Proceedings of the XLVII Italian
Society of Agricultural Genetics - SIGA Annual Congress
Verona,
Italy - 24/27 September, 2003
ISBN 88-900622-4-X
Poster
Abstract - 3.21
TOBACCO
PLANTS AS BIOFACTORIES FOR THE PRODUCTION OF AN ANTI-HIV VACCINE
E. FERRAIOLO*,
L. BUONAGURO**, F.M. BUONAGURO**, M.L. TORNESELLO**, L. MONTI*, N. SCOTTI*, S.
GRILLO*, T. CARDI*
*)
CNR-IGV, Institute of Plant Genetics, Res. Div. Portici
**)
Viral Oncology, Ist. Naz. Tumori “Fond. G. Pascale”, Napoli
tobacco,
biopharmaceuticals, biofactories, vaccines, transgenic plants
Tobacco,
an important crop in various Italian Regions, is an attractive plant for the
production of Plant Made Pharmaceuticals, since it is a no-food crop and thus
there is no risk of cross contamination of the food chain. It has a large
biomass and very good response to various biotechnological approaches. Further,
its cultivation for the traditional use as drug, subsidized by European Union,
does no longer remunerate farmers and it is under close scrutiny.
In
Western Countries, the HIV, the well known agent of the “Acquired
ImmunoDeficiency Syndrome” (AIDS), is presently controlled by
anti-retroviral therapeutic strategies (HAART). Nevertheless, the definitive
control of the disease is still difficult due to the high frequency of
“escape mutants” and the unavailability of the HAART therapy in
developing countries. An alternative strategy relies on the development of
preventive anti-HIV vaccines. A promising approach is based on the property of
the capsid protein Gag to auto-assemble and form Virus-Like Particles (VLPs)
displaying several viral epitopes on the surface. Encouraging results have been
already obtained by producing VLPs expressing gag-pol-nef and env genes in the baculovirus/insect cell
expression system (Buonaguro L. et al. 2001 Antiviral Res. 49, 35-47; Buonaguro
L. et al. 2002 Antiviral Res. 54, 189-201).
In
order to test the feasibility to produce a similar range of viral antigens in
plant cells, we recently cloned a modified gag-pol-nef gene construct in an Agrobacterium-based plant expression vector, and the env
gene in a plant vector
suitable for biolistic transformation. In the former case selection was based
on a cointegrated nptII gene
conferring kanamycin resistance, whereas in the latter it depended on a hpt gene, determining hygromycin resistance,
on a separate plasmid. Expression of both transgenes was driven by the
constitutive 35S CaMV promoter.
We
obtained transgenic plants with both constructs and approaches. In experiments
with Agrobacterium tumefaciens, from
30 co-cultivated explants we
obtained 23
independent KanR shoots that were also positive to PCR analysis;
that is equivalent to a transformation frequency of 0.8 transgenic shoots per
explant. In co-transformation experiments using the biolistic approach, we
bombarded explants with different molar ratios of two plamids, carrying the hpt
and the env genes, respectively. Transformation frequencies ranged from 2.4 to 9.8 independent hpt+ shoots per bombarded
explant. Co-transformation frequencies varied from 33 to 57%. Best results,
both for the integration of the
marker gene and for the co-transformation efficiency, were obtained with a 1:8 (hpt:env) ratio between the two plasmids.
Further
molecular characterization (RT-PCR) of transgenic plants indicated that both gag
and env genes were transcribed in the tobacco
cells, albeit at relatively low levels. Plants with higher production of the
transcript could be selected. Analysis of protein expression is under way.