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

Author:
, , , , , ,
Year
: 2017
Abstract: 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 plantproduced

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. Agroinfiltration

expedites the process of recombinant antigens expression in plant tissues.

Accordingly, our results provide great opportunity for the exploration of transiently plantmanufactured

HA1 as vaccine candidate.
Keyword(s): Avian influenza,Gene expression,Plant-manufactured HA1,Recombinant protein
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