A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom
BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy...
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0041010114001512 https://hdl.handle.net/10669/30097 |
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RepoKERWA300972021-04-12T15:24:32Z A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom Castro, J. M. A. Oliveira, T. S. Silveira, C. R. F. Caporrino, M. C. Rodriguez, D. Moura Da Silva, Ana M. Ramos, O. H. P. Rucavado Romero, Alexandra Gutiérrez, José María Magalhães, G. S. Faquim Mauro, E. L. Fernandes, Irene Neutralizing antibody scFv Metalloproteinase BaP1 Hemorrhage Snake venom BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of recombinant antibody fragments for improving the neutralization of local tissue damage in snakebite envenoming. Fundação de Amparo à Pesquisa do Estado de São Paulo/[2012/01028-3]/FAPESP/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[PIPE/FAPESP 2004/08297-3]/FAPESP/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Universidad de Costa Rica//UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) 2017-06-12T21:56:24Z 2017-06-12T21:56:24Z 2014-09-01 artículo científico http://www.sciencedirect.com/science/article/pii/S0041010114001512 0041-0101 https://hdl.handle.net/10669/30097 10.1016/j.toxicon.2014.05.017 24887282 en_US application/pdf Toxicon; Volumen 87, 2014 |
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Universidad de Costa Rica |
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Repositorio KERWA |
| language |
Inglés |
| topic |
Neutralizing antibody scFv Metalloproteinase BaP1 Hemorrhage Snake venom |
| spellingShingle |
Neutralizing antibody scFv Metalloproteinase BaP1 Hemorrhage Snake venom Castro, J. M. A. Oliveira, T. S. Silveira, C. R. F. Caporrino, M. C. Rodriguez, D. Moura Da Silva, Ana M. Ramos, O. H. P. Rucavado Romero, Alexandra Gutiérrez, José María Magalhães, G. S. Faquim Mauro, E. L. Fernandes, Irene A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom |
| description |
BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of recombinant antibody fragments for improving the neutralization of local tissue damage in snakebite envenoming. |
| format |
Artículo |
| author |
Castro, J. M. A. Oliveira, T. S. Silveira, C. R. F. Caporrino, M. C. Rodriguez, D. Moura Da Silva, Ana M. Ramos, O. H. P. Rucavado Romero, Alexandra Gutiérrez, José María Magalhães, G. S. Faquim Mauro, E. L. Fernandes, Irene |
| author_facet |
Castro, J. M. A. Oliveira, T. S. Silveira, C. R. F. Caporrino, M. C. Rodriguez, D. Moura Da Silva, Ana M. Ramos, O. H. P. Rucavado Romero, Alexandra Gutiérrez, José María Magalhães, G. S. Faquim Mauro, E. L. Fernandes, Irene |
| author_sort |
Castro, J. M. A. |
| title |
A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom |
| title_short |
A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom |
| title_full |
A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom |
| title_fullStr |
A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom |
| title_full_unstemmed |
A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom |
| title_sort |
neutralizing recombinant single chain antibody, scfv, against bap1, a p-i hemorrhagic metalloproteinase from bothrops asper snake venom |
| publishDate |
2017 |
| url |
http://www.sciencedirect.com/science/article/pii/S0041010114001512 https://hdl.handle.net/10669/30097 |
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