Method for producing high-quality recombinant allergens in a plant

Abstract

A method for producing a recombinant protein in a plant, in particular a tobacco plant, preferably Nicotiana benthamiana , includes the following steps: a) culturing the plant aeroponically or hydroponically, preferably on mobile floats and under LED lighting; b) vacuum agroinfiltration of the plant obtained in a) by agrobacteria that include a DNA fragment coding for the recombinant protein; c) returning the plants to culture after step b), under the same conditions as for step a); and d) extracting and purifying the recombinant protein from the aerial portions of the plants produced in step c).

1 . A method for producing a recombinant allergen in a plant; the method comprising the following steps:
 a) culturing the plant aeroponically or hydroponically, then   b) agroinfiltrating the plant obtained in a) under vacuum with an agrobacteria comprising a DNA fragment coding for the recombinant allergen, then   c) returning the plants to culture, in the same conditions as for step a), then   d) extracting and purifying the recombinant allergen from the aerial parts of the plants produced in step c).   
     
     
         2 . The method of  claim 1 , characterized in that the plant is  Nicotiana benthamiana.    
     
     
         3 . The method of  claim 1 , wherein the recombinant allergen is selected from the group consisting of allergens of  Dermatophagoides farinae , allergens of  Dermatophagoides pteronyssinus , allergens of Euroglyphus manei, allergens of Acarus siro, allergens of  Blomia tropicalis , cockroach allergens, tree allergens, grass allergens, a pollen allergens, allergens of animals, allergens of molds, allergens of hevea latex and allergens responsible for food allergies. 
     
     
         4 . The method of  claim 1 , wherein the recombinant allergen is selected from Der f 10, Der f 11, Der f 13, Der f 14, Der f 15, Der f 16, Der f 17, Der f 18, Der f 2, Der f 2.0101, Der f 2.0102, Der f 2.0103, Der f 2.0104, Der f 2.0105, Der f 2.0106, Der f 2.0107, Der f 2.0108, Der f 2.0109, Der f 2.0110, Der f 2.0111, Der f 2.0112, Der f 2.0113, Der f 2.0114, Der f 2.0115, Der f 2.0116, Der f 2.0117, Der f 20, Der f 3, Der f 4, Der f 5, Der f 6, Der f 7, Der f 8, Der f 9 and Der f HSP70. 
     
     
         5 . The method of  claim 1 , wherein the recombinant allergen is an allergen of  Dermatophagoides pteronyssinus  selected from the group consisting of Der p 10, Der p 11, Der p 14, Der p 15, Der p 18, Der p 2, Der p 2.0101, Der p 2.0102, Der p 2.0103, Der p 2.0104, Der p 2.0105, Der p 2.0106, Der p 2.0107, Der p 2.0108, Der p 2.0109, Der p 2.0110, Der p 2.0111, Der p 2.0112, Der p 2.0113, Der p 20, Der p 21, Der p 3, Der p 4, Der p 5, Der p 6, Der p 7, Der p 8 and Der p 9. 
     
     
         6 . The method of  claim 1 , wherein the recombinant allergen is selected from Blo t 1, Blo t 5, Blo t 9, Blo t 10, Blo t 12 and Blo t 21. 
     
     
         7 . The method of  claim 1 , wherein agroinfiltrating is carried out under vacuum by the Venturi effect. 
     
     
         8 . The method of  claim 7 , wherein agroinfiltrating is carried out either by a step of putting the plant under vacuum for 2 minutes,
 or by putting the plant under vacuum, then breaking the vacuum and returning to atmospheric pressure, then putting under vacuum, followed finally by return to atmospheric pressure.