Clonagem, expressão e caracterização imunológica da proteína Pb40r presente na fração protetora F0 de paracoccidioides brasiliensis

Abstract

Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; its usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation marked by extreme variability among batches, prevents a proper standardization of the procedures. To overcome this lack of reproducibility, molecular biology tools were used to produce a recombinant 40-kDa-molecular-mass antigen from this fungus. This antigen was identified by Goes et al. (2005), through the AST strategy, as a homolog of Neurospora Crassa calcineurin B. This cDNA was cloned in pGEX4T-3 vector and was expressed in a prokaryotic system as a protein with 378 amino acids and approximately 40 kDa of molecular weight, denominated pb40r. This protein has two EF-hand binding calcium domains and the comparisons of the transcribed sequence to sequences in different gene banks reveal a homology to the EF-HAND domain protein of Neosartorya fischeri and Aspergillus clavatus. Enzyme-linked immunosorbent assay (ELISA) using mice immune serum samples against the recombinant protein, showed that pb40r is an immunogenic protein able to induce the production of a great quantity of IgG, IgG1 and IgG2a antibodies. Moreover, mice infected with P. brasiliensis are also able to recognize pb40r. They showed production of IgG, IgG1 and IgG2a against pb40r. A battery of 149 human serum samples, consisting of 72 sera samples from patients with several clinical forms of PCM, 61 sera sample from patients with diverse infections and 16 from healthy subjects, were all tested by ELISA using the purified pb40r. The sensitivity was 90.3%, when compared to the healthy subjects and the specificity for PCM patients was 81.25% while for patients with other diseases, it was 81.96%. A significant high cross-reaction was detected when sera from patients with leishmaniases was evaluated. These results indicate that the recombinant protein pb40r is a promising antigen to be used in immunodiagnosis of PCM.