title: Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi.
abstract: Cruzipain, the major proteinase of Trypanosoma 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 (L kappa) 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 in Escherichia coli cells. 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.
authors.affiliation: Institute of Biology and Experimental Medicine (IBYME), Buenos Aires, Argentina.
authors.forename: D
authors.initials: D
authors.lastname: Kaplan
authors.forename: C
authors.initials: C
authors.lastname: Baldi
authors.forename: M G
authors.initials: MG
authors.lastname: Chiaramonte
authors.forename: M M
authors.initials: MM
authors.lastname: Fernandez
authors.forename: M J
authors.initials: MJ
authors.lastname: Levin
authors.forename: E
authors.initials: E
authors.lastname: Malchiodi
authors.forename: A
authors.initials: A
authors.lastname: Baldi
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D000953:Antigens, Protozoan / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors / Q000138:chemical synthesis; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014349:Trypanosoma cruzi / Q000276:immunology*
chemical_list: D000953:Antigens, Protozoan; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C063214:cruzipain