Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi

Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important ro le in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag136B6 hybrido ma against cruzipain. This procedure involves the use of cDNAs obtained wit h the aid of a specific set of primers complementary to the complete light kappa chain (LK) and the first two do mains 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 in Escherichia coli cells. T he recombinant Fab molecule recognized cruzipain by ELISA, in a fashion sim ilar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specif ic anti-mouse IgG (Fab)a, indicated the immunoglobulin nature of the recomb inant product. Moreover, both the mAb anti-Ag136B6 and the soluble Fab frag ment described here react similarly with the intact parasite surface, as ob served in an indirect immunofluorescence assay. In conclusion, our recombin ant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosi s, antigen purification, and eventually treatment of Chagas-afflicted indiv iduals. (C) 1998 Academic Press.