Caracterização da antigenicidade e do efeito protetor em modelo murino de proteínas recombinantes de Erysipelothrix rhusiopathiae candidatas a antígenos vacinais
Fuzissaki, Yuri Nakau
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Erysipelothrix rhusiopathiae is a gram-positive, bacilliform, and encapsulated bacterium that causes swine erysipelas, a disease characterized by skin lesions and other symptoms such as septicemia, arthritis, and endocarditis in the affected animals. Vaccines from attenuated or inactive E. rhusiopathiae cells are available on the market, but they are not so effective to overcome the acute form of the disease. Also, they can aggravate the symptoms of the chronic form. E. rhusiopathiae extracellular proteins were detected as potentially antigenic. It has previously been cloned the coding sequences for 11 genes related to these proteins in the pJET1.2/blunt vector and pET28 expression vector. The present work aims to perform heterologous expression of four of these proteins, named P1, P3, P6, and P7, for functional characterization of their in vitro antigenicity and protection character against E. rhusiopathiae in a murine model. For this, the following was performed: production of recombinant forms of P1 and P7 proteins in Escherichia coli Rosetta strain (DE3) and of P3 and P6 proteins in BL21 (DE3) strain; purification of the four recombinant proteins by affinity and molecular exclusion chromatography. All recombinant proteins were successfully expressed in E. coli and were functionally evaluated for: in vitro antigenicity by Western blot and ELISA, using sera from swine immunized with the available commercial vaccine; protective effect in mice by challenging with E. rhusiopathiae and monitoring of the animals survival; in vitro antigenicity by ELISA using sera from mice immunized with recombinant proteins and challenged with E. rhusiopathiae. The results demonstrated that all proteins are antigenic in pigs and mice, but only P7 protein provides survival for at least one of the six challenged mice. This work leads to the conclusion that the immunoproteomic analysis was a useful tool for the discovery of new antigens of E. rhusiopathiae, however, the antigenic proteins have limited protection effect for the proposals of effective new vaccines. New studies need to be carried out in order to verify whether the protective effect of the recombinant proteins studied here can be enhanced by changing the dosage or administration routes, or even by a combination of P7 protein with other antigenic proteins for a greater protective efficacy against E. rhusiopathiae.
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