EP0910650A1 - Tollwut-glykoprotein g experimierende transgene pflanzen und durch diese herges tellte glykoprotein - Google Patents

Tollwut-glykoprotein g experimierende transgene pflanzen und durch diese herges tellte glykoprotein

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Publication number
EP0910650A1
EP0910650A1 EP97924066A EP97924066A EP0910650A1 EP 0910650 A1 EP0910650 A1 EP 0910650A1 EP 97924066 A EP97924066 A EP 97924066A EP 97924066 A EP97924066 A EP 97924066A EP 0910650 A1 EP0910650 A1 EP 0910650A1
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EP
European Patent Office
Prior art keywords
glycoprotein
virus
plant
rabies virus
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP97924066A
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English (en)
French (fr)
Inventor
Bertrand Merot
Sylvie Baudino
Véronique Gruber
Philippe Bournat
Philippe Lenee
Michel Emile Albert Riviere
Jean-Christophe Francis Audonnet
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MERIAL
Meristem Therapeutics SA
Original Assignee
BIOCEM
Merial SAS
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Filing date
Publication date
Application filed by BIOCEM, Merial SAS filed Critical BIOCEM
Publication of EP0910650A1 publication Critical patent/EP0910650A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a process for the production, by plant cells, of rabies glycoprotein G, or of a virus related to rabies.
  • the invention also relates to the glycoprotein thus obtained and to its use as a medicament, in particular as a human or veterinary vaccine.
  • the invention further relates to transformed plant cells, and transgenic plants, capable of producing the glycoprotein G of rabies.
  • the rabies virus is part of the family Rhabdoviridae. genus Lyssavirus. It is an RNA virus- whose genome contains approximately 12,000 nucleotides, coding for five structural proteins: the nucleocapsid protein "N", the matrix proteins “Mi” and “M2” / glycoprotein from envelope "G” and the protein "L” encoding the viral polymerase.
  • the viral envelope consists of a lipid bilayer containing spicules composed of a polymeric form of glycoprotein G. It is expressed in the form of a precursor having 524 amino acids, of which 19 constitute the N-terminal signal sequence hydrophobic. The N-terminal signal is cleaved to give rise to the mature glycoprotein of 505 amino acids.
  • the molecular weight of mature monomeric rabies glycoprotein is 66 kDa.
  • this form of glycoprotein is the so-called “insoluble” form, that is to say that it comprises a C-terminal transmembrane domain allowing the glycoprotein to bind in the viral envelope.
  • the “soluble” form of glycoprotein has a molecular weight of around 60 kDa and is distinguished from the insoluble form by the absence of about 58 C-terminal amino acids. This form of glycoprotein, being devoid of transmembrane segment, can no longer associate with the viral envelope.
  • Glycoprotein is the major antigen of the virus, responsible for the induction of neutralizing antibodies and protective immunity. Only the "insoluble" form is protective, although there are no antigenic determinants in the C-terminal (Dietzschold B. et al., 1983).
  • rabies glycoprotein G has been widely studied and used in the development of subunit vaccines.
  • the cDNAs of different strains of the rabies virus have been cloned (Anilionis A. et al., 1981; Yelverton et al., 1983) and expressed in different cellular hosts.
  • Yelverton et al. (1983) described the expression of the rabies glycoprotein gene (CVS strain) in E. coli.
  • the antigenic activity of the expression product is only 2 to 3% compared to the natural glycoprotein.
  • Kieny et al. (1984) described the expression of glycoprotein G in cells of higher eukaryotes, infected with a vaccinia virus, recombinant and replicative.
  • rabies glycoprotein (CVS strain) having a molecular weight of 63 and 65 kDa (Préhaud et al., 1989), and that of the Mokola virus, virus related to the rabies virus (Tordo N. and al., 1993), was obtained in insect cells infected with a recombinant baculovirus vector.
  • the technical problem which the present invention proposes to solve is to produce, in a plant cell, the glycoprotein of the rabies virus, or of a virus related to the rabies virus, conforming to the “insoluble” form of the glycoprotein. natural, especially with regard to its molecular weight.
  • the present inventors that the replacement of the endogenous N-terminal signal peptide of rabies glycoprotein, by a heterologous N-terminal signal peptide, makes it possible to obtain glycoproteins having a higher molecular weight than those obtained in plants before.
  • the present inventors have therefore used, for the transformation of the plant cell, a chimeric coding sequence comprising the coding sequence of the rabies glycoprotein gene, in which the sequence of the natural N-terminal signal peptide has been replaced by a sequence coding for a heterologous N-terminal signal peptide.
  • the present invention relates to a process for the production of the rabies virus glycoprotein G, or of a virus related to the rabies virus, characterized by: i) the introduction, into a plant cell, of a nucleic acid molecule comprising a chimeric coding sequence, comprising on the one hand a sequence coding for said mature viral protein G, or for an analogous protein, and on the other hand, a sequence coding for an other N-terminal signal peptide as that naturally associated with the viral protein G, the introduction of the chimeric coding sequence allowing the expression in the cell of the protein G; ii) the multiplication, in the form of cell culture, of the transformed cells, or the regeneration of whole transgenic or chimeric plants from these cells; iii) optionally the extraction and purification of the glycoprotein G from the cells or plant tissues thus obtained.
  • rabies virus or virus related to the rabies virus
  • serotype i corresponds to the rabies virus itself and includes different strains of the rabies virus, for example CVS, HEP, ERA, PM. There is normally at least 90% homology in the amino acid sequence between the different strains of rabies.
  • the other serotypes correspond to viruses related to the rabies virus (“rabies - related viruses”) and are exemplified by the “Lagos bat” virus (serotype 2); Mokola virus (serotype 3); Duvenhage virus (serotype 4). All rabies viruses are pathogenic to mammals, including humans, and give rise to rabies encephalitis.
  • glycoprotein G means the glycoprotein constituting the spicules of the viral envelope. According to the invention, it is normally in monomeric form but can also exist in polymeric form, for example in homodimer. According to some authors, glycoprotein G is also called the “surface antigen” of the virus, as well as “haemagglutinin”, because of its ability to cause the agglutination of erythrocytes.
  • the first step of the invention consists in the introduction, into a plant cell, of a nucleic acid molecule comprising a chimeric coding sequence.
  • a “chimeric coding sequence” means a nucleic acid sequence, coding for an amino acid chain, consisting of peptide fragments of different origins. In this case, it is a sequence comprising, on the one hand, a sequence encoding the mature viral glycoprotein G, that is to say devoid of the N-terminal signal naturally associated with the protein, and d 'somewhere else, a sequence coding for a heterologous N-terminal signal peptide to glycoprotein.
  • sequence coding for mature glycoprotein G mention may be made of that of rabies glycoprotein, CVS strain, the nucleic acid and amino acid sequence of which have been described by Yelverton et al. (1983), or that of the ERA strain, the sequence of which has been described by Anilionis et al. (1981), and modified by Kieny et al. (1984).
  • the mature protein lacks the 19 N-terminal amino acid sequence.
  • Other sequences are described by N. Tordo et al., Virol., 1993, 194, 59-69.
  • the nucleic acid coding for mature glycoprotein G is obtained by eliminating the part of the sequence which codes for the N-terminal signal peptide.
  • the coding sequence can code for a protein “analogous” to glycoprotein G.
  • Analogous proteins are proteins which normally have at least 90% homology with natural glycoprotein, and which also have functional and immunological homology with glycoprotein natural.
  • sequence coding for the endogenous N-terminal signal peptide is therefore replaced by a heterologous signal.
  • a sequence coding for a heterologous N-terminal signal peptide mention may be made of a plant signal peptide (or “pre-peptide”), or originating from a plant virus, or from a microorganism, by exempts yeast.
  • N-terminal signal peptide is meant the peptide responsible for addressing the nascent protein in the endoplasmic reticulum.
  • the addressing of proteins is based on the same principle as in animal cells. From chromosomal DNA, the gene is transcribed into messenger RNA, then translated into protein at the ribosome level. If the nascent protein has an N-terminal signal peptide or prepeptide, it enters the endoplasmic reticulum where a number of post-translational maturations take place, in particular the cleavage of the signal peptide, the N-glycosylations leading to polymannosidic glycans, and the formation of disulfide bridges.
  • the sequence coding for the prepeptide responsible for addressing the protein in the endoplasmic reticulum, is part of the chimeric coding sequence. It is normally a hydrophobic N-terminal signal peptide having between 10 and 40 amino acids and being of animal or vegetable origin. Preferably, it is a prepeptide of plant origin, for example that of sporamine, barley lectin, plant extensin (pEXT), ⁇ -mating (factor, proteins plants involved in defense against microorganisms (PRla and PRS, "pathogenesis related proteins").
  • the introduction into the plant cell of the nucleic acid molecule, comprising the chimeric coding sequence, is carried out so that expression of the sequence can be obtained.
  • the chimeric coding sequence must be found, in the genome of the plant, under the control of transcriptional regulatory sequences recognized by the plant cell.
  • regulatory sequences in particular a transcription initiation signal (promoter) and a transcription termination signal (comprising the polyadenylation signal), may be endogenous to the plant, in which case, the insertion of the chimeric coding sequence must be done by homologous recombination.
  • the transforming nucleic acid comprises, on each end, a sequence homologous to the sequences which adjoin the desired site of insertion into the genome.
  • the transcription regulatory sequences can be included in the nucleic acid molecule comprising the chimeric coding sequence.
  • the nucleic acid molecule constitutes a chimeric gene which is also part of the invention.
  • the regulatory sequences include one or more promoters of plant origin or from Agrobacterium tumefaciens or from a plant virus. It can be a constitutive promoter, for example 35S or double 35S of CaMV, NOS, OCS, or specific promoters of certain tissues such as grain, or specific of certain phases of plant development.
  • promoters of the napin gene and of the acyl carrier protein (ACP) EP-A-0255378, as well as the promoters of the AT2S genes of Arabidopsis thaliana, ie ie the promoters PAT2S1, PAT2S2, PAT2S3 and PAT2S4 (Krebbers et al., Plant Physiol., 1988, vol. 87, pages 859-866).
  • the cruciferin or phaseoline promoter the pGEA1 and pGEA6 promoters Arabidopsis promoters of the "EM, Early Methionine labeled protein" type gene which are highly expressed during the desiccation phases of the seed.
  • the termination regulatory sequences are of plant, viral, or bacterial origin, for example 35S, NOS, etc.
  • the chimeric coding sequence comprises, in addition to the N-terminal signal peptide part and the part coding for the mature envelope protein, other addressing signals, for example an endoplasmic retention signal or a vacuolar addressing signal.
  • the endoplasmic retention signal consists of the peptides KDEL, SEKDEL or HKDEL. These signals are normally found at the C-terminus of the protein and remain on the mature protein. The presence of this signal on the proteins of the invention is advantageous for several reasons: on the one hand, the retention of the protein in the endoplasmic reticulum tends to increase the yields of recombinant proteins. On the other hand, the maturation of polymannosic glycosylation into complex glycans does not take place; the protein therefore retains polymannosic glycosylation, minimizing the risk of undesirable immunological reactions when the protein is administered to humans as a medicine.
  • the glycoprotein therefore comprises the amino acid sequence of the protein G, a signal of the KDEL type, at least one glycan of the polymannosic type, and is free of glycans of the complex type.
  • this type of protein can also be obtained by using plant mutants incapable of manufacturing N-acetyl glucosaminyl transferase (von Schaewen et al., Plant Physiol. 1993 102: 1109-1118), and therefore incapable of produce complex glycans.
  • the protein of the invention can, in addition to the prepeptide, also include a vacuolar addressing signal or "propeptide".
  • a vacuolar addressing signal or "propeptide” In the presence of such a signal, the protein is addressed to the vacuoles of the aqueous tissues, for example the leaves, as well as to the protein bodies of the reserve tissues, for example seeds, tubers and roots.
  • the targeting of the protein to the protein bodies of the seed is particularly interesting because of the capacity of the seed to accumulate proteins, up to 40% of the proteins relative to the dry matter, in cellular organelles derived from vacuoles. , called protein bodies and because of the possibility of storing for several years the seeds containing the recombinant proteins in the dehydrated state.
  • propeptide use may be made of a signal of animal or vegetable origin, plant signals being particularly preferred, for example proposporamine, or barley lectin.
  • the propeptide can be N-terminal ("N-terminal targeting peptide” or NTTP), or C terminal (CTTP) or can consist of a sequence internal to the protein. Since the propeptide is normally cleaved upon entry of the protein into the vacuole, it is not present in the mature protein. It has been found that the use of the "propeptide", in combination with a prepeptide, is particularly advantageous, and gives rise to glycoproteins having a molecular weight in accordance with the natural molecule.
  • a sequence coding for an agent allowing the selection of the transformed cells is introduced into the plant cell at the same time as the sequences coding for the glycoprotein.
  • the gene coding for the selection agent can be a chimeric gene made up of regulatory sequences recognized by the plant in association with the coding sequence of the selection agent, for example NPT I, NPT II, dhfr, etc.
  • the chimeric selection gene can be part of the same vector as that coding for glycoprotein. Alternatively, it can be carried by an independent vector and introduced by co-transformation.
  • all known means for transforming the nuclear genome can be used, for example Agrobacterium, electroporation, fusion of protoplasts, bombardment with a particle gun, or penetration of DNA into cells such as pollen, microspore, seed and immature embryo, viral vectors such as Geminiviruses or satellite viruses.
  • Agrobacterium tumefaciens and rhizogenes are the preferred means.
  • the sequence of the invention is introduced into an appropriate vector with all the necessary regulatory sequences such as promoters, terminators, etc., as well as any sequence necessary for selecting the transformants.
  • the transformed cells are selected by virtue of the presence of the gene coding for the selection agent. These cells are then subjected to a step of multiplication by cell culture, where they are regenerated into transgenic or chimeric plants.
  • a biomass capable of producing glycoprotein G in large quantity.
  • They can be cultures of plant cells in vitro, for example in a liquid medium.
  • Different culture modes ("batch", “fed batch” or continuous) for this type of cells are currently being studied. Batch cultures are comparable to those carried out in an Erlenmeyer flask insofar as the medium is not renewed, the cells thus have only a limited quantity of nutritive elements.
  • the "fed batch” culture corresponds to a "batch” culture with a programmed feeding in substrate.
  • the cells are continuously supplied with nutritive medium. An equal volume of the biomass-medium mixture is removed in order to keep the volume of the reactor constant.
  • biomass densities of around 10 to 30 g of dry weight per liter of culture can be obtained, for species such as Nicotiana tabacum, Vinca rosea and Catharanthus roseus.
  • the cells of the invention can also be immobilized, which makes it possible to obtain a constant and prolonged production of the glycoprotein.
  • immobilization method mention may be made of immobilization in alginate or agar beads, inside polyurethane foam, or even in hollow fibers.
  • the cells of the invention can also be root cultures.
  • the roots cultivated in vitro, in liquid medium, are called “Hairy roots", they are roots transformed by the bacterium Agrobacterium rhizo ⁇ enes.
  • glycoprotein of the invention By culturing plant cells, it is possible to regenerate chimeric or transgenic plants from transformed explants, by using techniques known per se.
  • the process of the invention may or may not include a step for recovering glycoprotein G.
  • the process of the invention gives rise to plant cells, or to transgenic plants capable of produce glycoprotein G.
  • Cells or plants are in themselves a source of the vaccinating protein and can be administered as such to animals or humans by ingestion.
  • the glycoprotein G is recovered by extraction from the plant material, and optionally purified.
  • the extraction step comprises the solubilization of the glycoprotein, associated with the cell membranes, with a detergent. More particularly, the plant material, for example, leaves, seeds, roots, etc., is ground in liquid nitrogen. The homogenate is then suspended in an appropriate buffer, added with a detergent, preferably nonionic.
  • the detergent is used in concentrations of 0.1 to 2%, preferably 0.1 to 0.5%. It can for example be
  • Triton X-100 Triton X-100, or NP40 or ⁇ -octyl glucopyranoside.
  • the use of detergent releases glycoprotein from cell membranes, while retaining its integrity and immunogenic power.
  • the homogenate thus obtained is centrifuged, the “solubilized” glycoprotein then being present in the supernatant. Normally, the supernatant is filtered.
  • the plant extract corresponding to the supernatant can be used as such as a source of vaccinating protein, or can be subjected to one or more purification step (s).
  • the detergent can be eliminated or reduced.
  • the extract of plant material for example tobacco leaves or rapeseed
  • the fraction corresponding to the retentate or to the dead volume is then subjected to an ion exchange chromatography on a QAE Sephadex A50 Pharmacia support equilibrated beforehand in ethylene diamine acetic acid buffer pH 8.1. After washing with this same pad, the glycoprotein is eluted with a 0.6 M sodium acetate solution (P. Atanasiu et al., 1976).
  • the invention also relates to the glycoprotein G capable of being obtained by implementing the method of the invention.
  • the invention relates to a glycoprotein characterized in that:
  • glycoprotein 1 • invention is recognized by antibodies specific to the glycoprotein of rabies virus or related to rabies. They can be polyclonal or monoclonal antibodies against the virus, or against glycoprotein purified from the virus.
  • the glycoprotein of the invention has a molecular weight of approximately 66 kDa.
  • “approximately” means the variability resulting from measurement techniques. More particularly, the glycoprotein of the invention has a molecular weight of 66 kDa on average when several Western Blot tests are carried out, for example, at least 3 or 5 tests, under the same conditions.
  • the molecular weight can be between 65 and 80 kDa, for example 66 to 67 kDa.
  • the molecular weight is an apparent molecular weight, determined by electrophoresis on polyacrylamide gel under denaturing conditions, according to the technique of Laemmli et al. (1970).
  • the polyacrylamide gel is a 10 or 12.5% gel, but lower concentrations can be used to obtain a clearer resolution of the areas between 60 and 65 kDa.
  • the glycoprotein of the invention is highly insoluble.
  • the term “insoluble” means that the molecule is not soluble in the exracellular medium, but is found associated with cell membranes. Indeed, it has been found that the presence of detergent, for example SDS, Triton X-100, is essential to extract and dissolve the glycoprotein.
  • the “insoluble” nature of glycoprotein is linked to the presence of the C-terminal transmembrane domain. The presence of the C-terminal region, i.e., the region located approximately 40 to 60 amino acids from the carboxy end of the glycoprotein, is important to obtain a protective response when the glycoprotein is used as vaccine.
  • the glycoprotein of the invention is indirectly insoluble by its association with cell membranes. In the absence of a detergent, it cannot be in the supernatant of cell cultures producing glycoprotein.
  • the glycoprotein of the invention is glycosylated by at least one glycan of the polymannosidic type, and / or by at least one glycan of the complex type comprising within its structure one or more xylose residues ⁇ -1,2, and / or a or several residues of fucose linked in ⁇ -1,3, and being free of sialic acid residues.
  • glycosylation of glycoproteins produced in plant cells is different from those produced in animal cells. Although the glycosylation steps leading to the polymannosidic glycans are carried out identically in plants and in mammals, the maturation of the polymannosidic glycans into complex glycans is very different.
  • the N-glycans of vegetable glycoproteins differ mainly from mammalian glycans by the absence of sialic acid, also known as N-acetylneuraminic acid, and the presence of a residue of ⁇ 1,2 xylose and a fucose residue linked in ⁇ 1,3 to the GlcNAc residue proximal to the "core" (Driouich et al., Regard sur la biochemimie, 1993, vol. 3, pages 33-42).
  • Glycosylation plays an important role in the establishment of the spatial structure of the protein, in protection against proteolytic attacks and in the mechanisms of immune recognition.
  • the protein of the invention is glycosylated at at least two natural N-glycosylation sites.
  • the glycosylation is preferably at positions 247 and 319.
  • the glycosylation is of mannosidic, polymannosidic or complex type, or a mixture of both.
  • the mannosidic glucan (s) has the GlcNAc2-Manl structure.
  • the polymannosidic glucan (s) has the structure GlcNAc2-Man2-9, for example GlcNAc2-Man9, GlcNAc2-Man8, GlcNAc2-Man6 or GlcNAc2-Man5, or GlcNAc2-Man3.
  • the glucan (s) of complex type are biantennary and have a basic structure GlcNAc2-Man3 with which are optionally associated residues of xylose (Xyl), fucose (Fuc) and possibly galactose (Gai) or N-acetylglucosamine (GlcNAc ). They are normally free of sialic acid residues, this compound having not hitherto been detected in plant cells.
  • the complex glycan is of the "phytohemagglutinin” (PHA) type consisting of a structure
  • GlcNAc2Man3 whose ⁇ -linked mannose carries a residue of ⁇ 1,2-xylose and whose proximal GlcNAc carries a residue of ⁇ 1.3 fucose. This kind of structure is frequent for glycoproteins of vacuolar or extracellular localization.
  • the 1,3-linked ⁇ -fucose residue may possibly be absent.
  • the complex glycan can also be of the "Laccase” type composed of a basic structure GlcNAc2 (Fuc) Man3 (Xyl) associated with two side chains.
  • Each side chain consists of a residue of ⁇ 1.2 GlcNAc to which is linked a residue of ⁇ 1.6 fucose and a residue of ⁇ 1.4 galactose.
  • the glycoprotein of the invention can be glycosylated at the three natural sites or only at one or two of them.
  • This type of glycosylation exists in identical form in plants and animals. Therefore, the appearance of an adverse immunological reaction is avoided.
  • This type of glycosylation is obtained by the use of an N-terminal signal peptide in association with an endoplasmic retention signal.
  • glycoproteins carrying exclusively glycans of the complex type for example two or three glycans of the PHA type: GlcNAc2 (Fuc) Man3 (Xyl).
  • the carbohydrate part normally represents between 2 and 30%, for example 5 to 20% of the total mass of the glycoprotein of the invention, the natural molecule comprising 11% of glycosylation.
  • composition of glycoprotein glycans can be determined by digestion of oligosaccharides with N-glycanase, as described by Tuchiya K. et al. (1992).
  • the glycoprotein of the invention is capable of inducing the formation of neutralizing antibodies and protective immunity against the rabies virus or against a virus related to the rabies virus.
  • glycoprotein The protective character of glycoprotein is demonstrated by the administration, to an animal, of the vaccine containing glycoprotein, followed by a virulent test with the rabies virus.
  • the level of neutralizing antibodies produced in animals is determined by a test such as the RFFIT test ("Rapid Fluorescence Focus Inhibition Test" described by Smith et al. (1973).
  • the glycoprotein of the invention may include one or more of the addressing signals described above.
  • the only peptide signal left is the HKDEL-type endoplasmic retention, the other addressing signals being cleaved in the cell.
  • Glycoprotein with an endoplasmic retention signal has a molecular weight greater than 66 kDa due to the presence of additional amino acids.
  • the protein part of the glycoprotein of the invention may correspond to that of natural glycoprotein, or it may be a protein "analogous" to the natural protein.
  • an "analogous" protein means a protein which has at least 90% homology with the reference molecule. It is for example a rabies glycoprotein of which certain amino acids have been replaced, deleted or inserted. The modifications preferably take place on the N-terminal side of the molecule.
  • Analogous proteins are also insoluble, recognized by antibodies specific to rabies glycoprotein or to a virus related to the rabies virus and capable of inducing the formation of neutralizing antibodies.
  • the invention also relates to the nucleic acids, RNA or DNA, used in the production of glycoprotein.
  • the invention relates to a nucleic acid comprising a chimeric coding sequence comprising on the one hand a sequence coding for the mature protein G of the rabies virus or of a virus related to the rabies virus, or for a similar protein , and on the other hand, a sequence coding for an N-terminal signal peptide other than that naturally associated with the virus, or the sequences complementary to the above-mentioned coding sequences.
  • the regulatory sequences described above, as well as the sequences encoding the addressing signals are, if appropriate, also included in the nucleic acid molecule.
  • the invention further relates to the vectors and plasmids used for the introduction of the nucleic acid into plant cells. They may be Ti plasmids of A ⁇ robacterium. or viral vectors such as Geminiviruses or CaMV.
  • the invention also relates to transformed plant cells capable of producing the glycoprotein of the invention. These cells are in the form of cell culture, as indicated above, or are part of transgenic or chimeric plants.
  • Angiosperms comprising monocots and dicots. More particularly, mention may be made of tobacco, species belonging to botanical families such as legumes (for example beans, peas, etc.), crucifers (for example cabbage, radishes, rapeseed, etc.) , nightshade (for example tomatoes, potatoes, etc.), cucurbits (for example melon, zucchini, cucumber), chenopodiaceae (for example vegetable beet), umbelliferae (for example carrots, celery, etc.). Mention may also be made of cereals such as wheat, corn, barley, triticale and rice, and oilseeds such as sunflower and soybeans.
  • legumes for example beans, peas, etc.
  • crucifers for example cabbage, radishes, rapeseed, etc.
  • nightshade for example tomatoes, potatoes, etc.
  • cucurbits for example melon, zucchini, cucumber
  • chenopodiaceae for example vegetable beet
  • the invention also relates to the seeds of transgenic plants capable of producing rabies glycoprotein G, as well as their progeny.
  • the invention also relates to the use of glycoprotein G as a therapeutic product, in particular as a vaccine against infection by rabies, or by related viruses.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising: one or more glycoprotein (s) according to the invention, or - cells according to the invention, or
  • the pharmaceutical composition contains at least 1 ⁇ g of glycoprotein.
  • it contains 1 ⁇ g to 1 mg, or preferably 10 ⁇ g to 200 ⁇ g, or preferably 25 to 100 mg, of purified glycoprotein, per dose, for example 200 or 300 ⁇ g.
  • the glycoprotein is used in a form other than purified, for example in the form of an extract from the plant, or the plant itself, the quantity administered must be adjusted so that the dose of active principle reaches at least 100 ⁇ g.
  • the excipient used in the composition is an excipient normally used for rabies vaccines, for example alumina gel.
  • the vaccine is formulated for oral or parenteral administration. It can also take the form of a food product produced from the plant material of the invention.
  • the invention also includes a method of immunizing mammals, including humans, against infection with this rabies virus, or with a virus related to the rabies virus, comprising administering a glycoprotein of the invention, or a plant material comprising this glycoprotein.
  • Lane 1 Reference rabies glycoprotein G reconstruction in tobacco juice
  • Lane 4 FFeeuuiilllleess of tobacco transformed with pBIOCllO;
  • Lane 5 F Feeuuiilllleess of unprocessed tobacco
  • Lane 7 MMaarrqqueueuurr cde molecular weight.
  • Lane 1 Reference rabies glycoprotein G
  • Lane 3 Reference rabies glycoprotein G reconstruction in corn callus juice
  • Lane 1 Reconstruction of rabies glycoprotein G reference in tobacco leaf juice
  • Lane 2 Unprocessed tobacco leaf
  • Lane 3 Tobacco leaf transformed with pBIOCllO (PPS-RabG);
  • Lane 4 Tobacco leaf transformed with pBI0C104 (PS-RabG);
  • Lane 5 Molecular weight marker.
  • rabies glycoprotein G required the following regulatory sequences:
  • the constitutive double 35S promoter (pd35S) of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987). or the chimeric super-promoter (pSP; Ni et al., 1995). It consists of the fusion of the triple repetition of a transcriptional activating element of the promoter of the octopine synthase gene from Agrobacterium tumefaciens, a transcriptional activating element of the promoter of the mannopine synthase gene and the mannopine synthase promoter. Agrobacterium tumefaciens;
  • polyA 35S terminator which corresponds to the 3 ′ non-coding region of the sequence of the double-stranded DNA virus circular of the cauliflower mosaic producing the 35S transcript (Franck et al., 1980).
  • the plasmid pGA492 was doubly digested with Sac1 (restriction site of the polylinker) and by Seal (restriction site present in the sequence of the cat gene) and then subjected to the action of the enzyme T4 DNA polymerase (Biolabs) according to the manufacturer's recommendations.
  • Ligation of the modified plasmid (20 ng) was carried out in a 10 ⁇ l reaction medium containing 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham); 2.5 U of T4 DNA ligase enzyme (Amersharo) at 14 ° C for 16 hours.
  • the HindIII restriction site of the plasmid DNA of the retained clone was modified into an EcoRI restriction site using a phosphorylated HindIII-EcoRI adapter (Stratagene Cloning Systems).
  • 500 ng of plasmid DNA of the retained clone were digested with HindIII, dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations and coprecipitated in the presence of 1500 ng of DNA adapter HindIII-EcoRI, 1/10 volume of 3M sodium acetate pH4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min.
  • the ligation reaction mixture was digested with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with ethanol 70%, dried, then ligated as described above.
  • the resulting binary plasmid which only has the last 9 amino acids of the coding sequence for the cat gene and whose EcoRI site is unique, was called pBI0C4.
  • the expression cassette consisting of the pd35S promoter and the polyA 35S terminator, was isolated from the plasmid pJIT163 ⁇ .
  • the plasmid pJIT163 ⁇ derives from the plasmid pJIT163 which itself derives from the plasmid pJIT60 (Guerineau and Mullineaux, 1993).
  • Plasmid pJIT163 has an ATG codon between the HindIII and SalI sites of the polylinker.
  • the plasmid DNA pJIT163 was digested twice with HindIII and SalI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, subjected to the action of the enzyme Klenow (Biolabs) according to the manufacturer's recommendations, deproteinized by extraction with 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) then 1 volume of chloroform: isoamyl alcohol ( 24: 1), precipitated in the presence of 1/10 volume of 3M sodium acetate pH4.8 and 2.5 volumes of absolute ethanol at - 80 ° C for 30 min.
  • DH5 ⁇ previously made competent, have been transformed (Hanahan, 1983).
  • the expression cassette consisting of the pd35S promoter and of the polyA 35S terminator (SacI-Xhol fragment) the plasmid DNA of the clone pJIT163 ⁇ retained was digested with SacI and Xhol.
  • Sacl-Xhol fragment carrying the expression cassette, was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 of volume of 3M acetate. sodium pH4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with 70% ethanol, dried, then subjected to the action of the enzyme Mung Bean Nuclease (Biolabs) according to the manufacturer's recommendations.
  • This purified insert (200 ng) was cloned into the plasmid DNA of pBIOC4 (20 ng) digested with EcoRI, treated with the enzyme Mung Bean Nuclease and dephosphorylated by the enzyme alkaline phosphatase from calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
  • the ligation reaction was carried out in 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the PvuII - Sali fragment subjected to the action of Klénow containing the pSP promoter was isolated from the plasmid pBISNl (Ni and al., 1995), purified by electrophoresis on 1% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to the plasmid DNA of pBIOC ⁇ l doubly digested with Kpnl and EcoRI subjected to the action of T4 DNA polymerase and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
  • the plasmid pBIOC ⁇ l corresponds to pB10C21 whose Xbal site has been deleted. To do this, the plasmid pB10C21 was digested with Xbal then subjected to the action of Klenow and ligated by action of T4 DNA ligase.
  • the ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of DNA fragments carrying pSP described above in a 20 ⁇ l reaction medium in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ) and 2 ⁇ l of 50% polyethylene glycol 8000, and 5 ⁇ of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • T4 DNA ligase x 10 buffer Amersham
  • 50% polyethylene glycol 8000 50% polyethylene glycol 8000
  • 5 ⁇ of T4 DNA ligase enzyme Amersham
  • Rabies glycoprotein G isolated from the ERA strain, is naturally synthesized in the form of a precursor.
  • the mature protein RabG is made up of 505 amino acids. Its signal peptide is composed of 19 amino acids.
  • the cDNA coding for the envelope glycoprotein of the rabies virus, strain ERA was amplified by PCR according to the usual methods, using the following two oligonucleotides:
  • Oligo 1 (30 sea) 5 'AAA GGA TCC ATG GTT CCT CAG GCT CTC CTG 3'
  • Oligo 2 (30 sea) 5 AAA CTG CAG TCA CAG TCT GGT CTC ACC CCC 3 '
  • the 1.69 kb PCR fragment was digested with BamHI and Pst1 and then cloned into the vector pBlueScript previously digested with BamHI and Pstl, thus giving the plasmid pPBO10.
  • the complementary DNA of the RabG precursor is contained in the plasmid pPBO10. It was used for the construction of the binary plasmids pBIOC104 and PBIOC105 containing the sequence coding for PS-RabG, pBIOCl07 and pBIOC108 containing the sequence coding for BL-RabG and pBIOCllO and pBIOClll containing the sequence coding for PPS-RabG where the sequence coding for RabG is preceded by the sequence coding for a signal peptide, PS and BL, and for an N-terminal prepropeptide (PPS i.e. a signal peptide followed by N-terminal vacuolar addressing sequences) of plant origin respectively.
  • PPS N-terminal prepropeptide
  • the PS and PPS sequences consisting respectively of 23 and 37 amino acids, are those of a reserve protein for tuberous sweetpotato roots: sporamine A (Murakami et al., 1986; Matsuoka and Nakamura, 1991).
  • the BL sequence composed of 26 amino acids, is that of barley lectin (Lerner and Raikhel, 1989).
  • cDNA RabG was modified at its 3 'end by PCR-directed mutagenesis using 2 oligodeoxynucleotides, 5' CTC AGG AGT TGA CTT GGG 3 '(containing the unique HincII site in the plasmid pPBO10) and 5' CCG GAT CCT CAC AGT CTG GTC TCA C 3 '(containing the unique BamHI site in the plasmid pPBO10).
  • the PCR amplification of the HincII-BamHI fragment was carried out in 100 ⁇ l of reaction medium comprising 10 ⁇ l of Taq DNA polymerase x10 buffer (500 mM KC1, 100 mM Tris-HCl, pH9.0 and 1% Triton x100), 6 ⁇ l 25 mM MgCl2, 3 ⁇ l of 10 mM dNTP (dATP, dCTP, dGTP and dTTP), 100 ⁇ M of each of the 2 oligodeoxynucleotides described above, 5 ng of template DNA (vector pPBO10), 2.5 U of Taq DNA polymerase (Promega) and 2 drops of petrolatum oil.
  • reaction medium comprising 10 ⁇ l of Taq DNA polymerase x10 buffer (500 mM KC1, 100 mM Tris-HCl, pH9.0 and 1% Triton x100), 6 ⁇ l 25 mM MgCl2, 3 ⁇ l of 10
  • the DNA was denatured at 94 ° C for 5 min., Subjected to 30 cycles each consisting of 1 min. denaturation at 94 ° C, 1 min. hybridization at 44 ° C and 1 min. elongation at 72 ° C, then the elongation at 72 ° C was continued for 5 min.
  • This PCR reaction was carried out in the "DNA Thermal Cycler" machine from PERKIN ELMER CETUS. The oil was removed by extraction with chloroform. Then, the DNA fragments of the reaction medium were precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min.
  • T7 TM marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
  • the resulting plasmid was called pBI ⁇ C102.
  • the plasmid pBIOC102 was digested twice with BglII and HindIII in order to suppress the sequence coding for the natural signal peptide of rabies glycoprotein G and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL).
  • This sequence has been replaced by that coding for the signal peptide PS of 23 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC) merged with that coding for the first 9 codons of the mature RabG protein ("PS-9 first codons of mature RabG").
  • the sequence "first PS-9 mature RabG codons" was amplified by PCR from the plasmid pMAT103 (Matuoka and Nakamura, 1991) using the 2 oligodeoxynucleotides, 5 'cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3' (containing the unique BglII and additional EcoRI sites in the plasmid pPBO10) and 5 'GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGA ATG GGC TGG ATT GGG CAG G 3' (containing the unique HindIII site in the plasmid pPBO10) by following the PCR amplification protocol described
  • the hybridization temperature was 40 ° C.
  • the DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min ., washed with 70% ethanol, dried, then ligated with plasmi DNA pBIOClOl plate doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried.
  • the ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer. (Amersham) and -4.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
  • the EcoRI fragment carrying the sequence PS-RabG was isolated by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to precipitation. alcoholic, dried, and ligated to the plasmid DNA of pBI0C21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
  • the ligation was carried out with 100 ng of dephosphorylated pBIOC21 vector and 50 ng of DNA fragments containing PS-RabG, described above, in a 10 ⁇ l reaction medium in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • T4 DNA ligase x 10 buffer Amersham
  • Amersham 2.5 U of T4 DNA ligase enzyme
  • the nucleic acid sequence of the fragment coding for the recombinant protein PS-RabG was verified by sequencing using the T7 TM sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
  • the plasmid of the binary vector pBIOC104 was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
  • pBIOClOS is similar to that of pBI0C104 except that the EcoRI fragment carrying the sequence PS-RabG was cloned at the Xbal site of pBIOC82.
  • the EcoRI sites of the insert and Xbal of the vector were subjected to the action of Klenow. The usual cloning methods were applied.
  • the plasmid pBIOC103 was digested twice with BglII and HindIII in order to suppress the sequence coding for the signal peptide PS and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL).
  • This sequence has been replaced by that coding for the BL amino acid signal peptide of 26 amino acids (ATG AAG ATG ATG AGC ACC AGG GCC CTC GCT CTC GGC GCG GCC GCC GTC CTC GCC TTC GCG GCG GCG ACC GCG CAC GCC) merged with that coding for the first 9 codons of the mature RabG protein ("BL-9 first codons of mature RabG").
  • the sequence "BL-9 first codons of mature RabG” was amplified by PCR from the plasmid BLc3 (Lerner and Raikhel, 1989) using the 2 oligodeoxynucleotides, 5 'ggagatctgaattoaacaATG AAG ATG ATG AGC ACC AGG 3' (containing the unique BglII and additional EcoRI sites in the plasmid pBIOC102) and 5 'AGG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GTC CGC GGT CGC CGC G 3' (containing the unique HindIII site in the plasmid pBIOCl02) PCR amplification protocol described above.
  • the ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer. (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
  • the sequences coding for BL and mature RabG were cloned maintaining their open reading phases (ie, so that they constitute a single open reading phase)
  • the cleavage sequence between the BL and mature RabG sequences is Ala-Lys.
  • the resulting plasmid was called pBIOClO ⁇ .
  • the EcoRI fragment carrying the sequence BL-RabG was isolated by enzymatic digestion by EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried, and ligated to the plasmid DNA of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme calf intestine alkaline phosphatase (Boehringer Mannheim) according to the manufacturer's recommendations.
  • the ligation was carried out with 100 ng of dephosphorylated pBI0C21 vector and 50 ng of DNA fragments containing BL-RabG, described above, in a 10 ⁇ l reaction medium in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • T4 DNA ligase x 10 buffer Amersham
  • Amersham 2.5 U of T4 DNA ligase enzyme
  • the nucleic acid sequence of the fragment coding for the recombinant protein BL-RabG was verified by sequencing using the T7 TM sequencing kit sold by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
  • the plasmid DNA of the binary vector pBIOCl07 was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
  • pBIOClo ⁇ is similar to that of pBIOC107 except that the EcoRI fragment carrying the sequence BL-RabG was cloned at the Xbal site of pBIOC82.
  • the EcoRI sites of the insert and Xbal of the vector were subject to the action of Klenow. The usual cloning methods were applied.
  • the plasmid pBIOC102 was digested twice with BglII and HindIII in order to suppress the sequence coding for the natural signal peptide of rabies glycoprotein G and the first 9 amino acids of the mature RabG protein (KFPIYTIPDKL).
  • This sequence has been replaced by that coding for the PPS signal peptide of 37 amino acids (ATG AAA GCC TTC ACA CTC GCT CTC TTC TTA GCT CTT TCC CTC TAT CTC CTG CCC AAT CCA GCC CAT TCC AGG TTC AAT CCC ATC CGC CTC CCC ACC ACA CAC GAA CCC GCC) merged with that coding for the first 9 codons of the mature RabG protein ("PS-9 first codons of mature RabG").
  • the sequence "PPS-9 first codons of mature RabG” was amplified by PCR from the plasmid pMAT103 (Matuoka and Nakamura, 1991) using the 2 oligodeoxynucleotides, 5 'cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3 • (containing the unique BglII and additional EcoRI sites in the plasmid pPBO10) and 5 'GG AAG CTT GTC TGG GAT CGT GTA AAT AGG GAA TTT GGC GGG TTC GTG TGT GGT GGG GAG G 3' (containing the unique HindIII site in the plasmid pPBO10) following the PCR amplification protocol described above.
  • the hybridization temperature was 40 ° C.
  • the DNA fragments resulting from the PCR amplification were purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 by volume of 3M sodium acetate pH 4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min., centrifuged at 12000 g for 30 min., washed with ethanol 70%, dried, and then ligated to the plasmid DNA of pBIOClOl doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried.
  • the ligation was carried out with 100 ng of the vector and 50 ng of digested DNA fragments resulting from the PCR amplification, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer. (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
  • the EcoRI fragment carrying the PPS-RabG sequence was isolated by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to precipitation. alcoholic, dried, and ligated to the plasmid DNA of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
  • Ligation was carried out with 100 ng of pPHOC21 dephosphorylated vector and 50 ng of DNA fragments containing PPS-RabG, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 enzyme DNA ligase (Amersham) at 14 ° C for 16 hours.
  • T4 DNA ligase x 10 buffer Amersham
  • T4 enzyme DNA ligase Amersham
  • the plasmid DNA of the binary vector pBIOC10O was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the clone retained was verified by enzymatic digestion of the plasmid DNA introduced.
  • pBIOC111 is similar to that of pBIOC11O except that the EcoRI fragment carrying the sequence PS-RabG was cloned at the Xbal site of pBIOC82.
  • the EcoRI sites of the insert and Xbal of the vector were subjected to the action of Klenow. The usual cloning methods were applied. II. CONSTRUCTION OF CHEMICAL GENES ENCODING THE RECOMBINANT PROTEIN OF G RABIC GLYCOPROTEIN AND ALLOWING EXPRESSION IN CANNED SEEDS.
  • rapeseed of the viral gene coding for rabies glycoprotein G required the following regulatory sequences:
  • the transcription terminator sequence polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the transcript 35S (Franck et al., 1980); the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 • non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
  • the plasmid pBI221-CRURSP is derived from pBI221 (marketed by Clontech) by replacing the 35S promoter with the pCRU promoter.
  • the "EcoRI treated with Klenow - BamHI" fragment carrying the pCRU promoter was purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to the plasmid DNA of pJIT163 (described in paragraph I.) digested with Kpnl, treated with T4 DNA Polymerase (Biolabs) according to the manufacturer's recommendations, then digested with BamHI, purified by electrophoresis on 0.8% agarose gel , electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
  • the ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of DNA fragments "EcoRI treated with Klenow - BamHI" described above in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of buffer T4 DNA ligase x 10 (Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 -and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the ligation was carried out with 20 ng of the dephosphorylated vector described above and 200 ng of XhoI-EcoRI DNA fragments described above in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for
  • the plasmid pBSII-pGEAID was obtained in two stages: - on the one hand, the Sacl-EcoRI fragment carrying tNOS (terminator of the nopaline synthase gene) of Agrobacterium tumefaciens, treated with the T4 DNA polymerase enzyme (Biolabs) according to the manufacturer's recommendations and purified, was cloned at EcoRV site of pBSIISK-t- marketed by Stratagene, dephosphorylated by the enzyme alkaline phosphatase of calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
  • the ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing tNOS described above in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham), of 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • T7 TM marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
  • the resulting plasmid was called pBSII-tNOS.
  • the ligation was carried out with 100 ng of the vector described above and 50 ng of DNA fragments described above in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the ligation was carried out with 20 ng of dephosphorylated pSCV1.2 and 200 ng of fragments carrying the expression cassette "pGEAlD - tNOS", in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer ( Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the EcoRI-BamHI fragment treated with Klenow, containing the promoter pGEA6 was isolated from the plasmid pGUS2-pGEA6.
  • the DNA fragment between the AccI site and the sequences upstream of the ATG of the GEA6 gene of the clone pGUS-2-pGEA6 was therefore amplified by PCR using the 2 oligonucleotides: 5 ′ AAGTACGGCCACTACCACG 3 • and 5 'CCCGGGGATCCTGGCTC 3'.
  • the hybridization temperature has been adapted.
  • the PCR amplified fragment was digested with AccI and BamHI, purified by electrophoresis on 2% agarose gel, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M sodium acetate pH4, 8 and 2.5 volumes of absolute ethanol at -80 ° C for 30 min.
  • the modified pBIOC21 plasmid was obtained by double digestion with Klenow-treated HindIII and Kpnl of pBIOC21 to delete the fragment carrying the pd35S promoter and replace it with the Kpnl-EcoRV fragment carrying the polylinker composed of the Kpnl-XhoI-SalI-ClaI sites -HindlII-BamHI-Smal-EcoRI- EcoRV from pBSIISK +.
  • the ligation was carried out with 20 ng of dephosphorylated modified pBIOC21 and 200 ng of EcoRI-BamHI DNA fragments, described above, in a 20 ⁇ l reaction medium in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the expression cassette "pGEA6D - t35S” was isolated from pBIOC92.
  • This expression cassette carried by the Kpnl-EcoRV fragment treated with the T4 DNA polymerase enzyme (Biolabs) according to the manufacturer's recommendations and purified, was cloned at the SmaI site of pSCV1.2 dephosphorylated by the enzyme alkaline phosphatase d ' calf intestine (Boehringer Mannheim) according to the manufacturer's recommendations.
  • the ligation was carried out with 20 ng of pSCV1.2 dephosphorylated and 200 ng of fragments carrying the expression cassette "pGEAlD - tNOS", in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the bacteria, Escherichia coli DH5cc, made competent beforehand, have been transformed (Hanahan, 1983).
  • the plasmid pBIOC109 described previously in I.c, contains the EcoRI fragment carrying the sequence PPS-RabG.
  • pd35S double constitutive promoter 35S of CaMV (cauliflower mosaic virus). It corresponds to a duplication of the transcription activating sequences located upstream of the TATA element of the natural 35S promoter (Kay et al., 1987);
  • the transcription terminator sequence polyA 35S terminator, which corresponds to the 3 ′ non-coding region of the sequence of the circular double-stranded DNA virus of the cauliflower mosaic producing the 35S transcript (Franck and al., 1980); the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 'non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
  • the plasmid pBIOC11 ⁇ where the sequence coding for PS-RabG is placed under the control of pAR-IAR has been obtained by cloning of the EcoRI fragment carrying the sequence coding for PS-RabG at the "Ncol and Sali" sites of pBSII-pAR-IAR-tNOS.
  • the EcoRI fragment carrying the sequence coding for PS-RabG was isolated from pBIOC103 by enzymatic digestion with EcoRI, purified by electrophoresis on 0.8% agarose gel, electroeluted, subjected to alcoholic precipitation, dried and then treated with the Klenow enzyme.
  • the plasmid pBSII-pAR-IAR-tNOS was digested twice with Sali and Ncol, purified, treated with the enzyme Mung Bean Nuclease (Biolabs) and dephosphorylated by the enzyme alkaline phosphatase of calf (Boehringer Mannheim) .
  • the ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing the sequence coding for RS-LGC, described above, in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase buffer.
  • x 10 (Amersham), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the plasmid pBSII-pAR-IAR-tNOS results from cloning at the sites "Eco0109I treated with Klenow and Kpnl" of pBSII-tNOS of the SnaBI-Kpnl fragment carrying the sequence corresponding to "pAR-IAR-start of the coding sequence for the gene gus "isolated from the plasmid pActl-F4.
  • the ligation was carried out with 100 ng of vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 ⁇ l digested in the presence of 1 ⁇ l of T4 DNA ligase buffer x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the bacteria, Escherichia coli DH5 ⁇ made previously competent, were transformed (Hanahan, 1983).
  • the plasmid pBSII-tNOS was obtained by cloning at the dephosphorylated EcoRV site of pBSIISK + marketed by Stratagene, from the Sacl-EcoRI fragment carrying the tNOS sequence isolated from pBI121 marketed by Clontech by double enzymatic digestion with Sacl and EcoRV, subjected to purification by electrophoresis on 2% agarose gel, and treated with the T4 DNA polymerase enzyme.
  • the ligation was carried out with 20 ng of the dephosphorylated vector and 200 ng of DNA fragments containing the tNOS sequence, described above, in a reaction medium of 20 ⁇ l in the presence of 2 ⁇ l of T4 DNA ligase x 10 buffer (Amersham ), 2 ⁇ l of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the plasmid pBIOC117 where the sequence coding for PS-RabG is placed under the control of pd35S was obtained by cloning at the "Kpnl and Xbal" sites of the plasmid pBSIISK + marketed by Stratagene, of the Kpnl-Xbal fragment carrying the sequence corresponding to "pd35S- PS-RabG "isolated from pBIOC103.
  • the ligation was carried out with 100 ng of vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 ⁇ l digested in the presence of 1 ⁇ l of T4 buffer DNA ligase x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C. for 16 hours
  • the plasmid DNA of the clones obtained selected from
  • 50 ⁇ g / ml ampicillin was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
  • the plasmid p ⁇ 63 results from the cloning of p ⁇ zein at the HindIII and Xbal sites of a plasmid pUC18 containing, between its HindIII and EcoRI sites, the expression cassette "p35S-gus-tNOS" of pBI221 marketed by Clontech. It allows an expression in the albumen of corn seeds.
  • polyA NOS terminator which corresponds to the 3 'non-coding region of the nopaline synthase gene of the plasmid Ti of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
  • PS-RabG is placed under the control of p ⁇ zein, was obtained by cloning at the "Sacl sites treated with the enzyme T4 DNA polymerase and BamHI" of the plasmid p ⁇ 63, of the fragment "BamHI treated with Klenow - BglII” isolated from pBIOC103 .
  • the ligation was carried out with 100 ng of the vector and 50 ng of DNA fragments, described above, in a reaction medium of 10 ⁇ l in the presence of 1 ⁇ l of T4 DNA ligase x 10 buffer (Amersham) and of 2, 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.
  • the plasmid pBIOC119 results from the substitution of the BamHI-HincII fragment of pBIOC11 ⁇ by the BamHI-HincII fragment carrying the sequence coding for KDEL placed before the stop codon obtained by PCR amplification according to the methods described above.
  • the 2 oligodeoxynucleotides used during this reaction were: 5 'GGT CTC AGG AGT TGA CTT GGG TCT CCC GAA CTG GG 3' (single HincII site) and 5 'CCC GGA TCC TCA TAG CTC ATC TTT CAG TCT GGT CTC ACC CCC ACT C 3 '(sequence coding for KDEL and unique BamHI site).
  • the hybridization temperature was 50 ° C.
  • the cloning was carried out as described previously.
  • the tobacco plants used for the transformation experiments are cultivated in vitro on the basic medium of Murashige and Skoog (1962) supplemented with the vitamins of Gamborg et al. (1968, Sigma reference M0404), sucrose at 20g / L and agar (Merck) at 8g / L.
  • the pH of the medium is adjusted to 5.8 with a potassium hydroxide solution before autoclaving at 120 ⁇ C for 20 min.
  • Tobacco seedlings are transplanted by cutting internodes every 30 days on this MS20 multiplication medium.
  • the transformation technique used is derived from that of Horsch et al. (1985).
  • a preculture of Agrobacterium tumefaciens strain LBA4404 containing the binary plasmids is carried out for 48 h at 2 ⁇ ° C with stirring, in LB medium supplemented with appropriate antibiotics (rifampicin and tetracycline).
  • the preculture is then diluted to 50th in the same medium and cultivated under the same conditions.
  • the culture is centrifuged (10 min, 3000 rpm), the bacteria are taken up in an equivalent volume of liquid MS30 medium (30 g / L sucrose) and this suspension is diluted with 10eme.
  • Explants of approximately 1 cm 2 are cut from the leaves of the seedlings described above. They are then placed in contact with the bacterial suspension for 1 h, then quickly dried on filter paper and placed on a coculture medium (solid MS30). .
  • the explants are transferred to petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200mg / L), a bacteriostatic, augmentin (400mg / L) and the hormones necessary for bud induction (BAP, lmg / L and ANA, 0, lmg / L).
  • the explants are subcultured on the same medium after 2 weeks of culture. After 2 new weeks, the buds are subcultured in Petri dishes on the development medium composed of the MS20 medium supplemented with kanamycin and augmentin. After 15 days, the buds are transplanted into pots on the same medium, the concentration of kanamycin has been halved. It takes about 20 days to take root, at the end of which the seedlings can be cloned by cutting internodes or emerging in the greenhouse.
  • the spring rapeseeds (Brassica napus cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a 15% Domestos solution. After 4 rinses with sterile water, the seeds are put to germinate, at a rate of 20 seeds per 7 cm diameter jars by 10 cm high, on Murashige and Skoog mineral medium (Sigma, reference M 5519) with 30 g / 1 of sucrose and solidified with 5 g / 1 of agargel. These pots are placed in a culture chamber at 26 ° C with a photoperiod of 16h / ⁇ h and under a light intensity of the order of 80 ⁇ E m "" 2 S -1 . After 5 days of germination, the cotyledons are removed sterile by cutting each petiole about 1 mm above the cotyledon node.
  • a culture of Agrobacterium tumefaciens strain LBA4404, containing the plasmid pBI0C114 is cultivated in a 50 ml Erlenmeyer flask for 36 h at 28 ° C. in 10 ml of YT bacterial medium supplemented with the antibiotics useful for the selection of the strain used.
  • This preculture is used to seed at 1% a new bacterial culture carried out under the same conditions. After 14 h the culture is centrifuged for 15 min at 3000 rpm and the bacteria are taken up in an equivalent volume of liquid germination medium. This suspension is distributed in petri dishes 5 cm in diameter at a rate of 5 ml / box.
  • the severed end of the petiole is immersed for a few seconds in the agrobacteria solution thus prepared, then the petiole is pressed a few millimeters into the regeneration medium.
  • This medium has the same basic composition as the germination medium with, in addition, 4 mg / 1 of benzylamino-purine, a phytohormone promoting the neoformation of buds. Twelve explants (cotyledon with petiole) are cultured in a 9 cm diameter petri dish (Greiner, reference 664102).
  • the explants are subcultured in phytatray boxes (Sigma, reference P1552) containing the above medium supplemented with a selective agent: 45 mg / 1 of kanamycin sulfate (Sigma, reference K4000) and a bacteriostatics: mixture of 1/6 (by weight) of potassium salt of clavulanic acid and 5/6 ammonium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1. Twice in succession, 3 weeks apart, the explants are transplanted sterile onto new medium under the same conditions.
  • a selective agent 45 mg / 1 of kanamycin sulfate
  • a bacteriostatics mixture of 1/6 (by weight) of potassium salt of clavulanic acid and 5/6 ammonium salt of amoxicillin (Augmentin injectable) at a rate of 600 mg / 1.
  • the green buds that appear at the end of the second or third transplanting are separated from the explant and cultured individually in transparent pots 5 cm in diameter and 10 cm high containing a medium identical to the above but free of BAP. After 3 weeks of culture, the stem of the transformed bud is cut off and the bud is transplanted into a pot of fresh medium. After three to four weeks the roots are sufficiently developed to allow acclimatization of the seedling to the phytotron. Buds that are not green or rooted are removed. These seedlings are then transplanted into 7 cm side pots filled with potting soil (standard NF U44551: 40% brown peat, 30% sifted heather and 30% sand) saturated with water.
  • standard NF U44551 40% brown peat, 30% sifted heather and 30% sand
  • the seedlings are repotted in 12 cm diameter pots filled with the same soil enriched with late fertilizer (Osmocote, at a rate of 4g / 1 of potting soil) then transported to a greenhouse (class S2) regulated at ⁇ ° C, with two daily waterings of 2 minutes.
  • a greenhouse class S2
  • the pods When the pods have reached maturity, they are harvested, dried and then beaten. The seeds obtained are used for the determination of biochemical activity.
  • the selection of the transgenic progeny is done by germination on a medium containing kanamycin sulfate at a rate of 100 to 150 mg / 1 (depending on the genotypes). Conditions The operating procedures are identical to those described at the beginning of this document except that the germinations are carried out in glass tubes with only one seed per tube. Only the seedlings developing secondary roots during the first three weeks are acclimatized in a phytotron before being transferred to the greenhouse.
  • calluses are obtained from immature embryos of genotype HI, II or (A188 x B73) according to the method and on the media described by Armstrong (Malze Handbook, 1994, M. Freeling, V. Walbot Eds., Pages 665- 671). The calluses thus obtained are multiplied and maintained by successive subcultures every fortnight on the initiation medium.
  • Seedlings are then regenerated from these calluses by modifying the hormonal and osmatic balance of the cells according to the method described by Vain et al. (Plant Cell Tissue and Organ Culture, 1969,! £: 143-151). These plants are then acclimatized in the greenhouse where they can be crossed or self-fertilized. b) Use of the particle gun for the genetic transformation of corn:
  • the plasmids carrying the genes to be introduced are purified on a Qiagen® column, following the manufacturer's instructions. They are then precipitated on tungsten particles (M10) following the protocol described by Klein (Nature, 1987, 327: 70-73). The particles thus coated are projected towards the target cells using the cannon and according to the protocol described by J. Finer (Plant Cell Report, 1992, ⁇ : 323-328).
  • the callus boxes thus bombarded are then sealed using Scellofrais® and then grown in the dark at 27 ° C.
  • the first subculture takes place 24 hours later, then every fortnight for 3 months on medium identical to the initiation medium supplemented with a selective agent, the nature and concentration of which may vary depending on the gene used (see paragraph 3).
  • the selective agents which can be used generally consist of active compounds of certain herbicides (Basta®, Round up®) or certain antibiotics (Hygromycin, Kanamycin ).
  • Calls are obtained after 3 months, or sometimes earlier, calluses whose growth is not inhibited by the selection agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene.
  • the frequency of obtaining such calluses is approximately 0.8 cal per bombarded box.
  • calluses are identified, individualized, amplified and then cultivated so as to regenerate seedlings (see paragraph a). In order to avoid any interference with untransformed cells, all of these operations are carried out on culture media containing the selective agent.
  • the plants thus regenerated are acclimatized and then cultivated in a greenhouse where they can be crossed or self-fertilized.
  • the homogenate thus obtained is immediately centrifuged at 4 ° C for 10 min at 10,000 rpm.
  • extraction is carried out at the rate of 100 mg of seeds for 4 ml of buffer.
  • extraction is carried out at a rate of 0.5 g in 1 ml of buffer.
  • Rabies glycoprotein G is demonstrated by "Western blots" (Renart and Sandoval, 1984) and assayed by an ELISA test.
  • the proteins extracted according to the above protocol are denatured by heating at 95 ° C for 5 min, in the presence of 50 mM pH 6.8 Tris HCl buffer; SDS 4%; BSH 1%; Sucrose 20% and bromophenol blue 0.01%.
  • the proteins are then separated by electrophoresis, on a polyacrylamide gel under denaturing conditions at 8%, 10% or 12.5%, according to the technique of Laemmli U.K. (1970) at the rate of 50 ⁇ g of total proteins per sample.
  • the proteins are transferred to a nitrocellulose membrane.
  • An anti-rabies G glycoprotein antibody obtained in sheep is used as a probe and the revelation is carried out by means of an anti-sheep IgG antibody margined with alkaline phosphatase.
  • the tests were carried out on the transformed plants with the following constructs: pBIOC104, pBIOC10O, pBIOC114, pBIOC116.
  • the control protein migrates in the form of 2 bands of apparent molecular weights 66 and 61 KDa.
  • the lightest form corresponds to a protein, truncated at the C-terminal end (Wunner et al, 1983), more soluble than the complete protein.
  • the following protocol was used: extraction of the glycoprotein: the samples of the leaves or seeds, taken on ice on the day of the test, are ground at a rate of 1 g for 4 ml of extraction buffer for the leaves and 100 mg for 4 ml of extraction buffer for the seeds.
  • the amount of total protein is then determined by the Bradford method and all the extracts are brought to a concentration of 5 ⁇ g / ⁇ l. These extracts are diluted extemporaneously to 1/1000.
  • the reference range (glycoprotein G: 0 to 20 mg / ml) is prepared in an extract of ground unprocessed plant, standardized and diluted to 1/1000 in the same way.
  • the assay is then carried out by direct reading on a standard curve carried out under the same conditions, with purified glyco-protein G.
  • glycoprotein G varies from 1.5 to 0.4 ⁇ g / ml for the different extracts tested.
  • the homogenate thus obtained is immediately centrifuged at 4 ° C for 10 min at 13000 g. Then, the supernatant is removed and filtered through Miracloth (CALBIOCHEM). A determination of the soluble proteins is then carried out by the method of Bradford (1976).
  • Rabies glycoprotein G is demonstrated by immunodetection of the "Western blot” type (Renart and Sandoval, 1984).
  • the proteins extracted according to the above protocol are denatured by heating at 95 ° C. for 5 min, in the presence of 50 mM Tris-HCl buffer, pH 6.8, 4% SDS, 1% BSH, 20% sucrose and bromophenol blue 0 , 01%.
  • the proteins are then separated by electrophoresis on a polyacrylamide gel under denaturing conditions at 8% or 10%, according to the technique of Laemmli (1970) at the rate of 30 ⁇ g of soluble proteins per sample.
  • the proteins are transferred to a PVDF (polyvinylidene difluoride) membrane.
  • PVDF polyvinylidene difluoride
  • the control reference rabies glycoprotein G is characterized by 2 bands of apparent molecular masses of 66 and 61 KDa.
  • the form lighter corresponds to a protein truncated at the C-terminal end (Wunner et al, 1983), more soluble than the complete protein. No band corresponding to rabies glycoprotein G is detected in the unprocessed corn extracts.
  • the modification of the cDNA encoding the rabies glycoprotein G was carried out according to the LbPCR technique by modifying the 5 '[fragment BglII (agatct) - Xhol (ctcgag)], central [fragment Xhol - Aatll (gacgtc)] and 3' [fragment Aatll - BamHI (ggatcc)]
  • the LbPCR technique consists of an LCR ligation step (Barany F. PNAS USA 1991, 88, 189-193) to produce continuous single-stranded DNA using "sense" oligodeoxynucleotides and a PCR step to obtain double-stranded DNA.
  • modified parts were verified by sequencing at using the Amersham Sequenase PCR product sequencing kit according to the manufacturer's recommendations Then, either the modified 5 'part, or the modified 5' and central parts, or the modified 5 ', central and 3' parts, were introduced into pBIOC109 by enzymatic digestions (restriction enzymes delimiting the fragments)
  • the 5 ′ part was constituted using the following oligodeoxynucleotides
  • the 5 ′ part amplified by PCR was doubly digested with BglII and Xhol, then cloned to replace the BglII - Xhol fragment of pBIOC109 according to the methods already described previously.
  • the resulting plasmid was called pBIOC109-5'M This plasmid was used in genetic transformation of corn
  • the central part was formed using the following oligodeoxynucleotides.
  • the central part amplified by PCR was doubly digested with Xhol and Aatll, then cloned to replace the Xhol - Aatll fragment of pBIOC109-5'M according to the methods already described above.
  • the resulting plasmid was called pBIOC109-5'CM. This plasmid was used in genetic transformation of corn.
  • the 3 ′ part was constituted using the following oligodeoxynucleotides:
  • the isolated fragments were ligated to the plasmid DNA of pBIOC21, pBIOC28, pBIOC82, pBIOC91 and pBIOC1 12 digested with EcoRI and dephosphorylated. The usual cloning methods were applied.
  • the plasmid DNA of the resulting binary plasmids was introduced by direct transformation into the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). Genetic transformation has been completed.
  • the invention therefore particularly extends to methods, to glycoproteins and their uses, to nucleic acids, transformed plant cells, chimeric or transgenic plants, seeds of transgenic plants, monoclonal or polyclonal antibodies, pharmaceutical compositions and products.
  • foodstuffs according to the invention characterized in that the coding sequence, allowing expression in the cell of the protein G, has been subjected to modifications to increase the expression, for example has been subjected to a "revegetation" according to example E above.

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EP97924066A 1996-05-09 1997-05-07 Tollwut-glykoprotein g experimierende transgene pflanzen und durch diese herges tellte glykoprotein Withdrawn EP0910650A1 (de)

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PCT/FR1997/000827 WO1997043428A1 (fr) 1996-05-09 1997-05-07 Plantes transgeniques exprimant la glycoproteine g de la rage, et glycoproteines ainsi obtenues

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