Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co-transfected cell line.
Recombinant rabies virus glycoprotein (rRVGP) was expressed in Drosophila melanogaster Schneider 2 (S2) cells. The cDNA encoding the entire RVGP gene was cloned in an expression plasmid under the control of the constitutive actin promoter (Ac), which was co-transfected into S2 cells together with a hygromycin selection plasmid. Selected S2 cell populations (S2AcRVGP) had a decreased ability to grow and consume substrates, when compared to the non-transfected cells (S2). They were shown, by PCR, to express the RVGP gene and mRNA and, by immunoblotting, to synthesize the rRVGP in its expected molecular mass of 65 kDa. ELISA kinetic studies showed the rRVGP expression in cell lysates and supernatants attaining concentrations of 300 microg/L. By flow cytometry analysis, about 30% of the cells in the co-transfected populations were shown to express the rRVGP. Cell populations selected by limiting dilution expressed higher rRVGP yields. Mice immunized with rRVGP were shown to synthesize antibodies against rabies virus and be protected against experimental infection with rabies virus. The data presented here show that S2 cells can be suitable hosts for the rRVGP expression, allowing its synthesis in a high degree of physical and biological integrity.
Yokomizo
Adriana Y
AY
Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, Brasil.
Jorge
Soraia A C
SA
Astray
Renato M
RM
Fernandes
Irene
I
Ribeiro
Orlando G
OG
Horton
Denise S P Q
DS
Tonso
Aldo
A
Tordo
Noel
N
Pereira
Carlos A
CA
D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011820:Rabies virus / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins