A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera

• Published in: Journal of biotechnology 2010-02, Vol.145 (4), p.377-386
• Main Authors: Madesis, P., Osathanunkul, M., Georgopoulou, U., Gisby, M.F., Mudd, E.A., Nianiou, I., Tsitoura, P., Mavromara, P., Tsaftaris, A., Day, A.
• Format: Article
• Language: English
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Chloroplast transformation; Chloroplasts - genetics; DNA topology and gene expression; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fundamental and applied biological sciences. Psychology; HCV; Hepacivirus - genetics; Hepacivirus - immunology; Hepatitis; Hepatitis Antibodies - blood; Hepatitis Antibodies - immunology; Hepatitis C - blood; Hepatitis C - immunology; Hepatitis C virus; Humans; Molecular Sequence Data; Nicotiana - genetics; Open Reading Frames - genetics; Peptides - genetics; Peptides - immunology; Plant Leaves - metabolism; Plants, Genetically Modified; Plasmids - genetics; RNA, Viral - genetics; RNA, Viral - metabolism; Serum - immunology; Temperature; Toxic protein; Viral Core Proteins - genetics; Viral Core Proteins - immunology
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2009.12.001

Hepatitis C virus (HCV) is a major disease agent affecting ∼3% of the world's population. Expression in plant chloroplasts enables low-cost production of the conserved HCV core protein used in diagnostic tests to combat virus spread in developing countries with high infection rates. The bactericidal activity of the 21 kDa precore protein hinders cloning the core gene in plastid expression cassettes, which are active in bacteria due to the similarities between bacterial and plastid promoters and ribosome binding sites. This was overcome by using a topology-dependent expression cassette containing tandem rrn and psbA plastid promoters, whose activity was shown to be dependent on temperature. The viral core gene and a codon-optimised gene encoding a C-terminal truncated 16 kDa core polypeptide were expressed in tobacco chloroplasts. The codon-optimised gene increased monocistronic core mRNA levels by at least 2-fold and core polypeptides by over 5-fold, relative to the native viral gene. Expression of the 16 kDa core polypeptide was stable in leaves of different ages. Anti-core antibodies in HCV-infected human sera were detected by the 16 kDa core polypeptide in total leaf protein fractionated on Western blots providing a first step towards developing a chloroplast-based HCV diagnostic method.