Abstract
Cruzipain, the major proteinase ofTrypanosoma cruzi,plays an important role in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain. This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (Lκ) and the first two domains of the IgG1 heavy chain (VH/CH1). These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed inEscherichia colicells. The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product. Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals.
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