Rabies virus glycoprotein expression in Drosophila S2 cells. I: Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression

Autor(es)

Nobre Lemos, Marcos Alexandre ^[1] ; dos Santos, Alexandra Souza ^[1] ; Astray, Renato Mancini ^[1] ; Pereira, Carlos Augusto ^[1] ; Calil Jorge, Soraia Attie ^[1]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Lab Imunol Viral, BR-05503900 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Biotechnology; v. 143, n. 2, p. 103-110, AUG 20 2009.
• Citações Web of Science: 16

Resumo

The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids under the control of the inductive metallothionein promoter. They were designed in order to bear or not a secretion signal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2 cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin. Cell cultures were examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP. All cell populations were shown to express the RVGPmRNA upon induction. S2MtRVGPHy cell population. transfected with one vector that contains RGPV gene and selection gene, was shown to express higher amounts of RVGP as evaluated by flow cytometry (similar to 52%) and ELISA (0.64 mu g/10(7) cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy(+) (5.5 mu g/107 cells at day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MtRVGPHy(+) (8.4 mu g/10(7) cells at day 7). SF900II medium leading to a higher S2MtRVGPHy(+) cell growth allowed a higher final RVGP synthesis in this cell culture. RVGP synthesis maybe optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed. (C) 2009 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 05/51746-6 - Expressao da proteina viral (gvp - raiva) ou da proteina fluorescente (egfp) em celulas de drosophila melanogaster.
• Beneficiário: Marcos Alexandre Nobre Lemos
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 02/09482-3 - Expressão de genes heterólogos em células de dípteros: biologia molecular e engenharia de processos
• Beneficiário: Carlos Augusto Pereira
• Modalidade de apoio: Auxílio à Pesquisa - Temático