Development of a Novel Recombinogenic Technique for Chloroplast Transformation and its Use for Production of human papillomavirus E7 Protein in Plants
Project goals
Our aim is to develop more efficient method for plant chloroplast transformation using biolistic bombardment with linear DNA vectors together with chloroplast cassettes expressing bacteriophage lambda Red proteins. The idea stems from experiments with targeted insertion mutagenesis in yeast, cyanobacteria, and E. coli, where DNA ends promote recombination, and lambda Red proteins Exo, Beta, and Gam are very efficient in short recombinogenic ends protection and processing. Linear vectors can be generatedconveniently as PCR products with synthetic primer ends that are homologous to sequence targets in the chloroplast genome. Chloroplast vectors will carry marker genes and also human papillomavirus oncogene E7 in the form of highly immunogenic but nononcogenic fusion E7GGG/GUS. It is our goal to obtain transplastomic plants producing this antigen with potential use as therapeutic vaccine. Viral antigens produced in plants will be assayed with antibodies and tested in mice.
Keywords
transgenic plantschloroplast transformationlambda Red recombinationhuman papilloma viruscervical carcinoma
Public support
Provider
Academy of Sciences of the Czech Republic
Programme
Grants of distinctly investigative character focused on the sphere of research pursued at present particularly in the Academy of Sciences of the Czech Republic
Call for proposals
Výzkumné granty 9 (SAV02009-A)
Main participants
—
Contest type
VS - Public tender
Contract ID
IAA500960903
Alternative language
Project name in Czech
Vývoj nové rekombinační technologie k transformaci chloroplastů a její využití pro produkci E7 proteinu lidského papilomaviru v rostlinách
Annotation in Czech
Je navržen vývoj nové metody transformace chloroplastů využívající lineární DNA vektory aplikované biolisticky spolu s chloroplastovými kazetami exprimujícími rekombinační geny Red bakteriofága lambda. Idea vychází z pokusů o cílenou inserční mutageneziv kvasinkách, sinicích a E. coli, kde konce DNA podporují výrazně homologní rekombinaci a lambdové proteiny Exo, Beta a Gam velmi účinně chrání a upravují konce DNA, které pak i při velmi krátké homologii výrazně podporují rekombinaci. Lineární vektory mohou být připraveny jako produkty PCR reakce se syntetickými primery na koncích, homologními s cílovou sekvencí v chloroplastovém genomu. Chloroplastové vektory ponesou kromě markerových genů také onkogen E7 z lidského papilomaviru ve formě neonkogenní,ale vysoce immunogenní fuze E7GGG/GUS. Cilem je získat transformanty produkující antigen využitelný jako součást terapeutické vakciny. Virové antigeny produkované v rostlinách budou stanovovány protilátkami a testovány na myších.
Scientific branches
R&D category
ZV - Basic research
CEP classification - main branch
EI - Biotechnology and bionics
CEP - secondary branch
GC - Plant growing, crop rotation
CEP - another secondary branch
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20801 - Environmental biotechnology
20802 - Bioremediation, diagnostic biotechnologies (DNA chips and biosensing devices) in environmental management
20803 - Environmental biotechnology related ethics
20901 - Industrial biotechnology
20902 - Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation
20903 - Bioproducts (products that are manufactured using biological material as feedstock) biomaterials, bioplastics, biofuels, bioderived bulk and fine chemicals, bio-derived novel materials
30401 - Health-related biotechnology
30402 - Technologies involving the manipulation of cells, tissues, organs or the whole organism (assisted reproduction)
30403 - Technologies involving identifying the functioning of DNA, proteins and enzymes and how they influence the onset of disease and maintenance of well-being (gene-based diagnostics and therapeutic interventions [pharmacogenomics, gene-based therapeutics])
30404 - Biomaterials (as related to medical implants, devices, sensors)
30405 - Medical biotechnology related ethics
40106 - Agronomy, plant breeding and plant protection; (Agricultural biotechnology to be 4.4)
40401 - Agricultural biotechnology and food biotechnology
40402 - GM technology (crops and livestock), livestock cloning, marker assisted selection, diagnostics (DNA chips and biosensing devices for the early/accurate detection of diseases) biomass feedstock production technologies, biopharming
40403 - Agricultural biotechnology related ethics
Completed project evaluation
Provider evaluation
U - Uspěl podle zadání (s publikovanými či patentovanými výsledky atd.)
Project results evaluation
Tobacco and Chlamydomonas strains were prepared with chloroplasts transformed by semisynt. vectors carrying human papilloma virus E7 oncogen. Algal extracts were used for mice immunization where E7-antibodies were induced but not cytotoxic lymphocytes.
Solution timeline
Realization period - beginning
Jan 1, 2009
Realization period - end
Dec 31, 2012
Project status
U - Finished project
Latest support payment
Feb 29, 2012
Data delivery to CEP
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data delivery code
CEP13-AV0-IA-U/02:2
Data delivery date
Feb 12, 2014
Finance
Total approved costs
4,613 thou. CZK
Public financial support
4,613 thou. CZK
Other public sources
0 thou. CZK
Non public and foreign sources
0 thou. CZK
Basic information
Recognised costs
4 613 CZK thou.
Public support
4 613 CZK thou.
100%
Provider
Academy of Sciences of the Czech Republic
CEP
EI - Biotechnology and bionics
Solution period
01. 01. 2009 - 31. 12. 2012