Expressão e purificação de uma proteína de Leptospira spp. selecionada in silico e sua aplicação no diagnóstico

Abstract

Leptospirosis is a zoonotic and cosmopolitan disease caused by bacteria of the Leptospira genus. There are two classification systems for this bacterium: one based on serogroups and serovars, which rely on antigenic determinants Leptospira interrogans (pathogenic), comprising more than 250 serovars and 23 serogroups, and Leptospira biflexa (saprophytic). The second is the genetic classification, in which Leptospira species are divided into 21 genomospecies with more than 300 serovars distributed across 28 serogroups, categorized as pathogenic, intermediate, and non-pathogenic or saprophytic. Leptospira interrogans belongs to the pathogenic group. Knowledge of the most effective measures to prevent leptospirosis in mammals must be constantly revised, as the disease remains prevalent in various regions worldwide. Therefore, studies utilizing recombinant proteins to enhance new diagnostic and preventive methods against the disease are necessary. The objective of the present study was to express and purify a recombinant protein from Leptospira interrogans serovar Copenhageni strain M20, which was selected in silico, and to use it as an antigen in an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of IgG antibodies against Leptospira spp. in serum samples from dogs with leptospirosis. The expression of the selected recombinant protein was carried out using Escherichia coli BL21, followed by purification of the expressed product using the AKTA Purifier 100 FPLC system with a Nickel HisTrap HP column (5 × 5 mL). Serum samples from dogs with and without leptospirosis were subjected to the microscopic agglutination test (MAT). The indirect ELISA test was standardized using the recombinant protein as an antigen to differentiate between reactive and non-reactive animals for Leptospira spp. In the MAT, dogs predominantly reacted to the Icterohaemorrhagiae serogroup, with serovar Copenhageni being the most prevalent, as 53.8% (7/13) of the seroreactive animals tested positive. The ELISA results, using the selected recombinant protein as an antigen, showed a statistically significant difference in optical densities (OD) between the groups [t (24) = 2.53; p = 0.018], indicating that the mean OD of seroreactive positive dogs was significantly higher than that of negative dogs. This ELISA demonstrated a sensitivity of 84.62% and a specificity of 61.54%. The expression and purification of the recombinant protein were achieved using a plasmid containing the in silico-selected chimeric protein from Leptospira interrogans Copenhageni strain M20. The indirect ELISA test developed in this study, which utilized the purified recombinant protein as an antigen, was able to detect IgG antibodies against Leptospira spp. in serum samples from dogs with leptospirosis.