Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications

Abstract

Background:Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for “transient-expression” that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp.Objectives:The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco.Materials and Methods:A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays.Results:The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of “GGTAAG” splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants with better outcomes for PVX, compared to pBI121 vector (0.022% versus 0.019% of the total soluble protein). The plant-derived HCVcp (pHCVcp) could properly identify the HCVcp antibody in HCV-infected human sera compared to Escherichia coli-derived HCVcp (eHCVcp), indicating its potential for diagnostic/immunization applications.Conclusions:By employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of HCVcp in tobacco with proper antigenic properties could be possible.

Transient Expression of Biologically Active Anti-rabies Virus Monoclonal Antibody in Tobacco Leaves

“…The difference between the values of S1 and S2 samples is due to the fact that each transient expression experiment is an independent transformation event. The yield of the plant-made MAb (0.014-0.019% of TSP) was similar to that reported previously for HCVcp ( 48 ) using a similar method and higher than that reported for human growth hormone peptide (0.002% of TSP) ( 52 ). The higher yield obtained in our study compared to the latter report might be due to the codon optimization and/or co-agroinfiltration of the p19 silencing suppressor used in our approach.…”

“…Mohammadzadeh et al . ( 48 ) have reported that co-infiltration of p19 could enhance the expression levels of plant-produced hepatitis C virus core protein (HCVcp) up to about 5-folds. The use of pCAMBIA1304 containing gus:gfp fusion vector allowed us to use the GFP as a visual marker for evaluation of the expressed genes located on T-DNA.…”

Heterologous Expression of Hepatitis C Virus Core Protein in Oil Seeds of Brassica napus L.

“…The plasmid was also used to express N-terminally 6xHis-tagged HCVcp protein in BL21-AI strain of E. coli by arabinose induction, as previously described ( 12 ). The HCVcp N-121 coding sequence was optimized to enhance the efficiency of gene expression in dicotyledonous plants according to our previous study ( 36 ). For this purpose, several modifications were considered including: I) Codon optimization according to codon usage table of B. napus ; II) Removal of (plant) mRNA destabilizing sequences from native HCVcp coding sequence; III) The addition of Kozak (GCCACCATGGC) sequence ( 37 ) and hexahistidine (6 × His)-tag for nickel affinity purification at 5’ site; IV) The addition of nucleotides encoding Endoplasmic Reticulum Retrieval Signal KDEL at the 3’ end ( 38 ); V) The restriction sites for Cfr9I and SacI were designed at both ends of the gene for directional cloning into the same sites of plant expression binary vector pBI1400 ( 33 ).…”

Transient expression of virus-like particles in plants: a promising platform for rapid vaccine production

“…In the first version vectors, some viral regions were present in T-DNA in addition to the cloned genes such as the promoter. The weak point of the first vectors such as PBI121 for transient expression was the fact that the expression yield was very low [40][41][42] (Fig. 2B).…”