MALCHIODI Emilio Luis
Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi.
KAPLAN D, BALDI C, CHIARAMONTE MG, FERNANDEZ MM, LEVIN MJ, MALCHIODI E, BALDI A.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Año: 1998 vol. 253 p. 53 - 58
Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important role in the biology of this parasite. This study reports the development of a recombinantFab antibody, using RNA isolated from the anti-Ag163B6 hybridoma againstcruzipain. This procedure involves the use of cDNAs obtained with the aid of aspecific set of primers complementary to the complete light kappa chain (L kappa)and the first two domains of the IgG1 heavy chain (VH/CH1). These products weresubsequently cloned in the pComb3 system, from which the gIII gene had beenremoved, and expressed in Escherichia coli cells. The recombinant Fab moleculerecognized cruzipain by ELISA, in a fashion similar to the original mAbanti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicatedthe immunoglobulin nature of the recombinant product. Moreover, both the mAbanti-Ag163B6 and the soluble Fab fragment described here react similarly with theintact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of thismolecule for diagnosis, antigen purification, and eventually treatment ofChagas-afflicted individuals