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 RVGP mRNA 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 ( approximately 52%) and ELISA (0.64 microg/10(7)cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy(+) (5.5 microg/10(7)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 microg/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 may be optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed.
Assuntos
Clonagem Molecular/métodos , Drosophila melanogaster/metabolismo , Glicoproteínas/biossíntese , Vírus da Raiva/genética , Proteínas Virais/biossíntese , Análise de Variância , Animais , Butiratos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Drosophila melanogaster/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glicoproteínas/genética , Isobutiratos , Cinética , Microscopia Confocal , Plasmídeos/genética , Proteínas Virais/genéticaRESUMO
The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids underthe control of the inductive metallothionein promoter. Theywere designed in order to bear or not a secretionsignal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin.Cell cultureswere examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP.All cell populationswere shownto express the RVGPmRNA upon induction. S2MtRVGPHy cell population,transfected with one vector that contains RGPV gene and selection gene, was shown to express higheramounts of RVGP as evaluated by flow cytometry (¡52%) and ELISA (0.64 g/107 cells at day 7). Subpopulationselection allowed a higher RVGP expression, specially for the S2MtRVGPHy+ (5.5 g/107 cellsat day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an evenhigher RVGP synthesis by S2MtRVGPHy+ (8.4 g/107 cells at day 7). SF900II medium leading to a higherS2MtRVGPHy+cell growthalloweda higher finalRVGPsynthesis in this cell culture.RVGPsynthesismay beoptimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposureand culture medium employed.
