Autoexamen de detección rápida del virus del Papiloma Humano
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Fecha
2022
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Editor
Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
El cáncer cérvico uterino (CCU) es el segundo tipo de cáncer más prevalente en mujeres a nivel
mundial. El 99% de los casos se relacionan con cepas oncogénicas del Virus del Papiloma
Humano (VPH). El VPH-16 es considerado una cepa de alto riesgo y es responsable del 60% de
los casos de CCU a nivel mundial, siendo los países con menores ingresos los más afectados
debido al limitado acceso a exámenes preventivos y tratamientos. En el mercado actual existen
métodos de detección para esta enfermedad, siendo la metodología más utilizada el Papanicolau
junto a un PCR. Esto implica un proceso extremadamente invasivo e incómodo además de
elevados tiempos de espera para la entrega de resultados o necesidad de recursos monetarios
para hacerse el examen. Como solución a este problema se propuso el desarrollo de un
autoexamen para el VPH-16 que consta de una tira de flujo lateral que detecta en orina la
molécula oncogénica del VPH. Para esto, se diseñaron 6 secuencias recombinantes para la
expresión de Nanobodies Anti-VPH16E7 y de la oncoproteína E7, 3 de cada una. Una poseía la
secuencia sola, otra con His-Tag y otra con His-Tag y enteroquinasa. Se introdujeron en E. Coli
para su expresión inducida por IPTG y se purificaron en columnas de níquel con afinidad por Histag.
El resultado fue sometido a un ensayo Dot-blot y finalmente a un BN Page para determinar
si existe afinidad entre el nanobody y la oncoproteína. Se logró confirmar la correcta expresión y
purificación del Anti-VPH16E7 gracias al Dot-blot realizado, sin embargo, no se logró comprobar
la afinidad de Anti-VPH16E7 por la oncoproteína mediante el BN-page.
Cervical uterine cancer (CUC) is the second most prevalent type of cancer in women worldwide. 99% of cases are related to oncogenic strains of the Human Papillomavirus (HPV). HPV-16 is considered a high-risk strain and is responsible for 60% of CCU cases worldwide, with lowerincome countries being the most affected due to limited access to preventive tests and treatments. In the current market there are detection methods for this disease, the most widely used methodology being the Pap smear paired with a PCR, however, this implies an extremely invasive and uncomfortable process as well as long waiting times for the delivery of results or the need for the monetary resources to take the test. As a solution to this problem, the development of a selftest for HPV-16 was proposed, which consists of a lateral flow strip that detects the oncogenic molecule of HPV in urine. For this, 6 recombinant sequences were designed for the expression of Nanobodies Anti-HPV16E7 and for E7 oncoprotein, 3 of each. One had the sequence alone, the other with His-tag and another one with His Tag and enterokinase. They were introduced into E. coli for their IPTG-induced expression and purified on His-tag affinity nickel columns. The result was subjected to a Dot-blot assay and finally to a BN Page to determine if there is an affinity between the nanobody and the oncoprotein. The correct expression and purification of Anti- HPV16E7 was confirmed. Thanks to the Dot-blot carried out, however, it was not possible to verify the affinity of Anti-HPV16E7 for the oncoprotein by means of BN-Page.
Cervical uterine cancer (CUC) is the second most prevalent type of cancer in women worldwide. 99% of cases are related to oncogenic strains of the Human Papillomavirus (HPV). HPV-16 is considered a high-risk strain and is responsible for 60% of CCU cases worldwide, with lowerincome countries being the most affected due to limited access to preventive tests and treatments. In the current market there are detection methods for this disease, the most widely used methodology being the Pap smear paired with a PCR, however, this implies an extremely invasive and uncomfortable process as well as long waiting times for the delivery of results or the need for the monetary resources to take the test. As a solution to this problem, the development of a selftest for HPV-16 was proposed, which consists of a lateral flow strip that detects the oncogenic molecule of HPV in urine. For this, 6 recombinant sequences were designed for the expression of Nanobodies Anti-HPV16E7 and for E7 oncoprotein, 3 of each. One had the sequence alone, the other with His-tag and another one with His Tag and enterokinase. They were introduced into E. coli for their IPTG-induced expression and purified on His-tag affinity nickel columns. The result was subjected to a Dot-blot assay and finally to a BN Page to determine if there is an affinity between the nanobody and the oncoprotein. The correct expression and purification of Anti- HPV16E7 was confirmed. Thanks to the Dot-blot carried out, however, it was not possible to verify the affinity of Anti-HPV16E7 for the oncoprotein by means of BN-Page.
Notas
Tesis (Ingeniero en Biotecnología)
Palabras clave
Cáncer del Cuello Uterino, Papillomavirus, Mortalidad, Prevención y Control, Diagnóstico, Chile