Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi
| Autor: | Carolina Baldi, Dan Kaplan, Mariano J. Levin, Alberto Baldi, Emilio L. Malchiodi, Marisa M. Fernández, M. G. Chiaramonte |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
medicine.drug_class
Trypanosoma cruzi Blotting Western Genetic Vectors Molecular Sequence Data Biophysics Protozoan Proteins Cruzipain Antigens Protozoan Enzyme-Linked Immunosorbent Assay Biology Monoclonal antibody Biochemistry law.invention Antigen-Antibody Reactions Immunoglobulin Fab Fragments Antigen law medicine Animals Cloning Molecular Fluorescent Antibody Technique Indirect Molecular Biology Gene Nucleic acid sequence RNA Cell Biology Molecular biology Recombinant Proteins Cysteine Endopeptidases Immunoglobulin G Recombinant DNA biology.protein Immunoglobulin Light Chains Antibody Immunoglobulin Heavy Chains |
| Zdroj: | Biochemical and biophysical research communications. 253(1) |
| ISSN: | 0006-291X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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