Generation of a stable cell line overexpressing the furin enzyme

• Main Author: Tan, Cherie Kun Chu
• Other Authors: Gough Au
• Format: Final Year Project
• Language: English
• Published: 2018
• Subjects: DRNTU::Science
• Online Access: http://hdl.handle.net/10356/74849
• Institution: Nanyang Technological University

Dengue virus (DENV) is a global threat where it is estimated that 390 million dengue infections occur annually. One of the challenges in DENV research is the production of a homogenous population of DENV particles. Furin is an important protease that cleaves the premembrane (prM) of immature DENV to pr peptide and M protein, forming mature DENV particles. However, inefficient furin-mediated cleavage produces heterogeneous mixtures of DENV virions. The goal of this project was to generate a stably transfected cell line expressing high levels of human furin to produce a more homogenous DENV population. Furin was cloned from U937-DC-SIGN cells and modified to include a V5 epitope tag. This gene was cloned into a commercially available pcDNA3.1(+) vector and transfected into Vero and HEK293T cells. Expression of furin-V5 protein in transfected HEK293T cells was confirmed by dot blot and western blot probed with an anti-V5-HRP antibody. The results from this project have contributed to the establishment of a cell line expressing furin and further selection is ongoing to generate the stable cell line. Further experiments are required to test the functional expression of furin and whether the cell line results in higher levels of mature DENV particles post infection.