Structural and antigenic analysis of the yellow head virus nucleocapsid protein p20.
Yellow head virus (YHV) is an invertebrate nidovirus that is highly pathogenic for marine shrimp. Nucleotide sequence analysis indicated that the YHV ORF2 gene encodes a basic protein (pI = 9.9) of 146 amino acids with a predicted molecular weight of 16,325.5 Da. The deduced amino acid sequence indicated a predominance of basic (15.1%), acidic (9.6%) and hydrophilic polar (34.3%) residues and a high proportion proline and glycine residues (16.4%). The ORF2 gene was cloned and expressed in Escherichia coli as a M(r) = 21 kDa His(6)-protein that reacted with YHV nucleoprotein (p20) monoclonal antibody. Segments representing the four linear quadrants of the nucleoprotein were also expressed in E. coli as GST-fusion proteins. Immunoblot analysis using YHV polyclonal rabbit antiserum indicated the presence of linear epitopes in all except the V(37)-Q(74) quadrant. Immunoblot analysis of the GST-fusion proteins and C-terminally truncated segments of the nucleoprotein allowed mapping of YHV monoclonal antibodies Y19, Y20 and YII4 to linear epitopes in the acidic domain between amino acids I(116) and E(137). The full-length nucleoprotein was expressed at high level in E. coli and was easily purified in quantity from the soluble cell fraction by Ni(+)-NTA affinity chromatography.
Sittidilokratna
Nusra
N
National Center for Genetic Engineering and Biotechnology, BIOTEC, National Science and Technology Development Agency, NSTDA, Phathumthani 12120, Thailand. nusra@biotec.or.th
Phetchampai
Natthida
N
Boonsaeng
Vichai
V
Walker
Peter J
PJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D003001:Cloning, Molecular; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D015151:Immunoblotting; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D016366:Open Reading Frames; D033561:Penaeidae / Q000821:virology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D046828:Roniviridae / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D017422:Sequence Analysis, DNA
D000596:Amino Acids; D018985:Epitopes, B-Lymphocyte; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins