title: Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.
abstract: BACKGROUND
  Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition.
  
  
  OBJECTIVE
  We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine.
  
  
  METHODS
  A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector.
  
  
  RESULTS
  Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants.
  
  
  CONCLUSIONS
  The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.
authors.affiliation: a Department of Molecular Biology .
authors.forename: Sara
authors.initials: S
authors.lastname: Mohammadzadeh
authors.affiliation: b Department of Virology .
authors.forename: Farzin
authors.initials: F
authors.lastname: Roohvand
authors.affiliation: c Department of Hepatitis and AIDS , and.
authors.forename: Arash
authors.initials: A
authors.lastname: Memarnejadian
authors.affiliation: a Department of Molecular Biology .
authors.forename: Anis
authors.initials: A
authors.lastname: Jafari
authors.affiliation: d Department of Immunology , Pasteur Institute of Iran , Tehran , Iran , and.
authors.forename: Soheila
authors.initials: S
authors.lastname: Ajdary
authors.affiliation: e Department of Plant Biotechnology , National Institute of Genetic Engineering and Biotechnology , Tehran , Iran.
authors.forename: Ali-Hatef
authors.initials: AH
authors.lastname: Salmanian
authors.affiliation: a Department of Molecular Biology .
authors.forename: Parastoo
authors.initials: P
authors.lastname: Ehsani
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018506:Gene Expression Regulation, Plant; D016174:Hepacivirus / Q000378:metabolism*; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism* / Q000821:virology; D014026:Nicotiana / Q000378:metabolism* / Q000821:virology; D017864:Tombusvirus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D006514:Hepatitis B Surface Antigens; C493885:P19 protein, tomato bushy stunt virus; D014764:Viral Proteins
keywords: Agroinfiltration; HCV; Nicotiana tabacum; PVX; polyepitope; transient expression