Engineering chloroplasts to accumulate antigenic protein(s) to immunize poultry birds against infectious bursal disease

Abstract

Poultry industry is a dynamic sub-division of agriculture sector throughout the world as well as in Pakistan owing to rising global demand for this more affordable animal protein than red meat, due to exponential population growth and rapid urbanization in developing regions. However, the major problems encountered by the industry are infectious viral diseases; mainly infectious bursal disease (IBD), causing severe production and economic losses. Currently, live, attenuated or killed classical virus strains are used as vaccines to immunize birds but still outbreaks are reported even in vaccinated flocks probably owing to difference in antigenic structure between vaccine and field vvIBDV strains. Also, the current vaccine production systems including microbial, stable nuclear and transient plant viral technologies are not feasible in terms of cost and biosafety. So, as a preliminary step for development of clean quality and cost-effective recombinant subunit vaccine, a VP2 gene along with engineered choloroplast based Prrn promoter and PsbA terminator was synthesized commercially. Phylogenetic analysis and multiple sequence alignment with representative strains of different geographic origin confirmed the antigenic structure similarity of synthetic VP2 protein with those of circulating vvIBDV strains. Synthetic VP2 gene together with FLARE-S gene cassette was flanked by tobacco chloroplast flanking sequences through sequential cloning leading to development of final chloroplast transformation construct. Fully expanded 4-6 weeks old tobacco leaves were bombarded using a gene gun for chloroplast transformation. Initially, putative transplastomic lines regenerated on spectinomycin selection medium were selected by visual detection through gfp fluorescence as transplastomic sectors in a chimeric leaf tissue fluoresced green under UV light. These fluoresced sectors not only revealed heteroplasmic state of transgenic plants but also confirmed the successful integration of marker gene into tobacco chloroplasts. Putative transplastomic shoots were then subjected to additional rounds of selection and regeneration for developing homoplasmic clones. The genetic analysis of putative transplastomic plants through polymerase chain reaction confirmed the presence of VP2 as well as aada and gfp genes in the plastome. Integration of cassettes into inverted repeat region was confirmed using forward primer specific to aada gene and reverse primer specific to plastome. Further, development of an efficient regeneration system for soybean via somatic embryogenesis was attempted using 2 and 7 days old mature cotyledons from in-vitro germinated seedlings of a local ‘Faisal’ and an exotic ‘Jack’ cultivar with the intention to develop edible VP2 subunit vaccine as soybean is a chief constituent of poultry feed. It was observed that 7 days old mature cotyledonary explants of cultivar Jack were more responsive towards somatic embryogenesis than 2 days old ones and those of cultivar Faisal after callus induction on D20 medium directly. However, no regeneration was observed in either case indicating the need for further experimental investigation