Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.
The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.
Meulenberg
J J
JJ
Department of Virology, Institute for Animal Science and Health (ID-DLO), Lelystad, The Netherlands. J.J.M.Meulenberg@id.agro.nl
Bende
R J
RJ
Pol
J M
JM
Wensvoort
G
G
Moormann
R J
RJ
D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics*; D000918:Antibody Specificity; D018018:Arterivirus / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D007114:Immunization; D007313:Insecta / Q000166:cytology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000235:genetics; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000276:immunology / Q000821:virology; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins