Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas
ilustraciones, diagramas, gráficas, tablas
- Autores:
-
Riascos Orjuela, Laura Estefanía
- Tipo de recurso:
- Fecha de publicación:
- 2023
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/85255
- Palabra clave:
- 570 - Biología::572 - Bioquímica
Anticuerpos Monoclonales
Inmunización
Antibodies, Monoclonal
Immunization
Phage display
librerías de fagos
Spike
SARS-CoV-2
anticuerpos recombinantes
Phage display
phage libraries
Spike
recombinant antibodies
SARS-CoV-2
- Rights
- openAccess
- License
- Atribución-NoComercial 4.0 Internacional
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| dc.title.spa.fl_str_mv |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| dc.title.translated.eng.fl_str_mv |
Application of the phage display system for the production of monoclonal antibodies: An approach to the development of tools for protein detection |
| title |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| spellingShingle |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas 570 - Biología::572 - Bioquímica Anticuerpos Monoclonales Inmunización Antibodies, Monoclonal Immunization Phage display librerías de fagos Spike SARS-CoV-2 anticuerpos recombinantes Phage display phage libraries Spike recombinant antibodies SARS-CoV-2 |
| title_short |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_full |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_fullStr |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_full_unstemmed |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| title_sort |
Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínas |
| dc.creator.fl_str_mv |
Riascos Orjuela, Laura Estefanía |
| dc.contributor.advisor.none.fl_str_mv |
Ramírez Hernández, María Helena |
| dc.contributor.author.none.fl_str_mv |
Riascos Orjuela, Laura Estefanía |
| dc.contributor.researchgroup.spa.fl_str_mv |
Laboratorio de Investigaciones Básica en Bioquímica - LIBBIQ |
| dc.subject.ddc.spa.fl_str_mv |
570 - Biología::572 - Bioquímica |
| topic |
570 - Biología::572 - Bioquímica Anticuerpos Monoclonales Inmunización Antibodies, Monoclonal Immunization Phage display librerías de fagos Spike SARS-CoV-2 anticuerpos recombinantes Phage display phage libraries Spike recombinant antibodies SARS-CoV-2 |
| dc.subject.decs.spa.fl_str_mv |
Anticuerpos Monoclonales Inmunización |
| dc.subject.decs.eng.fl_str_mv |
Antibodies, Monoclonal Immunization |
| dc.subject.proposal.spa.fl_str_mv |
Phage display librerías de fagos Spike SARS-CoV-2 anticuerpos recombinantes |
| dc.subject.proposal.eng.fl_str_mv |
Phage display phage libraries Spike recombinant antibodies SARS-CoV-2 |
| description |
ilustraciones, diagramas, gráficas, tablas |
| publishDate |
2023 |
| dc.date.issued.none.fl_str_mv |
2023-10-27 |
| dc.date.accessioned.none.fl_str_mv |
2024-01-12T19:44:48Z |
| dc.date.available.none.fl_str_mv |
2024-01-12T19:44:48Z |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Maestría |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/masterThesis |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/TM |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://repositorio.unal.edu.co/handle/unal/85255 |
| dc.identifier.instname.spa.fl_str_mv |
Universidad Nacional de Colombia |
| dc.identifier.reponame.spa.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| dc.identifier.repourl.spa.fl_str_mv |
https://repositorio.unal.edu.co/ |
| url |
https://repositorio.unal.edu.co/handle/unal/85255 https://repositorio.unal.edu.co/ |
| identifier_str_mv |
Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia |
| dc.language.iso.spa.fl_str_mv |
spa |
| language |
spa |
| dc.relation.references.spa.fl_str_mv |
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Available from: http://dx.doi.org/10.1016/j.bbrc.2010.04.040 |
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Atribución-NoComercial 4.0 Internacional |
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xx, 86 páginas |
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Universidad Nacional de Colombia |
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Bogotá - Ciencias - Maestría en Ciencias - Bioquímica |
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Facultad de Ciencias |
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Bogotá, Colombia |
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Universidad Nacional de Colombia - Sede Bogotá |
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Atribución-NoComercial 4.0 InternacionalDerechos reservados al autor, 2023http://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Ramírez Hernández, María Helena3cd22161e24a39009214e3120ed6f39bRiascos Orjuela, Laura Estefaníaa3ea0703489648ac0a8a7739285f45d6Laboratorio de Investigaciones Básica en Bioquímica - LIBBIQ2024-01-12T19:44:48Z2024-01-12T19:44:48Z2023-10-27https://repositorio.unal.edu.co/handle/unal/85255Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, diagramas, gráficas, tablasLa tecnología phage display se ha constituido como una alternativa para la producción de anticuerpos recombinantes monoclonales (rmAbs) de alta calidad. En este trabajo, empleando el modelo aviar, se realizó una primera aproximación a la producción local de herramientas de importancia para la investigación científica en Colombia como los rmAbs desde IgYs para la detección de proteínas clínicamente importantes. En este caso, se utilizaron como antígenos los dominios N-terminal (NTD) y el dominio de unión al receptor (RBD) de la proteína Spike (S) de SARS-CoV-2. Estos dominios se expresaron en el sistema heterólogo de E. coli y se purificaron a partir de cuerpos de inclusión. Las proteínas recombinantes obtenidas (6xHis-SUMO-NTD y 6xHis-SUMO-RBD) fueron inoculadas en gallinas Hy-Line Brown siguiendo un esquema de inmunización previamente estandarizado. Durante el proceso de inmunización, se recolectaron huevos y sangrías con el objetivo de evaluar los anticuerpos policlonales (pAbs) allí presentes. Esto, permitió establecer que los IgYs de los últimos sueros de cada animal permitieron la detección de hasta 15,6ng de 6xHis-SUMO-NTD y 7,8ng de 6xHis-SUMO-RBD respectivamente. Los ensayos de especificidad evidenciaron el reconocimiento cruzado de otras proteínas recombinantes que cuentan con las etiquetas 6xHis o 6xHis-SUMO, indicando que en el proceso de inmunización se generaron anticuerpos contra dichas etiquetas. Con base en esto, se estableció que los dominios NTD y RBD expresados pueden funcionar como proteínas transportadoras o carrier de las etiquetas que se desempeñarían como haptenos. Por otro lado, una vez se finalizó el esquema de inmunización, se sacrificaron los animales para obtener los bazos a fin de extraer ARN. Luego, se sintetizó ADN complementario (ADNc) desde el cuál se amplificaron las regiones encargadas de codificar las cadenas variables ligeras (VL) y pesadas (VH) de las IgYs. Estas regiones fueron clonadas en el fásmido pSEX81 que cuenta con la secuencia codificante de una proteína de cobertura (pIII) del bacteriófago M13 a la cual se acoplan VL y VH. De esta forma, se construyeron librerías de un tamaño de 6,25x106 cfu para NTD y de 3,75x106 cfu para RBD. Células E. coli TG1 fueron transformadas por electroporación con los constructos obtenidos (pSEX81-ScFv) para el antígeno 6xHis-SUMO-RBD e infectadas con el hiperfago M13K07ΔPIII. Finalmente, después de llevar a cabo las rondas de biopanning, se obtuvo la librería a partir de la cual se han aislado clones específicos que reconocen a 6xHis-SUMO-RBD que podrán ser caracterizados y empleados en la producción de los rmAbs. (Texto tomado de la fuente)Phage display technology has become an alternative to produce high-quality recombinant monoclonal antibodies (rmAbs). In this work, using the avian model, we made a first approach to the local production of important tools for scientific research in Colombia such as rmAbs from IgYs for the detection of clinically important proteins. In this case, the N-terminal domains (NTD) and receptor-binding domain (RBD) of the SARS-CoV-2 Spike (S) protein were used as antigens. These domains were expressed in the E. coli heterologous system and purified from inclusion bodies. The recombinant proteins obtained (6xHis-SUMO-NTD and 6xHis-SUMO-RBD) were inoculated into Hy-Line Brown hens following a previously standardized immunization scheme. During the immunization process, eggs and sera were collected in order to evaluate the polyclonal antibodies (pAbs) present there. This allowed us to establish that the IgYs from the last collected sera of each animal allowed the detection of up to 15.6ng of 6xHis-SUMO-NTD and 7.8ng of 6xHis-SUMO-RBD respectively. The specificity assays evidenced the cross recognition of other recombinant proteins that have the 6xHis or 6xHis-SUMO tags, indicating that antibodies against these tags were generated in the immunization process. Based on this, it was established that the expressed NTD and RBD domains can function as transporter proteins or carriers of the tags that would act as haptens. On the other hand, once the immunization scheme was completed, the animals were sacrificed to obtain the spleens in order to extract RNA. Then, complementary DNA (cDNA) was synthesized from which the coding regions for the variable light (VL) and heavy (VH) chains of the IgYs were amplified. These regions were cloned in the phasmid pSEX81 that has the coding sequence for a coat protein (pIII) of the M13 bacteriophage to which VL and VH are coupled. In this way, libraries with a size of 6.25x106 cfu for NTD and 3.75x106 cfu for RBD were built. E. coli TG1 cells were transformed by electroporation with the constructs obtained (pSEX81-ScFv) for the 6xHis-SUMO-RBD antigen and infected with the M13K07ΔPIII hyperphage. Finally, after carrying out the rounds of biopanning, a library was obtained from which specific clones that recognize 6xHis-SUMO-RBD have been isolated, which can be characterized and used in the production of rmAbs.MaestríaMagíster en Ciencias - BioquímicaDesarrollo de herramientas biotecnológicasxx, 86 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - BioquímicaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología::572 - BioquímicaAnticuerpos MonoclonalesInmunizaciónAntibodies, MonoclonalImmunizationPhage displaylibrerías de fagosSpikeSARS-CoV-2anticuerpos recombinantesPhage displayphage librariesSpikerecombinant antibodiesSARS-CoV-2Aplicación del sistema phage display para la producción de anticuerpos monoclonales: Una aproximación al desarrollo de herramientas para la detección de proteínasApplication of the phage display system for the production of monoclonal antibodies: An approach to the development of tools for protein detectionTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMAbbas A, Lichtman A, Pillai S. 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Available from: http://dx.doi.org/10.1016/j.bbrc.2010.04.040EstudiantesInvestigadoresMaestrosLICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/85255/1/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD51ORIGINAL1026291353.2023.pdf1026291353.2023.pdfTesis de Maestría en Ciencias - Bioquímicaapplication/pdf4309578https://repositorio.unal.edu.co/bitstream/unal/85255/2/1026291353.2023.pdffb21d71bc06f776696b4ba07746d27a3MD52THUMBNAIL1026291353.2023.pdf.jpg1026291353.2023.pdf.jpgGenerated Thumbnailimage/jpeg5535https://repositorio.unal.edu.co/bitstream/unal/85255/3/1026291353.2023.pdf.jpg5740bceb486ece06c3f643590b682171MD53unal/85255oai:repositorio.unal.edu.co:unal/852552024-08-20 23:10:51.488Repositorio Institucional Universidad Nacional de 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