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.

In the present study, the growth and key metabolic features of a gene-transfected Drosophila melanogaster (fruitfly) S2 (Schneider 2) cell population (S2AcRVGP cells), cultured in Sf900-II medium, have been evaluated to provide substantial support for the development of a bioprocess to produce RVGP (rabies-virus glycoprotein). Experimental cultures were grown both in a 100 ml Schott flask incubated in a shaker at 28 degrees C and 100 rev./min and in a 3 litre stirred-tank bioreactor at 28 degrees C, with increasing agitation. In small-scale culture, S2AcRVGP cells reached a maximum cell concentration of 1.13 x 10(7) cell/ml, presented a mu(max) (maximum specific growth rate) of 0.037 h(-1) and the growth was limited by oxygen deprivation. An early and remarkably long stationary phase was observed under hypoxia. Cell cultures grown in the bioreactor without oxygen limitation exhibited a maximum cell concentration of 2.2 x 10(7) cells/ml and mu(max) values as high as 0.048 h(-1). The main substrate consumed in order to reach such a high growth rate was the amino acid proline, which seems to play an important role as a source of metabolic energy in the culture of S2AcRVGP cells. Under conditions of hypoxia, the cells were able to survive for 15 h without apparent damage, recovering their previous metabolic activity.

Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 lg/1E7 cells) and SFX medium (7 lg/1E7 cells) in comparison to SF900II medium (0.6 lg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation.

The aim of this study was to achieve expression of recombinant rabies virus glycoprotein (rRVGP) in Drosophila S2 cells. For this, a cDNA coding for the selection hygromycin antibiotic and the cDNA encoding the RVGP protein under the control of the constitutive actin promoter (Ac) were cloned in an expression plasmid, which was transfected into S2 cells. S2 cell populations (S2AcRVGPHy) showed rRVGP expression in cell lysates, attaining concentrations up to 1.5 microg/10(7) cells (705 microg/L). Of the transfected cells, 20% were shown to express the rRVGP. Cell subpopulations selected by limiting dilution expressed higher rRVGP yields and 90% of the cells were shown to express the rRVGP. Cell populations re-selected by addition of hygromycin were shown to express 10 times higher rRVGP yields. The data presented here show that Drosophila S2 cells can be efficiently transfected with an expression/selection plasmid for rRVGP expression, allowing its synthesis with a high degree of physical and biological integrity. The importance of subpopulation selection was indicated by the increasing rRVGP yields during these procedures.

Estudo comparativo entre a eficiência de co-transfecção e transfecção de vetores portadores do gene da glicoproteína do vírus rábico (GPV) em células de Drosophila melanogaster S2 /