Expression and characterization of codon-optimized Crimean-Congo hemorrhagic fever virus Gn glycoprotein in insect cells.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a major cause of tick-borne viral hemorrhagic disease in the world. Despite of its importance as a deadly pathogen, there is currently no licensed vaccine against CCHF disease. The attachment glycoprotein of CCHFV (Gn) is a potentially important target for protective antiviral immune responses. To characterize the expression of recombinant CCHFV Gn in an insect-cell-based system, we developed a gene expression system expressing the full-length coding sequence under a polyhedron promoter in Sf9 cells using recombinant baculovirus. Recombinant Gn was purified by affinity chromatography, and the immunoreactivity of the protein was evaluated using sera from patients with confirmed CCHF infection. Codon-optimized Gn was successfully expressed, and the product had the expected molecular weight for CCHFV Gn glycoprotein of 37 kDa. In time course studies, the optimum expression of Gn occurred between 36 and 48 hours postinfection. The immunoreactivity of the recombinant protein in Western blot assay against human sera was positive and was similar to the results obtained with the anti-V5 tag antibody. Additionally, mice were subjected to subcutaneous injection with recombinant Gn, and the cellular and humoral immune response was monitored. The results showed that recombinant Gn protein was highly immunogenic and could elicit high titers of antigen-specific antibodies. Induction of the inflammatory cytokine interferon-gamma and the regulatory cytokine IL-10 was also detected. In conclusion, a recombinant baculovirus harboring CCHFV Gn was constructed and expressed in Sf9 host cells for the first time, and it was demonstrated that this approach is a suitable expression system for producing immunogenic CCHFV Gn protein without any biosafety concerns.
Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.|Department of Virology, WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Pasteur Institute, Tehran, Iran.
Mehdi
M
Rahpeyma
Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Alireza
A
Samarbaf-Zadeh
Department of Virology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Manoochehr
M
Makvandi
Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Ata A
AA
Ghadiri
National Infection Service, Public Health England, Porton Down, Wiltshire, UK.
Stuart D
SD
Dowall
Department of Influenza and Other Respiratory Viruses, Pasteur Institute of Iran, Pasteur Institute, Tehran, Iran. fotouhi@pasteur.ac.ir.
Fatemeh
F
Fotouhi
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003062:Codon; D016207:Cytokines / Q000378:metabolism; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D003225:Hemorrhagic Fever Virus, Crimean-Congo / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D061987:Sf9 Cells; D013154:Spleen / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D016207:Cytokines; D014764:Viral Proteins