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.
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.
Lemos
Marcos Alexandre Nobre
MA
Laboratório de Imunologia Viral, Instituto Butantan, Avenida Vital Brasil 1500, São Paulo, 05503-900, Brazil.
Santos
Alexandra Souza Dos
AS
Astray
Renato Mancini
RM
Pereira
Carlos Augusto
CA
Jorge
Soraia Attie Calil
SA
D000704:Analysis of Variance; D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D058610:Isobutyrates; D007700:Kinetics; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D011820:Rabies virus / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D002087:Butyrates; D003470:Culture Media; D006023:Glycoproteins; D058610:Isobutyrates; D014764:Viral Proteins; C020380:isobutyric acid