Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus.
The outer capsid polypeptide, VP2, represents the major neutralizing antigen of infectious pancreatic necrosis virus (IPNV). A 926-bp viral cDNA, encoding an N-terminal truncated VP2, was cloned into the pWR590 expression plasmid family resulting in a C-terminal extension of a truncated Escherichia coli beta-galactosidase (beta Gal) under the control of the lac promoter. When cells transformed by in-phase hybrid plasmids were induced by isopropylthiogalactoside, high levels of the 100-kDa beta Gal-VP2 fusion protein accumulated within 4 h after induction. The fusion protein reacted in Western blots both with rabbit anti-beta Gal and with neutralizing mouse anti-VP2 monoclonal antibody. Sera of rabbits immunized with semipurified fusion protein reacted with the VP2 polypeptide in Western blots and with intact purified virus in ELISA and also neutralized IPNV infectivity in a plaque-reduction assay. Out-of-phase hybrid plasmids did not produce the fusion protein but expressed a small amount of structurally discrete VP2-specific sequences probably by internal initiation of translation at an in-phase AUG codon near the 5' end of the VP2 gene.
Lawrence
W R
WR
Department of Microbiology, College of Biological Sciences, University of Guelph, Ontario, Canada.
Nagy
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E
Duncan
R
R
Krell
P
P
Dobos
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P
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012087:Reoviridae / Q000235:genetics* / Q000276:immunology; D015183:Restriction Mapping; D012474:Salmon / Q000382:microbiology; D001616:beta-Galactosidase / Q000235:genetics
D000956:Antigens, Viral; D036022:Capsid Proteins; D003062:Codon; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase