , Coelhos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Ovinos , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/prevenção & controle , Especificidade por SubstratoRESUMO
The present study examines pyruvate kinase-conjugated antibodies for potential use in ELISA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles. This arrangement was found to be suitable as solid support for presentation of antigens in sensitive bioluminescence assays. The nanoparticles were well characterized in terms of protein surface load and were used to establish the number of conjugate complexes needed to generate a detectable signal. Under the biochemical conditions employed here, the detection limit of the pyruvate kinase conjugate lies in the femtomole range.