J Bacteriol Virol.  2015 Dec;45(4):319-327. 10.4167/jbv.2015.45.4.319.

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Affiliations
  • 1Avian Diseases Division, Animal and Plant Quarantine Agency, Gyeonggi, Korea. kchoi0608@korea.kr
  • 2Food and Mouth Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gyeonggi, Korea.
  • 3Bacterial Disease Division, Animal and Plant Quarantine Agency, Gyeonggi, Korea.
  • 4R&D Center, Komipham Co., Gyeonggi, Korea.

Abstract

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2(13) HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.

Keyword

Avian paramyxovirus-6; Hemagglutinin-neuraminidase protein; Baculovirus expression system; Hemagglutination inhibition test

MeSH Terms

Avulavirus*
Baculoviridae*
Blotting, Western
Clone Cells
Cross Reactions
Diagnosis
Ducks
Glycosylation
Hemagglutination
HN Protein
Immune Sera
Insects
N-Acetylneuraminic Acid
Serologic Tests
Serotyping
Spodoptera
HN Protein
Immune Sera
N-Acetylneuraminic Acid

Figure

  • Figure 1. Agarose gel electrophoresis of recombinant plasmids digested with restriction enzymes of EcoR I and Hind III. M, DNA marker; Lane 1, pCR/APMV6HN; lane 2, pFB/APMV6HN.

  • Figure 2. Predicted amino acid sequence of HN protein gene of APMV-6/WB12-163FS and comparison with APMV-6 prototype strain (APMV-6/duck/Hong Kong/199/77). Black dot (●) represents putative N-linked glycosylation site. Putative sialic acid binding motif was expressed in a box.

  • Figure 3. Western blot analisis of recombinant APMV-6 HN protein using APMV-6 reference antiserum. P. protein markar; lane 1, SPF ECE fluid; lane 2, normal Sf9 cell lysate; lane 3, APMV-6/WB12-163Fs; lane 4, rAPMV-6 HN. Allow represents HN protein of APMV-6.

  • Figure 4. Measurement of NA activity of the rAPMV-6 HN protein expressed in insect cells.


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