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The influenza A virus is of global concern for the poultry industry, especially the H5 subtype as it has the potential to become highly pathogenic for poultry and mankind. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. The goal of the present study was to investigate the possibility of expressing the HA1 protein in Nicotiana tabacum via agroinfiltration. In this study, the Hemagglutinin type 1 (HA1) of a high pathogenic avian influenza virus of the H5N1 subtype was synthesized and transiently expressed in Nicotiana tabacum. To examine the possibility of expressing the HA1 protein in N. tabacum, a cDNA fragment encoding the HA1 gene was synthesized de novo, modified with a Kozak sequence, a C-terminal hexa-Histidine (6His) tag, and an endoplasmic retention signal (KDEL). The construct was cloned into vector and the resulting - HA1 plasmid was agro-infiltrated into N. tabacum. The relative gene expression of recombinant plant-produced HA1 was measured by quantitative real-time PCR. Guided by the gene expression profile, HA1 protein was extracted at 3 dpi and subsequently purified utilizing the 6His tag. A recombinant HA1 protein was immunogenically detected by conjugated polyhistidine antibody in western blot, dot blot and ELISA assay. In order to verify the right conformation of HA1 produced in plants, western blot was also done using mouse monoclonal anti-influenza A virus (H5N1/HA1) [2B7]. The results of Real Time PCR assay indicated that the foreign gene was transcribed in transfected leaves. Migration size of protein was detected at 45 kD by Western blotting and demonstrated no discrepancy compared to the positive control (HA1). ELISA results showed that the HA1 was expressed in the transfected leaves in high level as the yield of recombinant protein was 8.8 % of TSP and the yield of purified HA1 was 0.16 g purified protein per kg fresh weight of leaves. This is the first research about the transient expression of the tobacco-made HA1 protein where a synthetic sequence was used for its expression. Here, the efficacy of agro-infiltration for expression of HA1 antigen in tobacco was illustrated. Agro-infiltration expedites the process of recombinant antigens expression in plant tissues. Accordingly, our results provide great opportunity for the exploration of transiently plant-manufactured HA1 as vaccine candidate.

Keywords: Avian influenza, Gene expression, Plant-manufactured HA1, Recombinant protein

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