Expression of equine morbillivirus (EMV) matrix and fusion proteins and their evaluation as diagnostic reagents

• Published in: Archives of virology 1997-01, Vol.142 (11), p.2269-2279
• Main Authors: Wang, L.F, Gould, A.R, Selleck, P.W
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Antibodies, Viral - immunology; Antibody response; Antigens, Viral - genetics; Antigens, Viral - immunology; Biological and medical sciences; Blotting, Western; Cloning, Molecular; Enzymes; Equidae; Escherichia coli; Evaluation Studies as Topic; Fundamental and applied biological sciences. Psychology; Glutathione transferase; Histidine; Horses; Humans; Indicators and Reagents; M protein; Matrix protein; Microbiology; Morbillivirus; Morbillivirus - genetics; Morbillivirus - immunology; Morbillivirus Infections - blood; Morbillivirus Infections - diagnosis; Morbillivirus Infections - immunology; N-Terminus; neutralization tests; Paramyxoviridae - immunology; Proteins; Rabbits; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Techniques used in virology; Viral Fusion Proteins - genetics; Viral Fusion Proteins - immunology; Viral Matrix Proteins - genetics; Viral Matrix Proteins - immunology; viral structural proteins; Virology
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s007050050241

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.