Production of ngf in plant
OwnerPiera Galba, Carlo Massimo Pozzi +3inventors
InventorsPiera Galba, Carlo Massimo Pozzi, Maria Rosaria Stile, Enrico Puja, Elena Audia
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Abstract
The invention relates to genetically modified plants capable of producing a human recombinant protein Nerve Growth Factor, either in the form of pre-pro-protein or in the mature form and parts and differentiated and undifferentiated tissues thereof. The invention relates, also, to methods for the transformation of said plants in a transient way and methods for the transformation of said plants and tissues in a stable or transient way, methods for the recombinant protein purification from crude extract of proteins derived from plant tissue of said plants.
Claims15
A dicotyledon plant not grown for alimentary purposes or a plant tissue or a plant cell thereof which is stably transformed with an expression vector comprising a nucleic acid expression cassette under the control of a strong constitutive promoter, comprising a 5′UTR sequence, a nucleotide sequence coding for a plant leader sequence, a cDNA sequence coding for human pre-pro-NGF, a nucleotide sequence coding for a sequence that mediates the entry of a protein to which it is bound into the endoplasmic reticule, a 3′ UTR sequence, operatively linked, from 5′ to 3′, said plant being capable of constitutively expressing a human recombinant pre-pro-NGF.
A method for stably transforming dicotyledon plants comprising the following steps:
- cells from Agrobacterium tumefaciens are transformed with an expression vector comprising a nucleic acid expression cassette under the control of a strong constitutive promoter, comprising a 5′ UTR sequence, a nucleotide sequence coding for a plant leader sequence, a cDNA sequence coding for human pre-pro-NGF, a nucleotide sequence coding for a sequence that mediates the entry of a protein to which it is bound into the endoplasmic reticule, a 3′ UTR sequence, operatively linked, from 5′ to 3′;
- leaf discs from said plant are transformed with a solution comprising said Agrobacterium tumefaciens cells;
- formation of calluses from said leaf discs is induced;
- the calluses thus obtained are selected for the presence of said vector;
- the thus selected calluses are used for regenerating whole plants that are hemizygous for the cDNA coding for the human pre-pro-NGF;
- and
- the thus obtained plants are allowed to self fertilise up to the third generation so to generate plants that are stably transformed with said vector that are capable of constitutively expressing human recombinant pre-pro-NGF.
-12. (canceled)
-17. (canceled)
Dependencies are inferred from the claim text.