Adams, David S.
The purpose of this project was to develop several of the components required for an in vitro cell culture-based assay for detecting replication-competent dengue virus. This plan involved the stable transfection of a 293T cell-line with a recombinant plasmid encoding protease-cleavable dengue non-structural proteins NS4A/B-NS5 fused to a tetracycline transactivator (tTA). This same cell-line was also stably transfected with a reporter plasmid encoding green fluorescent protein (GFP) under the control of a tetracycline responsive element (TRE). Infection of this engineered cell line with replication-competent dengue virus results in the cleavage of the NS4A/B-NS5-tTA fusion protein by active NS3 protease, there by releasing free tTA, which activates expression of the GFP reporter, allowing for easy detection. To date, the NS4A/B-NS5-tTA plasmid has been partially constructed, while the TRE/GFP reporter plasmid has been successfully transfected into 293T cells.
Worcester Polytechnic Institute
Major Qualifying Project
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Biology and Biotechnology