The development of multiple expression vectors for high level synthesis of eukaryotic proteins: expression of LCMV-N and AcNPV polyhedrin protein by a recombinant baculovirus.
A copy of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV), in association with the coding region of lymphocytic choriomeningitis virus N protein (LCMV-N) and the relevant polyhedrin transcription termination signals, has been cloned into the unique EcoRV site of a plasmid representing an EcoRI derived fragment of the AcNPV genome. The cloning site is upstream (but in the opposite orientation) of the natural AcNPV polyhedrin gene. The derived pAcVC2 transfer vector has, therefore, both the normal polyhedrin gene and the LCMV-N gene each with its own copy of the polyhedrin transcriptional machinery. Co-transfection of Spodoptera frugiperda insect cells with the pAcVC2 plasmid together with infectious polyhedrin-negative AcNPV DNA resulted in the isolation of recombinant viruses that made polyhedrin protein as well as LCMV-N protein. Electron microscopy demonstrated the presence of occluded virus particles in the nucleus of the recombinant virus infected cells and aggregates of LCMV-N protein in the cytoplasm of the same cells. Unlike polyhedra-negative AcNPV recombinants, the occluded recombinants were potent infectious agents for the caterpillar Trichoplusia ni. The implications of these data are discussed in relation to the design of multiple eukaryotic expression vectors and recombinant baculovirus insecticides.
Emery
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NERC Institute of Virology, Oxford, UK.
Bishop
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D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D008854:Microscopy, Electron; D000077183:Occlusion Body Matrix Proteins; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D015678:Viral Structural Proteins
D004279:DNA, Viral; D000077183:Occlusion Body Matrix Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus