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Universidad Nacional Mayor De San Marcos Universidad Del Perú Universidad Nacional Mayor de San Marcos Universidad del Perú. Decana de América Facultad de Ciencias Biológicas Escuela Profesional de Genética y Biotecnología Expresión de anticuerpos recombinantes de un solo dominio de llama (Lama glama) y análisis de su capacidad neutralizante de la actividad hemorrágica de una fracción del veneno de la serpiente Bothrops atrox TESIS Para optar el Título Profesional de Biólogo Genetista Biotecnólogo AUTOR Walter Jhon LEIVA DURAN ASESORES Dr. Armando YARLEQUÉ CHOCAS Mg. Henri BAILÓN CALDERÓN Lima, Perú 2019 Reconocimiento - No Comercial - Compartir Igual - Sin restricciones adicionales https://creativecommons.org/licenses/by-nc-sa/4.0/ Usted puede distribuir, remezclar, retocar, y crear a partir del documento original de modo no comercial, siempre y cuando se dé crédito al autor del documento y se licencien las nuevas creaciones bajo las mismas condiciones. No se permite aplicar términos legales o medidas tecnológicas que restrinjan legalmente a otros a hacer cualquier cosa que permita esta licencia. Referencia bibliográfica Leiva, W. (2019). Expresión de anticuerpos recombinantes de un solo dominio de llama (Lama glama) y análisis de su capacidad neutralizante de la actividad hemorrágica de una fracción del veneno de la serpiente Bothrops atrox. Tesis para optar el título profesional de Biólogo Genetista Biotecnólogo. Escuela Profesional de Genética y Biotecnología, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú. HOJA DE METADATOS COMPLEMENTARIOS Código Orcid del autor (opcional): Código Orcid del asesor o asesores: Armando Yarleque Chocas 1) 0000-0002-8038-2162 Henri Bailón Calderón 2) 0000-0002-9593-6092 DNI del autor: 47546146 Grupo de Investigación: Laboratorio de Biotecnología y Biología Molecular Institución que financia parcial o totalmente la investigación: Instituto Nacional de Salud. Ubicación geográfica donde se desarrolló la investigación. Debe incluir localidades y coordenadas geográficas. Localidad: Av. Prolongación Defensores del Morro 2268, Cercado de Lima 15064, Lima, Perú. Coordenadas geográficas: -12.182789,-77.016880 Año o rango de años que la investigación abarcó: 2017-2019 DEDICATORIA A mi familia, a mi querida hija, a mis amados padres, a mis hermanos y amigos con los cuales siempre conté. AGRADECIMIENTOS Al Mg. Henri Bailón Calderón, Responsable del Laboratorio de Referencia Nacional de Biotecnología y Biología Molecular, mi maestro; gracias por la oportunidad brindada. A todos los integrantes del laboratorio por ser tan amables y trabajadores les doy las gracias, a Carlos Padilla, Omar Cáceres, Marco Galarza, Juana Choque, Silvia Capristano también gracias a los compañeros de los que aprendí sobre otras áreas de investigación Luis Jaramillo, Andrés, José, Gaby, Verónica, Eva, Cleydi, Astrid, Lizbeth. Al Dr. Armando Yarlequé Chocas, jefe del Laboratorio de Biología Molecular, asesor, profesor y, con el debido respeto, amigo; gracias por encaminarme rigurosamente en el desarrollo de este trabajo, por los conocimientos brindados, los momentos de compartir que me hicieron sentir bienvenido en su laboratorio. A los integrantes del laboratorio que también fueron mis profesores y maestros el Dr. Gustavo Sandoval, la Dra. Fanny Lazo, El Dr. Dan Vivas, La Dra. Edith Rodríguez, a los tesistas que también son amigos Andrés, Daniel, Lorgio. A los que ayudaron durante el proceso de realización de este trabajo en diferentes momentos, a Henri y Pamela del laboratorio de Entomología. Un agradecimiento especial al doctor Cisneros del Laboratorio de Referencia Nacional de Cultivo Celular del INS por la mentoria y su tiempo para explicar didácticamente los métodos de cultivo de células VERO. ABREVIATURAS ACD ácido cítrico dextrosa, anticoagulante ADN Ácido desoxirribonucleico ADNc ADN complementario APS Persulfato de amônio ARNm ácido ribonucleico mensajero DHM Dosis Hemorrágica Mínima EMEM Medio Mínimo Esencial de Eagle FAB Fragmento de unión al antígeno, por sus siglas en inglés fragment antigen FHP Fracción Hemorrágica Pesada HCAbs Anticuerpos de cadena pesada, por sus siglas en inglés Heavy-chaina antibody HEPES (ácido 4-(2-hidroxyetil)-1-piperazineethanesulfonico Ig Inmunoglobulina IPTG Isopropil-ß-D-1-tiogalactopiranósido kDa KiloDaltons mA miliamperios mg Miligramo min. Minutos mL Mililitro mM Milimolar Nb Nanoanticuerpo o anticuerpo de un solo dominio ng Nanogramo nm Nanometros OD600 Densidad óptica en la longitud de onda de 600 nm PBS Buffer fosfato salino pH Potencial de hidrógeno RT-PCR reacción en cadena de la polimerasa con transcriptasa inversa rpm revoluciones por minuto SVMP Metaloproteasa de veneno de serpiente TAE Tris-acetato-EDTA TCA Ácido tricloroacético TEMED tetrametiletilendiamino µL Microlitro µg Microgramo µM Micromolar V Voltios VH Región variable de anticuerpos convencionales VHH Región variable de anticuerpos de HCAbs Índice I. INTRODUCCIÓN ............................................................................................................... 1 II. MARCO TEÓRICO ............................................................................................................ 5 Producción de anticuerpos de un solo dominio ......................................................... 5 Llama ............................................................................................................................... 6 Anticuerpo....................................................................................................................... 6 Anticuerpos de un solo dominio (Nb) ........................................................................ 10 II.4.1) Definición ............................................................................................................... 10 II.4.2) Historia ................................................................................................................... 10 II.4.3) Características y estructura ................................................................................ 11 II.4.4) Usos y aplicaciones de los anticuerpos de un solo dominio .......................... 19 Ofidismo y Salud Pública ............................................................................................ 20 II.5.1) Bothrops atrox (LINNAEUS, 1758) .................................................................... 20 II.5.2) Veneno de Bothrops atrox .................................................................................. 21 II.5.3) Hemorragia y metaloproteinasas ....................................................................... 22 Producción de antiveneno en Perú y alternativas innovadoras ............................ 29 Sistema de expresión.................................................................................................. 30 II.7.1) Escherichia coli ..................................................................................................... 31 II.7.2) Cepa ....................................................................................................................... 31 II.7.3) Plásmido ................................................................................................................ 32 Ensayos In vitro. .......................................................................................................... 35 II.8.1) Células VERO ....................................................................................................... 35 II.8.2) Citotoxicidad .......................................................................................................... 36 II.8.3) Ensayo con Alamar Blue ..................................................................................... 36 III. ANTECEDENTES ........................................................................................................... 38 IV. HIPÓTESIS Y OBJETIVOS ........................................................................................... 41 Hipótesis ...................................................................................................................... 41 Objetivos ..................................................................................................................... 41 IV.2.1) Objetivos generales ............................................................................................ 41 IV.2.2) Objetivos específicos ......................................................................................... 41 V. MATERIALES Y MÉTODOS .......................................................................................... 42 Materiales ..................................................................................................................... 42 V.1.1) Material biológico ................................................................................................. 42 V.1.2) Equipos ................................................................................................................. 42 V.1.3) Material de laboratorio ......................................................................................... 43 Métodos ........................................................................................................................ 45 V.2.1) Expresión anticuerpos de un solo dominio ....................................................... 45 V.2.2) Purificación de anticuerpos de un solo dominio .............................................. 46 V.2.3) Evaluación de la pureza ...................................................................................... 47
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