Produzione di anticorpi ricombinanti in pianta per usi biofarmaceutici
Author(s)
Pugnali, Margherita
Date Issued
February 8, 2011
Type
Doctoral Thesis
Abstract
Currently the demand for pharmaceutical proteins for humans is steadily
increasing and these drugs have been produced by mammalian cells, thus
providing the biological features of the expected well-designed product.
However, this production is very expensive due to high costs of
construction and management of industrial fermenters needed for this
purpose. For this reason, other expression systems are studied and
evaluated, and a great alternative to the traditional system is represented by
the biological plant system.
The cultivation of plants is more economical than an industrial plant,
extraction and purification of the products is easier and above all, because
the plants do not host animal pathogens, such as viruses or prions, the plant
production ensures greater safety to human health. The plants are able to
operate post-posttranslational modification and they allow virtually the
expression of any gene and multiple genes simultaneously.
The use of plants as biofactory takes the name of Plant Molecular Farming.
The research reported tisi the present PhD work and in particular the
objective of the experimental work was the production of two single-chain
antibody fragments (scFvs) in plant. The scFv, called scFv800E6, specific
to the HER2 tumor antigen (ErbB2) over-expressed in 30% of cases of
breast cancer and the other one, called scFvADDLs, directed against the â
oligomers, main cause of degenerative in Alzheimer 's disease, were built
into the plant expression vectors, synthesized in plants and analysed in
various ways.
A further objective of the work, aiming to make the production of
recombinant proteins in plant even safer and less expensive, was to direct
the antibody fragments in root system to take advantage of the natural
process of plant’s rizosecretion. The rizosecretion is a continuous and
undestructive process and this allows an easier purification of protein of
interest, in addition to this type of production allows to use crop systems
like hydroponic and aeroponic, already available in the agroindustry and on
the market.
As regards scFv800E6, synthesized in the ENEA laboratories and
successfully expressed in bacteria, and in plants, transient and transgenic
systems, the work was focused exclusively to study its accumulation in root
and its possible rizosecretion in the plant growth medium.
In contrast, for the ADDLs (Amyloid Derived Diffusible Ligands), an
antibody produced at low yield exclusively in bacteria, in the laboratory of
Prof. A. Cattaneo, the objective was to assess the possibility of its
expression in plant system to increase the yield, basic issue to solve for any
hypothesis to develop this ScFv as a future drug.
As regards scFv800E6, the obtained results demonstrate that the transgene
is successfully delivered and the encoded protein is preferentially and
largely accumulated in roots. Preliminary analyses carried out on the
medium growth, don’t show the presence of antibody, perhaps due to a low
efficiency of secretion system or due to retention and storage of the ScFv in
the apoplastic space.
Concerning the ScFv-ADDLs, it was espressed for the first time, in a plant
system. The transgene ScFv-ADDLs is properly expressed in leaves of
transient plants and transgenic plants have been obtained. The positive
transgenic plants will be analyzed in the next future to assess the correct
protein expression of the ScFv-ADDLs.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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