Expressão de proteínas do vírus da dengue em células de inseto utilizando o sistema baculovírus de expressão
Dengue is the most common mosquito-borne viral disease of humans and its infection may evolve to a potentially lethal disease known as Dengue haemorrhagic fever (DHF). This disease is caused by a positive-sense, single-stranded, enveloped RNA virus that belongs to the genus Flavivirus within Flaviviridae and its genome is organized in a single ORF which encodes three structural proteins (capsid, pre-M, and envelope) and seven non-structural proteins (NS1, NS2a, NS12b, NS3, NS4a, NS4b, NS5). Among the viral proteins, the envelope protein is one of the most antigenic and NS1 play a possible role in complement-mediated cytolysis. Different heterologous expression systems may be used for the expression of viral antigens for vaccine and/or diagnosis development. Among these, the Baculovirus Expression System (BEV) is one of the most popular and efficient system. Baculoviruses have a circular, double stranded DNA genome and infect arthropodes, mainly insects. There are many advantages of using BEV, when compared to other expression systems, such as high expression levels and post-translational modifications that allows the expressed proteins to be correctly folded and biologically active. A Brazilian Dengue 1 isolate was used to amplify theenvelope gene alone or envelope plus NS1 gene by RT-PCR using specificoligonucleotides and cloned into the commercial transfer vector pFastBac1 (Invitrogen).This plasmid has site-specific regions which allows transposition of the chosen insertinto a baculovirus genome (bacmid) propagated in Escherichia coli. The recombinantbacmid DNA is used to obtain recombinant baculovirus and heterologous proteinexpression in virus-infected insect cells. Two different recombinant virus were obtained,vAcDE and vAcDENS1, and they were used to infect Spodoptera frugiperda larvae. Fatbody extracts of infected larvae (96 h p.i.) were analysed by SDS-PAGE and Western blot using an anti-Dengue antibody which detected a polypeptide around 70 kDa in both extracts. The size of this polypetide is different from the predicted size of the envelope protein (~50 kDa) and the fused envelope and NS1 proteins (~90kDa). However, transcriptional analysis confirmed that the genes were being transcribed in infected cells. We also used the vAcDENS1 virus in a membrane fusion assay and confirmed pH-dependent syncytium formation, which is a characteristic of the dengue virus envelope protein.. The NS1 gene alone was cloned under the control of hsp70 promoter and used to co-transfect insect cells with a plasmid containing the puromycin resistance gene, in order to produce stably transformed insect cells.
Advisor:Tatsuya Nagata; Rose Gomes Monnerat Solon de Pontes; Anamélia Lorenzetti Bocca; Rena D. de Oliveira Resende; Bergmann Morais Ribeiro
School:Universidade de Brasília
Source Type:Master's Thesis
Date of Publication:02/15/2007