SeqÃenciamento do genoma completo do dengue vÃrus sorotipo 1 do DF e expressÃo de proteÃnas virais ME em pichia pastoris

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

Dengue is one of the most important arboviruses (arthropod-borne virus) that belongs to the Flaviviridae viral family and a casual agent of Dengue disease in tropical and subtropical countries. Dengue virus (DENV) has four serotypes (1, 2, 3, and 4) by different reaction in serological tests. By the analysis of four DENV-1 isolates in the world, specific oligonucleotide primers were designated to clone by RT-PCR the whole genome of DENV-1 autochthones of the Federal District n 01021093 (DF-01) in the aim to reveal its sequence. To clone complete genome, the whole genome was amplified by RT-PCR dividing into two regions (5â- 6.8 kb and 3â- 5.0 kb) and cloned into pCR4 vector. The clones were sequenced and analyzed using âBlastNâ e âClustalWâ, and the isolate DF-01 showed much closer identity with Den1BR/90 isolate (The isolate of Rio de Janeiro, 1990). Aiming to prepare Dengue virus antigens for diagnostic, the viral proteins, membrane and envelop protein genes were subcloned to the yeast expression vector in pPICZα A (the construct named pPIC-ME), and a Pichia pastoris line (GS 115) was transformed and its integration was confirmed by PCR. The positive clones were selected and the protein expression was induced with methanol in the Petri dish for 72 hours at a 30 C and the colonies were transferred onto the nitrocellulose (Hybond-C) membrane. Colony-ELISA was performed using anti-HisTag monoclonal antibody and the secondary antibody of anti-mouse conjugated with alkaline phosphatase. Seven of 8 colonies showed the positive signals with tetrazolium precipitation, as the results of the recombinant protein expression by pPIC-ME transformation

ASSUNTO(S)

genetica molecular e de microorganismos seqÃenciamento dengue no distrito federal; seqÃÃncias de nucleotÃdeos pichia pastoris sequencing dengue

ACESSO AO ARTIGO

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