Genetic transformation and expression of hemagglutinin gene from avian influenza virus in carrot (Daucus carota)

Avian influenza is a disease of poultry that causes economic losses to poultry farms. Conventional vaccine production requires chicken embryos for virus proliferation with time-consuming and also affected by egg supply during the avian influenza virus outbreak. There are several advantages of plant cells as bio-factories for vaccine production, including low cost, low risk of contamination, and even edible. In this study, we chose carrot (Daucus carota) as a bioreactor to produce hemagglutinin (HA), an avian influenza virus antigen protein. HA gene linked with heat-labile enterotoxin B subunit (LTB) gene whose product plays the role of adjuvant protein was codon optimized with codon usage bias of carrot for high-level expression. The native and codon-optimized LTB-HA were transformed into carrots by Agrobacterium-mediated transformation. T-DNA insertion of two version constructs was confirmed by polymerase chain reaction. Integration of codon-optimized LTB-HA gene in all transgenic carrot lines was identified as a single copy by Southern blot analysis. LTB-HA protein expressed in carrot cells was determined through enzyme-linked immunoassay. Codon-optimized LTB-HA gene showed a better expression level than the native LTB-HA gene in transgenic carrots. The highest expression level of LTB-HA in codon-optimized LTB-HA-transformed carrot taproot reached up to 0.0108% of total soluble protein. This study is the first report about expression of HA antigen of H5N2 subtype AIV in a horticultural crop. Our transgenic carrots have successfully produced HA protein which shows great potential to become an edible vaccine against avian influenza virus.