Caracterização e avaliação da capacidade imunogênica da enolase de Conidiobolus lamprauges

Abstract

Enolase, an important glycolytic enzyme with increased expression at 37°C in Conidiobolus (C.) lamprauges, performs different functions in other organisms, among them, adhesion and invasion of hosts. Thus, the identification of enolase as an immunoreactive protein may contribute to the understanding of these diseases; provide the standardization of serological tests and also develop vaccines, thus bringing more effective treatments and reducing the death of animals. The objectives of this study were to characterize the enolase gene (eno) C. lamprauges and evaluate its immunogenic capacity towards naturally infected sheep. Eno gene amplification isolated from C. lamprauges by the oligonucleotides synthesized based on the sequence of the gene C. lamprauges resulted in a 1305pb fragment. The deduced aminoacid sequence comprised 434 residues with a predicted molecular weight of 47,2KDa. In the sequence analysis, we identified the signing of the enolase gene (LLLKVNQIGTVSES) corresponding to positions 342-345. In the analysis "in silico" probable regions were detected for epitopes to T and B lymphocytes. The sequence was cloned into the plasmid pFN6K (HQ) Flexi® Vector (Promega®) and the protein expressed in Escherichia coli BL21 (DE3) pLysS cells with IPTG induction. In SDS-PAGE and Western blot, the recombinant eno was detected with estimated molecular weight of 47kDa. Recombinant IgG anti-enolase was found is in all animals with conidiobolomycosis by means of the Western blot technique, demonstrating immunogenicity. In healthy sheep serum, used as a negative control, and serum from sheep with pythiosis used to evaluate cross-reactivity, bands were not detected. Therefore, this study demonstrated that recombinant enolase C. lamprauges is immunogenic against serum of naturally infected sheep with conidiobolomycosis. The detection of specific anti-enolase antibodies in animals shows that the protein is a promising candidate antigen for the development of effective vaccine against this disease.