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Gewählte Doctoral Thesis:

Syed Waqas Hassan (2012): Towards generation of transplastomic plants expressing antigens against human pathogens, for use as vaccins.
Doctoral Thesis - Abteilung für Pflanzenbau, BOKU-Universität für Bodenkultur, pp 94. UB BOKU obvsg FullText

Abstract:

To develop a new platform for vaccine production for low-income countries, the current study has investigated the generation of transplastomic plants expressing antigens against two pathogens: Human Papilloma virus (HPV) and Mycobacterium leprae. A modified HPV L1 gene with GST as a fusion protein was transformed into tobacco plastids. Expression of this recombinant protein was shown with an antigen capture ELISA by binding of conformation-specific antibodies. Assay confirmed correct assembly of the GST-L1 fusion protein which is necessary for positive immune reaction in future use. Southern Blot analysis confirmed homoplasmy of all transformants in all three generations T0-T3. The second transformation was performed with a vector covering the mmpI gene encoding a 35kDa protein which confers cross-protection against leprosy and Mycobacterium avium. The plant material was tested by PCR positively; however, the transformants did not reach homoplasmic state. One explanation for heteroplasmy could be a putative detrimental effect of constitutive expression of mmpI which might counter-select against transformed plant cells. To solve this problem a chemical induction system was tested which allows regulation of transgene expression according to a time schedule. This alternative was analyzed by expression of the GUS reporter gene under control of the promoter for a nuclear-encoded, chloroplast imported, ethanol-inducible T7 RNA polymerase in transplastomic plants. The MUG test confirmed inducible protein expression after application of 0.5% ethanol onto transformed plants. This result indicates the trans-activation system can later be used for the regulated expression of mmpI. Taken together, this data contribute another step forward towards the development of chloroplast-derived antigen expression to produce cost-efficient and easily available vaccines in low-income countries.

Betreuer: Kaul Hans-Peter

1. Berater: Lössl Andreas

2. Berater: Bürstmayr Hermann