Production of recombinant Potato mop-top virus coat protein in Escherichia coli and generation of antisera recognising native virus protein

• Published in: Journal of virological methods 2003-06, Vol.110 (1), p.91-97
• Main Authors: HELIAS, Valérie, JACQUOT, Emmanuel, GUILLET, Maryse, LE HINGRAT, Yves, GIBLOT-DUCRAY, Danièle
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Viral - biosynthesis; Antibodies, Viral - immunology; Biological and medical sciences; Blotting, Western; Capsid Proteins - biosynthesis; Capsid Proteins - genetics; Capsid Proteins - immunology; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Generalities. Techniques. Transmission, epidemiology, ecology. Antiviral substances, control; Immunization; Microbiology; Phytopathology. Animal pests. Plant and forest protection; Plant Diseases - virology; Plant Viruses - genetics; Plant Viruses - immunology; Plant Viruses - isolation & purification; Plant viruses and viroids; Rabbits; Recombinant Proteins - biosynthesis; Recombinant Proteins - immunology; RNA Viruses - genetics; RNA Viruses - immunology; RNA Viruses - isolation & purification; Sensitivity and Specificity; Solanum tuberosum - virology; Techniques used in virology; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(03)00105-8

Potato mop-top virus (PMTV, Pomovirus) is difficult to detect because it is unevenly distributed and present at low concentration in infected tissues. The production of PMTV-free seed relies on sensitive and specific detection methods of virus detection, including serological methods. The possibility of using a PMTV recombinant coat protein (CP) as an antigen for antiserum production was investigated. The region encoding the PMTV CP was inserted into pET3A, expressed in Escherichia coli, and the recombinant PMTV CP produced was used to raise antibodies in rabbits. Three antisera were produced. All recognised efficiently the recombinant CP in Western blot analysis and the most sensitive antiserum (H5003) detected native CP on Western blots and in ELISA. Thus, recombinant CP can be used as an alternative to purified virus for the production of specific antibodies against PMTV.