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To develop potential plant-derived vaccine, Canine Parvovirus (CPV) virion protein 2 (VP2) and Avian influenza virus A/H5N1 nucleoprotein (NP), were expressed in Esherichia coli and Nicotiana benthamiana, respectively. A full-length of VP2 gene was engineered to be expressed by a bacterial expression vector pET-28a, while a partial length of NP containing antigenic domain was constructed in pET28a vector. Both recombinant proteins fused with His-tag were purified from E. coli using Ni-NTA chromatography under denaturing conditions. The purified VP2 was injected to produce rabbit polyclonal antisera, and the purified NP was injected to produce polyclonal IgG from rat and IgY from chicken egg yolk. The full-length of VP2 gene and the antigenic domain of NP were also engineered to be expressed by a plant expression binary vector pPZP212 under the control of Cauliflower mosaic virus (CaMV) 35S promoter, and transformed with Agrobacterium. Therefore, both gene constructs were introduced into binary vector containing an expression cassette, CaMV 35S promoter, a TEV leader sequence, the PCR-amplified target gene fragments and a 35S terminator. Each gene construct was tested for its ability to express target protein after transient expression by agroinfiltration into N. benthamiana
leaves, and infected onto leaf discs for the production of transgenic plants. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the target cDNA into the N. benthamiana genome, as well as its transcription. The expression of each recombinant protein was then observed in transgenic plants qualitatively by Western blot and quantitatively by ELISA. The morphological characteristics, including shoot and root growth were investigated,
were shown no significant differences between non-transgenic and transgenic plants. Plant-expressed VP2 was strongly cross-reacted with rabbit polyclonal antisera against E. coli-produced recombinant VP2, and expressed as a 65 kDa band which is corresponding to the predicted molecular weight of recombinant protein from the cDNA sequence. The recombinant NP expressed in N. benthamiana was well cross-reacted with rat polyclonal antisera and chicken IgY against E. coli-produced NP. However, its size was 45 kDa which is much higher than the predicted molecular weight of recombinant protein from the cDNA sequence. It could be experienced the protein modification in plant cells, possibly by glycosylation. Both VP2 and NP genes were constructed in the same vector system under the same conditions, and probably both gene sequences of animal viruses contain the specific sequences which may not be favorable to be expressed in plants. Nevertheless, the expression of NP gene construct only undergoes the modification in plant. It suggests that a foreign gene expression in plant can be varied depending on the inserted gene source.