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

Prism 5.0 software (GraphPad, USA) was used for data handling and statistical analysis was carried out using Mann-Whitney non-parametric and ANOVA tests. Statistical significance was set at P ≤ 0.05. Figure 1 represents the nucleotide changes applied in the basal sequence of HCVcp to improve its codon utilization for efficient expression in plants. Other elements (Kozak, KEDEL, His-tag and restriction sites) were also included in HCVcp basal sequence in Tr-HCVcp ( Figure 1 ). In addition, to remove the plant mRNA destabilizing sequence "GGTAAG" (nucleotides 358-363) which is present in native HCVcp sequence, it was modified ( Figure 1 ). As shown in Figure 2 A, the corresponding nucleotide alterations increased the codon adaptation index (CAI) value from 0.65 to 0.85 and reduced the GC content from 62.62 to 51.05 (Figure 2 B) . Results of DNA sequencing (not shown) indicated that the Kozak (GCCACCATGGC) sequence (36) , harboring the start codon (ATG) at 5', KDEL nucleotides at 3′ end, the designed restriction sites and all nucleotide modifications, was properly located in the Tr-HCVcp sequence (Figure 1 Figure 3 represents the schematic of the constructed pBI-core (Figure 3 A) and PVX-core (Figure 3 B ) plant expression vectors harboring the Tr-HCVcp, located under the control of CaMV 35S promoter (in pBI-core) and CP promoter (in PVX-core), upstream of the NOS-Ter transcriptional terminator. The sequencing results (not shown) confirmed the presence of the cloned Tr-HCVcp gene in pBI-core and PVX-core expression vectors with no alterations in the nucleotides. The resulting plasmids were transferred into A. tumefaciens LBA4404 target hosts (in the case of PVX-core, A. tumefaciens LBA4404 harboring the pSOUP vector). The transformed Agrobacteria were screened for recombinant constructs using core-specific primers (F-Kozak-VX and R-core-VX) and the colony PCR assay indicated the expected 439-bp fragments (Figure 3 C) . One positive clone from each construct was subsequently used for agroinfiltration of N. tabacum leaves by p19 silencing-suppressor gene procedure as outlined in 3.3.