Clonagem and expression of protein EGFP in the region intergenic E/NS1 of cepa vaccine 17D of the virus of the Yellow Fever.
The Yellow Fever virus (YF) is a prototype member of the genus Flavivirus. The only available and licensed vaccine to this group of virus is the one against Yellow Fever. Thisvaccine is constituted by the vaccinal 17D strain virus. The general aim of this project was the establishment of a new strategy for expression of heterologous proteins in the intergenicregion E/NS1. The specific aims were the construction of two different recombinant Yellow Fever virus expressing EGFP and the characterization of their biological and genetic stabilityproprieties. This approach is based on functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region which were duplicated and fused to theheterologous gene, allowing the correct processing of the viral polyprotein precursor. The intergenic region E/NS1 was tested for tolerance of the insertion and heterologousprotein expression in the recombinant virus, through two constructions using the reporter gene encoding EGFP (Enhanced Green Fluorescent Protein), from Aequoria victoria. This gene has a protein product composed by 238 amino acids. In the two different recombinant FA vírus expressing EGFP, the heterolog gene was fused to a sequence corresponding to the 27 5?-terminal nucleotides from the NS1 protein gene and, at the C-terminal, to the complete or partial stem-anchor domains. Based on that, the present dissertation describes theconstructions of viruses expressing EGFP from Aequoria victoria in the E/NS1 intergenic region of the YF viral genome; the determination of their growth properties in cellmonolayers (viral growth kinetics and plaque phenotype), and the genetic stability of the viral samples after serial passages in cell monolayers through plaque phenotype analysis, RT-PCRand Flow Cytometry. The regeneration of the recombinant virus was successful and the expression and stability of the recombinant protein was confirmed through fluorescence detection and plaque phenotype analysis, suggesting that the cloned gene does not considerably influence the virusviability. The biological characterization of the growth kinetics of the recombinant virus shows that the genetic modifications introduced into the YF genome do not compromise viralstructure and function. These results are important since, till this moment, it has not been possible to stably express foreign proteins within the flavivirus genome due to insert deletionsfrom the Flavivirus genome.
Advisor:Ricardo Galler; Myrna Cristina Bonaldo
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Flavivirus DNA Intergênico Yellow Fever Vaccine Intergenic
Date of Publication:07/30/2007