Transient Expression of HA1 Antigen of H5N1 Influenza Virus in Tobacco (Nicotiana tabacum L.) via Agro-infiltration

Abstract

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