Hijacking tobacco hairy roots and leaves in order to produce IpaD antigen by means of different signal peptides
2017
Hajibehzad, ShahramShokrian | Mehrizi, FaribaAbooei | Honari, Hossein | Alizadeh, Houshang
IpaA, IpaB, IpaC, and IpaD are Shigella dysenteriae Ipa operon genes which collectively contribute in invasion to the epithelial cells of the human gut. Among them, IpaD has been demonstrated to play the most crucial role in shigellosis. Noteworthy, due to the more efficient, cost-effective and no need for advanced equipment in comparison with traditional systems, plant-based expression systems are considered as a novel strategy for production of recombinant proteins. As an aim of this research, attempts were carried out to examine and compare IpaD antigen production in three different plant-based platforms, including transgenic tobacco hairy roots and leaves as well as a transient based expression. Furthermore, different signal peptides (i.e. Zera® and Extensin) were also employed in order to improve the production level. Based on TAS-ELISA result, the highest yield of IpaD acquired by ER-derived protein bodies (Zera®) which was more than 1.29-fold higher as compared with apoplastic space based on TSP% in both transgenic tobacco hairy roots and leaves. Furthermore, transgenic tobacco hairy roots were more abundant than transgenic leaves averaging 0.52 ng of IpaD per μg TSP and with a maximum of 0.94 ng IpaD per μg TSP in the best-performing construct of pBI-ZeCIpaD. Totally, the results of quantitative RT-PCR and TAS-ELISA indicated that the best time point for the production of IpaD using agroinfiltration was 72 h post infiltration and during 72 to 96 hpi, expression levels descended rapidly. To the best of our knowledge, this is the first report representing and combining the potential effects of signal peptides and plant-based expression platforms on stably production of IpaD antigen in transgenic tobacco leaves and hairy roots.
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