Expression, characterization, and purification of a phosphorylated rabies nucleoprotein synthesized in insect cells by baculovirus vectors.
A baculovirus expression vector (AcNPV3) derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV) was prepared containing the complete coding region of the nucleoprotein (N) gene of rabies virus (Gif-sur-Yvette clone of the CVS strain). The gene was placed under the control of the AcNPV polyhedrin promoter and was expressed to high levels (66 mg N protein/liter of 2 x 10(9) cells) by the derived recombinant virus using a Spodoptera frugiperda cell line. Using available antisera, it was established that the antigenic characteristics of the N protein were similar by comparison with those of the native N protein of rabies virus. Characterization of the expressed protein established that, like the N protein of mammalian cell-grown CVS virus, the N protein was phosphorylated. The expressed rabies N protein induced antibodies in mice that reacted strongly with the rabies viral protein. The expressed nucleoprotein was recovered from the insect cells by differential centrifugation followed by ion exchange chromatography. The expressed rabies N protein represents a source of authentic protein suitable for virus diagnosis as well as structural studies.
Préhaud
C
C
NERC Institute of Virology and Environmental Microbiology, Oxford, United Kingdom.
Harris
R D
RD
Fulop
V
V
Koh
C L
CL
Wong
J
J
Flamand
A
A
Bishop
D H
DH
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology* / Q000648:ultrastructure; D009698:Nucleoproteins / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011820:Rabies virus / Q000235:genetics* / Q000648:ultrastructure
D004279:DNA, Viral; D009698:Nucleoproteins