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

Mohammadzadeh, Sara; Khabiri, Alireza; Roohvand, Farzin; Memarnejadian, Arash; Salmanian, Ali Hatef; Ajdary, Soheila; Ehsani, Parastoo

The pIVEX2.4a core plasmid encoding HCVcp N-121, used as both the source of the sequence for construction of plant-based expression vectors and for the expression of N-terminally 6xHis-tagged HCVcp protein in BL21-AI strain of Escherichia coli by arabinose induction (as positive control) was previously described (33, 34) . Several modifications for optimized (plant) codon-usage of HCVcp nucleotide sequence were considered, including: i) Codon optimization according to the codon adaptation index of nuclear-encoded genes of tobacco (35) , ii) Removal of (plant) mRNA destabilizing sequences from the native HCVcp coding sequence, iii) Addition of Kozak (GCCAC-CATGGC) sequence (36) and hexahistidine (6xHis)-tag for nickel affinity purification at the 5′ site, iv) Addition of nucleotides encoding endoplasmic reticulum retrieval signal (KDEL) at the 3′ end (37) , v) Addition of BamHI and SacI restriction sites at both ends of the gene for directional cloning into the same sites of the plant expression binary vector pBI121 ( Figure 1 ). The plant-optimized HCVcp gene for recombinant expression in tobacco (also termed Tr-HCVcp) was synthesized and delivered as a clone in pUC57 plasmid by ShineGene Molecular Biotech Inc. (Shanghai, China). As shown in Figure 3 A, the synthetic gene was subcloned into BamHI and SacI sites of the pBI121 vector (38) under the control of CaMV 35S promoter and upstream of the nopaline synthase transcriptional terminator (NOS-Ter). In this study, the PVX-GW vector (32) , which was kindly gifted by Dr. Cristiano Lacorte (EMBRAPA Recursos Geneticos e Biotecnologia, Brasília, Brazil), was used as the PVX-based viral vector. For cloning of the Tr-HCVcp sequence into PVX-GW vector, the synthetic gene was PCR amplified using a forward primer, F-Kozak-VX: 5΄-TAATATCGATCTCGAGCCACCATGGCTCATCACC-3΄ and a reverse primer, R-core-VX: 5΄-TAATGTCGACGGATCCT-CAGAGTTCGTCCTTCTTTC-3΄ harboring ClaI and SalI restriction sites (underlined sequences). PCR amplification was performed with 25 cycles at 94˚C for 30 seconds, 58˚C for 30 seconds, and 72˚C for 90 seconds and 72˚C for 15 minutes. The 439-bp amplicon was subsequently digested by ClaI and SalI enzymes and cloned into the same sites of PVX-GW vector under the control of duplicated PVX coat protein subgenomic promoter (CPP) (Figure 3 B ). All the recombinant constructs were confirmed by restriction and sequencing analyses using F-Kozak-VX and R-core-VX specific primers. All the cloning and molecular procedures were according to the standard protocols (39). The changes to the original sequence are shown by lower case letters. Location of the Kozak sequence, 6 xHis-tag, nucleotides encoding KDEL and restriction sites for BamHI and SacI are indicated. ATG and TGA denote the start and termination codons, respectively.