Development of a bi-cistronic baculovirus expression vector by the Rhopalosiphum padi virus 5' internal ribosome entry site.
A bi-cistronic baculovirus transfer vector was constructed based on the 5'UTR internal ribosome entry site (IRES) of the Rhopalosiphum padi virus (RhPV). Recombinant baculoviruses containing the red fluorescent protein gene and green fluorescent protein gene flanking the RhPV 5'UTR IRES can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. Quantization by fluorescence spectrophotometry of the fluorescent proteins produced in Sf21 cells indicated that the translational efficacy of the RhPV 5'UTR IRES was about 3-fold weaker than cap-dependent translation. We also demonstrated that recombinant baculoviruses containing the human interferon-gamma gene (IFN-gamma) and green fluorescent protein gene flanking the RhPV 5'UTR IRES can produce IFN-gamma proteins as well as green fluorescent proteins. These results suggest that the RhPV IRES can be used in the development of bi-cistronic baculovirus expression vectors for production of heterologous multiprotein complexes or can be combined with selection markers to facilitate applications of baculovirus expression systems.
Chen
Ying-Ju
YJ
Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan, ROC.
Chen
Wein-Shue
WS
Wu
Tzong-Yuan
TY
D020121:5' Untranslated Regions; D000818:Animals; D001042:Aphids / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D005796:Genes; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007313:Insecta; D007371:Interferon-gamma / Q000378:metabolism; D046912:Multiprotein Complexes; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D018411:Spodoptera
D020121:5' Untranslated Regions; D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma