Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity.
We have previously reported the cloning of the coding sequence for feline-specific interferon-gamma. Here, we describe the expression of this sequence in a baculovirus system and demonstrate the biological activity of the recombinant protein. The coding sequence for feline interferon was directionally cloned into the baculovirus transfer vector pAcCL29-1. Transfer vector and linearized wild-type AcMNPV (BacPAK6) were used to co-transfect Sf9 cells by calcium phosphate coprecipitation. Subsequently, wild-type and recombinant viruses were separated by plaque assay. Recombinant plaques were expanded and a master stock of virus is produced. Production of biologically active interferon-gamma from infected Sf9 cells was demonstrated using a standard cytopathic effect reduction assay, utilising vesicular stomatitis virus (VSV), and an MHC class II induction assay.
Argyle
D J
DJ
Division of Small Animal Clinical Studies, University of Glasgow Veterinary School, UK. da18X@udcf.gla.ac.uk
Harris
M
M
Lawrence
C
C
McBride
K
K
Barron
R
R
McGillivray
C
C
Onions
D E
DE
D000818:Animals; D002415:Cats / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D003588:Cytopathogenic Effect, Viral; D015870:Gene Expression; D005822:Genetic Vectors; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D018411:Spodoptera; D014721:Vesicular stomatitis Indiana virus / Q000472:pathogenicity
D000949:Histocompatibility Antigens Class II; D011994:Recombinant Proteins; D007371:Interferon-gamma