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 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 wit...
Guardado en:
| Autores principales: | , , , , , , |
|---|---|
| Formato: | JOUR |
| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_0006291X_v253_n1_p53_Kaplan |
| Aporte de: |
| id |
todo:paper_0006291X_v253_n1_p53_Kaplan |
|---|---|
| record_format |
dspace |
| spelling |
todo:paper_0006291X_v253_n1_p53_Kaplan2023-10-03T14:03:46Z Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi Kaplan, D. Baldi, C. Chiaramonte, M.G. Fernandez, M.M. Levin, M.J. Malchiodi, E. Baldi, A. complementary DNA cruzipain cysteine proteinase enzyme antibody immunoglobulin F(ab) fragment immunoglobulin heavy chain immunoglobulin kappa chain parasite antibody parasite antigen primer DNA recombinant antibody RNA animal cell antigen recognition article controlled study enzyme linked immunosorbent assay Escherichia coli gene expression system hybridoma immunofluorescence molecular cloning nonhuman nucleotide sequence priority journal Trypanosoma cruzi Animalia Escherichia coli Escherichia coli Trypanosoma Trypanosoma cruzi Trypanosoma cruzi 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 (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 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-Ag163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_0006291X_v253_n1_p53_Kaplan |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
complementary DNA cruzipain cysteine proteinase enzyme antibody immunoglobulin F(ab) fragment immunoglobulin heavy chain immunoglobulin kappa chain parasite antibody parasite antigen primer DNA recombinant antibody RNA animal cell antigen recognition article controlled study enzyme linked immunosorbent assay Escherichia coli gene expression system hybridoma immunofluorescence molecular cloning nonhuman nucleotide sequence priority journal Trypanosoma cruzi Animalia Escherichia coli Escherichia coli Trypanosoma Trypanosoma cruzi Trypanosoma cruzi |
| spellingShingle |
complementary DNA cruzipain cysteine proteinase enzyme antibody immunoglobulin F(ab) fragment immunoglobulin heavy chain immunoglobulin kappa chain parasite antibody parasite antigen primer DNA recombinant antibody RNA animal cell antigen recognition article controlled study enzyme linked immunosorbent assay Escherichia coli gene expression system hybridoma immunofluorescence molecular cloning nonhuman nucleotide sequence priority journal Trypanosoma cruzi Animalia Escherichia coli Escherichia coli Trypanosoma Trypanosoma cruzi Trypanosoma cruzi Kaplan, D. Baldi, C. Chiaramonte, M.G. Fernandez, M.M. Levin, M.J. Malchiodi, E. Baldi, A. Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi |
| topic_facet |
complementary DNA cruzipain cysteine proteinase enzyme antibody immunoglobulin F(ab) fragment immunoglobulin heavy chain immunoglobulin kappa chain parasite antibody parasite antigen primer DNA recombinant antibody RNA animal cell antigen recognition article controlled study enzyme linked immunosorbent assay Escherichia coli gene expression system hybridoma immunofluorescence molecular cloning nonhuman nucleotide sequence priority journal Trypanosoma cruzi Animalia Escherichia coli Escherichia coli Trypanosoma Trypanosoma cruzi Trypanosoma cruzi |
| description |
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 (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 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-Ag163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals. |
| format |
JOUR |
| author |
Kaplan, D. Baldi, C. Chiaramonte, M.G. Fernandez, M.M. Levin, M.J. Malchiodi, E. Baldi, A. |
| author_facet |
Kaplan, D. Baldi, C. Chiaramonte, M.G. Fernandez, M.M. Levin, M.J. Malchiodi, E. Baldi, A. |
| author_sort |
Kaplan, D. |
| title |
Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi |
| title_short |
Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi |
| title_full |
Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi |
| title_fullStr |
Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi |
| title_full_unstemmed |
Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi |
| title_sort |
expression of a recombinant fab antibody fragment against cruzipain, the major cysteine proteinase of trypanosoma cruzi |
| url |
http://hdl.handle.net/20.500.12110/paper_0006291X_v253_n1_p53_Kaplan |
| work_keys_str_mv |
AT kapland expressionofarecombinantfabantibodyfragmentagainstcruzipainthemajorcysteineproteinaseoftrypanosomacruzi AT baldic expressionofarecombinantfabantibodyfragmentagainstcruzipainthemajorcysteineproteinaseoftrypanosomacruzi AT chiaramontemg expressionofarecombinantfabantibodyfragmentagainstcruzipainthemajorcysteineproteinaseoftrypanosomacruzi AT fernandezmm expressionofarecombinantfabantibodyfragmentagainstcruzipainthemajorcysteineproteinaseoftrypanosomacruzi AT levinmj expressionofarecombinantfabantibodyfragmentagainstcruzipainthemajorcysteineproteinaseoftrypanosomacruzi AT malchiodie expressionofarecombinantfabantibodyfragmentagainstcruzipainthemajorcysteineproteinaseoftrypanosomacruzi AT baldia expressionofarecombinantfabantibodyfragmentagainstcruzipainthemajorcysteineproteinaseoftrypanosomacruzi |
| _version_ |
1807317818100678656 |