Expression of equine morbillivirus (EMV) matrix and fusion proteins and their evaluation as diagnostic reagents.
Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of the degradation pattern of recombinant M protein suggested that the N-terminus of the matrix protein might be more stable and antigenic than the C-terminal region. Therefore a third system was used to express a truncated M protein, composed of the N-terminal amino acid residues 1-197, with a (His)6-tag attached at the N-terminus. This recombinant protein [(His)6-Mtr], was stable but was also insoluble. After one-step affinity purification under denaturing conditions, (His)6-Mtr was used to monitor the antibody response to EMV infection by Western blot and ELISA. We obtained a 100% correlation between Western blot and virus neutralisation testing although the number of positive sera available for testing was very limited, which included seven horse, two rabbit and one human sera.
Wang
L F
LF
CSIRO Australian Animal Health Laboratory, Geelong, Victoria, Australia.
Gould
A R
AR
Selleck
P W
PW
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D018397:Equidae; D005069:Evaluation Studies as Topic; D006639:Histidine; D006736:Horses; D006801:Humans; D007202:Indicators and Reagents; D018103:Morbillivirus / Q000235:genetics / Q000276:immunology*; D018185:Morbillivirus Infections / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D010252:Paramyxoviridae / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014760:Viral Fusion Proteins / Q000235:genetics / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D014763:Viral Matrix Proteins; D006639:Histidine