CA2254115A1 - Transgenic plants expressing rabies glycoprotein g, and glycoproteins thus obtained - Google Patents

Abstract

The 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 molecule d nucleic acid 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 a signal peptide Nterminal other than that naturally associated with viral protein G, the introduction of the chimeric coding sequence allowing expression in the cell of 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.

Description

TRANSGENIC PLANTS EPRESSING GLYCOPROTEIN G FROM
RABIES, AND GLYCOPROTEINS THUS OBTAINED
The present invention relates to a method of production, by plant cells, of rabies G glycoprotein, or a virus related to rabies. The invention also relates to the glycoprotein thus obtained and its use as that medication, especially as a human vaccine or veterinary. The invention further relates to cells transformed plants, and transgenic plants, capable of producing the rabies G glycoprotein.
The rabies virus is part of the family of Rhabdoviridae, genus Lvssavirus. It is a virus RNA- whose genome contains around 12,000 nucleotides, encoding five structural proteins. protein of nucleocapsid "N", the matrix proteins "M1" and "M2", the envelope glycoprotein "G" and protein "L" encoding the viral polymerase.
The viral envelope consists of a bi-layer lipid containing spicules composed of a form polymer 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 rabies glycoprotein mature monomer is 66 kDa. This form of the glycoprotein is the so-called "insoluble" form, say it includes a C-terminal domain transmembrane allowing the glycoprotein to fix in the viral envelope. The "soluble" form of the glycoprotein has a molecular weight of around 60 kDa and differs from the insoluble form by the absence

2 of about 58 C-terminal amino acids. This form of the glycoprotein, being devoid of segment transmembrane, can no longer associate with the envelope viral.
Glycoprotein is the major antigen of virus, responsible for the induction of antibodies neutralizers and protective immunity. Only the "insoluble" form is protective, although there is no antigenic determinants in the C-terminal (Dietzschold H. et al., 1983).
For these reasons, the rabies G glycoprotein has been widely studied and used in development of subunit vaccines. DNA from different strains rabies virus have been cloned (Anilionis A. and al., 1981; Yelverton et al., 1983) and expressed in different cellular hosts.
For example, Yelverton et al. (1983) described expression of the rabies glycoprotein gene (strain CVS) in E. coli. The antigenic activity of the product expression is only 2 to 3% compared to the natural glycoprotein.
Kieny et al. (1984) described the expression of the glycoprotein G in eukaryotic cells superior, infected with a vaccinia virus, recombinant and replicative.
Expression of a 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. et al., 1993), was obtained from infected insect cells by a recombinant baculovirus vector.
Among these works, the use of hosts higher eukaryotes allowed to obtain recombinant glycoproteins capable of imparting protective immunity in vivo, orally or by intramuscular or intraperitoneal injection.

3 The use of transgenic plants for the production of rabies G glycoprotein also been considered. Indeed the production of protein immunization in plants has advantages considerable because the plants are more easier to grow than animal cells. In addition, the risk of contamination by viruses or by prions are eliminated when proteins are produced in plants.
McGarvey et al. (1995) described the cDNA transformation of cotyledons of tomatoes whole of the rabies virus glycoprotein G gene (ERA strain). Transgenic plants have been regenerated. The expression of a protein, recognized by rabies anti-glycoprotein antibodies, and appearing as two separate bands of 62 and 60 kDa on electrophoresis gel, was found in the leaves and in the fruits of transgenic plants.
The difference in molecular weight compared to the natural glycoprotein is probably linked, according to the authors, to a post-translational modification of the protein, in particular to a proteolytic cleavage or to a particular glycosylation.
To date, the production, in plants, rabies glycoprotein with molecular weight comparable to that of natural glycoprotein, has not been described.
The technical problem that we propose to solve the present invention is to produce, in a cell plant, the rabies virus glycoprotein, or a virus related to rabies virus, in accordance with “insoluble” form of the natural glycoprotein, especially with regard to its molecular weight.
Surprisingly, it was found by present inventors that replacing the peptide endogenous N-terminal signal of rabies glycoprotein,

4 by a heterologous N-terminal signal peptide, allows to obtain glycoproteins having a molecular weight higher than those obtained in plants before. The present inventors have therefore implemented work for the transformation of the plant cell, a chimeric coding sequence comprising the sequence coding for the rabies glycoprotein gene, in which the sequence of the N-terminal signal peptide natural has been replaced by a sequence coding for a heterologous N-terminal signal peptide.
More particularly, the present invention relates to a process for producing the rabies virus glycoprotein G, or a virus related to rabies virus, characterized by.
i) the introduction into a plant cell of a nucleic acid molecule comprising a sequence chimeric coding, comprising on the one hand a sequence encoding said mature viral protein G, or for an analogous protein, and on the other hand, a sequence encoding an N-terminal signal peptide other than the one naturally associated with viral protein G, the introduction of the chimeric coding sequence allowing expression in the cell of the protein G;
ii) multiplication, in the form of culture cell, transformed cells, or the regeneration of whole transgenic plants or chimeric from these cells;
iii) possibly the extraction and purification of G-glycoprotein from cells or tissues plants thus obtained.
In the context of the invention, the terms "Rabies virus, or virus related to the rabies virus rabies ”mean the viruses of the different serotypes of genus Lvssavirus (Bourhy et al., 1993). On time Lyssaviruses are divided into four separate serotypes. serotype 1 corresponds to the virus rabies proper and includes different rabies virus strains, e.g. CVS, HEP, ERA, PM. There is normally at least 90% homology at level of the amino acid sequence, between different strains of rabies. The other serotypes correspond to viruses related to rabies virus (“Rabies - related viruses”) and are exemplified by the "Lagos bat" virus (serotype 2); the virus Mokola (serotype 3); Duvenhage virus (serotype 4).
All rabies viruses are pathogenic to mammals, including humans, and give rise to a rabies encephalitis.
In the context of the present invention, the term "glycoprotein G" means glycoprotein constituting the spicules of the viral envelope. According to the invention, it is normally in the form monomeric but can also exist in form polymer, 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 agglutination of erythrocytes.
The first step of the invention consists of the introduction, into a plant cell, of a nucleic acid molecule comprising a sequence chimeric coding.
According to the invention, a "coding sequence chimeric "means a nucleic acid sequence, coding for a chain of amino acids, consisting peptide fragments of different origins. In the species, it is a sequence comprising, of a shares a sequence encoding the viral glycoprotein G
mature, i.e. lacking the N-terminal signal naturally associated with protein, and secondly, a sequence encoding an N-terminal signal peptide heterologous to glycoprotein.
As an example of a sequence coding for mature G glycoprotein, that of the rabies glycoprotein, CVS strain whose sequence in nucleic acid and amino acid have been described by Yelverton et al. (1983), or that of the strain ERA whose sequence has been described by Anilionis et al.
(1981), and modified by Kieny et al. (1984). The mature protein lacks the sequence of 19 N-terminal amino acids. Other sequences are described by N. Tordo et al., Virol., 1993, 194, 59-69.
The sequence of the G glycoprotein gene of the virus Mokola has been described by Tordo et al. (1993). The amino acid sequence of mature protein 20.
The nucleic acid coding for the glycoprotein G
mature is obtained by eliminating part of the sequence which codes for the N-terminal signal peptide.
The coding sequence can code for a protein "Analogous" to glycoprotein G. Proteins analogs are proteins that normally exhibit at least 90% homology with glycoprotein natural, and which also have a homology functional and immunological with glycoprotein natural.
According to the invention, the sequence coding for the endogenous N-terminal signal peptide is therefore replaced by a heterologous signal. As an example of a sequence encoding a heterologous N-terminal signal peptide, we can cite a signal peptide (or "pre-peptide") plant, or from a plant virus, or a microorganism, for example yeast.
“N-terminal signal peptide” means understand the peptide responsible for addressing the nascent protein in the endoplasmic reticulum.

In the plant cell, protein addressing is based on the same principle as in cells animal. From chromosomal DNA, the gene is transcribed into messenger RNA, then translated into protein at ribosome level. If the nascent protein has an N-terminal signal peptide or prepeptide, it enters the endoplasmic reticulum where a certain number of post-translational maturations, in particular the cleavage of the signal peptide, the N-glycosylations leading to glycans polymannosides, and the formation of bridges disulfides.
According to the invention, 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 of a hydrophobic N-terminal signal peptide having between and 40 amino acids and being of animal origin or vegetable. Preferably, it is a prepeptide of plant origin, for example that of sporamine, barley lectin, vegetable extensin (pEXT), a-mating (factor, vegetable proteins involved in defense against microorganisms (PRla and PRS, "pathogenesis related proteins").
The introduction into the plant cell of nucleic acid molecule, having the sequence chimeric coding, is carried out so that the expression of the sequence can be obtained. In in particular, the chimeric coding sequence must be find, in the genome of the plant, under control of sequences, regulating transcription, recognized by the plant cell.
These regulatory sequences, including a signal transcription initiation (promoter) and signal transcription termination (including signal polyadenylation), may be endogenous to plant, in which case the insertion of the coding sequence chimeric must be done by homologous recombination.
In this case, the transforming nucleic acid comprises, on each end, a sequence homologous to sequences which adjoin the desired insertion site in the genome.
Alternatively, the regulatory sequences of transcription can be included in the molecule of nucleic acid comprising the coding sequence chimerical. In this case, the nucleic acid molecule constitutes a chimeric gene which is also part of the invention. Regulatory sequences include one or more promoters of plant origin or from Agrobacterium tumefaciens or from a virus plant. It can be a constitutive promoter, for example example 35S or double 35S from CaMV, NOS, OCS, or specific promoters of certain tissues such as grain, or specific to certain phases of plant development.
As specific seed promoters, cite the promoter of the napin and acyl gene carrier protein (ACP) (EP-A-0255378), as well as promoters of the AT2S genes of Arabidopsis thaliana, that is to say the promoters PAT2S1, PAT2S2, PAT2S3 and PAT2S4 (Krebbers et al., Plant Physiol., 1988, vol.
87, pages 859-866). I1 is particularly preferred to use the cruciferin promoter or ~ the phaseoline, the pGEAl promoters and pGEA6 promoters "EM, Early Methionine" type Arabidopsis labeled protein "strongly expressed during desiccation phases of the seed.
I1 can be considered to use "enhancers"
to improve the efficiency of expression.
The termination regulatory sequences are of plant, viral or bacterial origin, by example of 35S, NOS etc.

According to a variant of the invention, the sequence chimeric coding includes, in addition to the peptide part N-terminal signal and the part encoding the protein mature envelope, other addressing signals, by 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 beneficial for several reasons. Firstly, protein retention in the reticulum endoplasmic tends to increase yields recombinant proteins. On the other hand, maturation of polymannosic glycosylation into glycans complex does not take place; the protein therefore retains the polymannosic glycosylation, minimizing the risk of adverse immunological reactions when the protein will be administered to humans as a medicine.
According to this variant of the invention, the glycoprotein therefore contains 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 complex type glycans. According to the invention, this type protein can also be obtained using plant mutants unable to make 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 an addressing signal vacuolar or "propeptide". In the presence of such signal, the protein is addressed to the vacuoles of aqueous tissue, e.g. leaves, as well as protein bodies in reserve tissue, for example seeds, tubers and roots. Addressing the protein to the protein bodies of the seed is particularly interesting because of the ability from seed to accumulate protein, up to 40%
proteins based on dry matter, in cellular organelles derived from vacuoles, called protein bodies and because of the possibility of store the seeds containing the dehydrated recombinant proteins.
As propeptide, one can use a signal of animal or vegetable origin, plant signals being particularly preferred, for example the pro-sporamine, or barley lectin. The propeptide can be N-terminal ("N-terminal targeting peptide" or NTTP), or C terminal (CTTP) or may consist of a protein internal sequence. To the extent that the propeptide is normally cleaved upon entry of the protein in the vacuole it is not present in the mature protein. it has been found that using "propeptide", in combination with a prepeptide, is particularly advantageous, and gives rise to glycoproteins having a molecular weight consistent with the natural molecule.
Preferably, an agent coding sequence allowing selection of 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 may be a gene chimeric consisting of regulatory sequences recognized by the plant in association with the sequence selection agent coding, e.g. 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 worn by an independent vector and introduced by co-transformation.

To introduce the different sequences heterologists in the plant cell, all means known to transform the nuclear genome can be used, for example Agrobacterium, electroporation, protoplast fusion, bombardment with particle gun, or penetration of DNA into cells like pollen, microspore, seed and the immature embryo, viral vectors such as Geminivirus or satellite viruses. Agrobacterium tumefaciens and rhizogenes constitute the means prefer. In this case, the sequence of the invention is introduced into an appropriate vector with all necessary regulatory sequences such as ~
promoters, terminators, etc ... as well as any sequence required to select the transformants.
After the cell transformation step plants, transformed cells are selected thanks to the presence of the gene coding for the agent selection. These cells are then subjected to a cell culture multiplication step, where they are regenerated into transgenic plants or chimerical.
When it comes to multiplication by crop cell in vitro, we obtain a biomass capable to produce glycoprotein G, in large quantities. I1 can be plant cell cultures in in vitro, for example in a liquid medium. Different modes culture ("batch", "fed batch" or continuous) to these types of cells are currently being studied. The batch cultures are comparable to those performed in an Erlenmeyer flask insofar as the medium is not renewed, the cells do not have only a limited amount of nutrients. The "batch fed" culture corresponds to a "batch" culture with a programmed feeding in substrate. For continuous culture, cells are continuously supplied with nutrient medium.
An equal volume of the biomass-medium mixture is stored in order to keep the volume of the reactor constant. The quantities of plant biomass that can be envisaged with bioreactor cultures are variable depending on the species plant, method of cultivation and type of bioreactor. Under certain conditions, densities biomass of about 10 to 30g dry weight per liter of culture can be obtained, for species like Nicotiana tabacum, Vinca roses and Catharanthus roseus.
The cells of the invention can also be immobilized, which makes it possible to obtain a production constant and prolonged glycoprotein. As immobilization method, we can cite immobilization in alginate beads, agar, to the interior of polyurethane foam, or even in hollow fibers.
The cells of the invention can also be root crops. Roots grown in in vitro, in liquid medium, are called "Hairy roots", these are roots transformed by the bacteria Aarobacterium rhizocrenes.
Instead of producing the glycoprotein from the invention by culturing plant cells, one can regenerate chimeric or transgenic plants-to from transformed explants, using techniques known per se.
The process of the invention may or may not include a glycoprotein G recovery stage.
the absence of a recovery step, the process of the invention gives rise to plant cells, or to transgenic plants capable of producing the glycoprotein G. Cells or plants constitute in themselves a source of the vaccinating protein and can be administered as is to the animal or to humans by ingestion.
According to another variant of the invention, the glycoprotein G is recovered by extraction of the vegetable matter, and possibly purified.
The extraction step comprises the solubilization of glycoprotein, associated with cell membranes, by a detergent. More specifically, the material vegetable, for example, leaves, seeds, roots etc., is ground in liquid nitrogen. Homogeneity is then suspended in an appropriate buffer, added a detergent, preferably non-ionic. Detergent is used in concentrations of 0.1 to 2%, preferably 0.1 to 0.5%. I1 can be for example of Triton X-100, or NP40 or (3-octyl glucopyranoside.
The use of detergent releases the glycoprotein from cell membranes, while maintaining its integrity and its immunogenic power. The homogenate thus obtained is centrifuged, the "solubilized" glycoprotein being then present in the supernatant. Normally, ie supernatant is filtered.
The plant extract corresponding to the supernatant can be used as such as a protein source vaccinating, or may be subjected to one or more purification step (sj. Optionally, the detergent can be eliminated or reduced. Preferably, the extract vegetable matter, for example tobacco leaves ~ or rapeseed, is dialyzed against an ethylene buffer diamine acetic acid pH 8.1, or is subjected to a gel filtration on Sephadex G25 Pharmacia support. The fraction corresponding to retentate or dead volume is then subjected to an exchange chromatography ion on QAE support Sephadex A50 Pharmacia balanced previously in ethylene diamine acetic acid buffer pH 8.1. After washing with this same pad, the glycoprotein is eluted by a 0.6 M acetate solution sodium (P. Atanasiu et al., 1976).
Other purification techniques are described in art, for example affinity chromatography using a purified monoclonal antibody coupled to a support Sepharose CL-48 activated with cyanogen bromide (B. Dietzschold et al., 1983); gel chromatography permeation on support Sepharose CL-4B (G. Coslett and al., 1980); electrofocusing technique (J. Cox and al., 1980).
The invention also relates to the glycoprotein G
likely to be obtained by implementing the method of the invention.
More particularly, the invention relates to a glycoprotein characterized in that.
- it is recognized by antibodies specific to the rabies virus or virus glycoprotein G
related to rabies virus;
- it has a molecular weight of 66 kDa approximately;
- it is insoluble;
- It is N-glycosylated by at least one glycan of polymannosidic type, and / or by at least one glycan complex type including within its structure a or more xylose residues ~ i-1,2, and / or one or several residues of fucose linked in oc-1,3, and being free of sialic acid residues.
The antigenic properties of glycoprotein of the invention are, at least in part, those of the natural corresponding glycoprotein. Indeed, the glycoprotein of the invention is recognized by antibodies specific to the rabies glycoprotein or a rabies-like virus. it could be of polyclonal or monoclonal antibodies against IS
the virus, or against the purified glycoprotein from of the virus.
The glycoprotein of the invention has a weight molecular of approximately 66 kDa. In the context of the present invention, "Approximately" means variability resulting from measurement techniques. More in particular, the glycoprotein of the invention has a molecular weight of 66 kDa on average when several Western Blot tests are carried out, by example, at least 3 or 5 tests, in the same conditions. For example the molecular weight can be between 65 and 80 kDa, for example 66 to 67 kDa. The weight molecular is an apparent molecular weight, determined by polyacrylamide gel electrophoresis under denaturing conditions, according to the technique of Laemmli et al. (1970). Preferably, the gel of polyacrylamide is a 10 or 12.5% gel, but lower concentrations can be used to get a sharper resolution of the areas between 60 and 65 kDa.
The glycoprotein of the invention is strongly insoluble. In the context of the invention, the term "Insoluble" means that the molecule is not soluble in the exracellular medium, but is found associated with cell membranes. Indeed, it was found that the presence of detergent, for example SDS, Triton X-100, is essential for extracting and dissolve the glycoprotein. The “insoluble” nature of glycoprotein is linked to the presence of the domain C-terminal transmembrane. The presence of the region C-terminal, i.e. the region lying about 40 to 60 amino acids from the carboxy end glycoprotein, is important for obtaining protective response when the glycoprotein is used as a vaccine.

The glycoprotein of the invention is shown indirectly insoluble by its association with cell membranes. In the absence of detergent, it can't be in the culture supernatant glycoprotein producing cells.
The glycoprotein of the invention is glycosylated with at least one glycan of the polymannosidic type, and / or by at least one complex type glycan comprising within its structure one or more xylose residues ~ i-1, 2, and / or one or more residues of fucose bound in a-1,3, and being free of residues sialic acid.
The glycosylation of glycoproteins produced in plant cells is different from those produced in animal cells. even though glycosylation steps leading to glycans polymannosidic be carried out so identical in plants and mammals, the maturation of polymannosidic glycans in -complex glycans is very different. The N-glycans of vegetable glycoproteins differ mainly mammalian glycans by the absence of sialic acid, also known as N-acetylneuraminic acid, and the presence of a residue of ~ i 1.2 xylose and a fucose residue bound in a 1.3 at the GlcNAc residue proximal to the "core" (Driouich et al., A look at biochemistry, 1993, vol. 3, pages 33-42).
Glycosylation plays an important role in establishment of the spatial structure of the protein, in protection against proteolytic attacks and in immune recognition mechanisms.
According to a preferred variant, the protein of the invention is glycosylated at at least two sites natural N-glycosylation. For example, for the strain ERA, glycosylation is preferably positions 247 and 319. Glycosylation is of the type mannosidic, polymannosidic or complex type, or a mixture of the two.
The mannosidic glucan (s) have GlcNAc2-Mani structure. The glucan (s) polymannoside (s) have the structure GlcNAc2-Man2-9, for example GlcNAc2-Man9, GlcNAc2-Man8, GlcNAc2-Man6 or GlcNAc2-Man5, or GlcNAc2-Mana.
The complex type glucan (s) are biantennaries and have a basic structure GlcNAc2-Mana to which are possibly associated residues of xylose (Xyl), fucose (Fuc) and possibly galactose (Gal) or N-acetyiglucosamine (GlcNAc). They are normally free of sialic acid residues, this compound not previously identified in plant cells.
Preferably, the complex glycan is of the type "phytohemagglutinin" (PHA) consisting of a structure "core" GlcNAc2Man3 whose mannose linked in ~ i carries a residue of ~ i 1,2-xylose and of which the proximal GlcNAc carries a residue of 1.3 fucose. This kind of structure is common for localization glycoproteins vacuolar or extracellular. The fucose residue has 1,3-liê may possibly be absent.
The complex glycan can also be of the type "Laccase" composed of a basic structure GlcNAc2 (Fuc) Man3 (Xyl) associated with two side chains.
Each side chain consists of a residue of (3 1,2 GlcNAc to which is bound a residue of 1,6 fucose and a residue of (3 1,4 galactose.
The glycoprotein of the invention can be glycosylated at three natural sites or only at one or two of them. Among the variants preferred, mention may be made of glycoproteins carrying exclusively polymannosidic glycans. This guy glycosylation exists in identical form in the plant and animal. Therefore, the appearance adverse reaction is avoided. This type of glycosylation is achieved through the use of an N-terminal signal peptide in association with a endoplasmic retention signal.
Mention may also be made of the glycoproteins carrying exclusively complex type glycans, for example example two or three glycans of 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 molecule natural with 11% glycosylation.
The composition of glycans of glycoprotein can be determined by digestion of oligosaccharides by N-glycanase, as described by Tuchiya K. et al. (1992).
The glycoprotein of the invention is capable induce the formation of neutralizing antibodies and protective immunity against the rabies virus or against a virus related to the rabies virus.
The protective character of glycoprotein is evidenced by the administration to an animal of glycoprotein-containing vaccine, followed by a virulent test with rabies virus. The rate 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 a or more of the addressing signals described above above. When it comes to mature glycoprotein, the only peptide signal remaining is the signal of endoplasmic retention of HKDEL type, the others addressing signals being cleaved in the cell. The glycoprotein with a retention signal endoplasmic has a molecular weight greater than 66 kDa due to the presence of amino acids additional.
The protein part of the glycoprotein the invention may correspond to that of the natural glycoprotein, or it can be a protein "analogous" to natural protein. In the context of the invention, an "analogous" protein means a protein that has at least 90%
of homology with the reference molecule. It is for example a rabies glycoprotein, some of which amino acids have been replaced, deleted or inserted.
The modifications take place preferably on the N- side.
terminal of the molecule. Analogous proteins are also insoluble, recognized by antibodies specific to rabies glycoprotein or to a rabies virus-like virus capable induce the formation of neutralizing antibodies.
The invention also relates to nucleic acids, RNA or DNA, used in the production of glycoprotein. In particular, the invention relates to a nucleic acid having a coding sequence chimeric comprising on the one hand a sequence coding for mature protein G of rabies virus or a virus related to the rabies virus, or for a protein analog, and on the other hand, a sequence encoding an N-terminal signal peptide other than the one naturally associated with the virus, or the sequences complementary to the above coding sequences.
The regulatory sequences described above, as well as the sequences coding for the signals addresses are, where applicable, also included in the nucleic acid molecule.

WO 97! 43428 PCT / FR97 / 00827 The invention further relates to the vectors and plasmids used for the introduction of the acid nucleic acid in plant cells. I1 can be of Actrobacterium Ti plasmids, or of viral vectors such as Geminiviruses or CaMV.
The invention also relates to plant cells transformed and able to produce glycoprotein of the invention. These cells are in the form of cell culture, as noted above, or do part of transgenic or chimeric plants.
As suitable plants, mention may be made of:
Angiosperms including monocots and dicotyledons. More particularly, we can cite the tobacco, species belonging to botanical families such as legumes (e.g. beans, peas, etc.), crucifers (for example cabbage, radish, rapeseed etc ...), nightshade (for example tomatoes, potatoes, etc.), cucurbits (e.g. melon, zucchini, cucumber), chenopodiaceae (e.g.
beetroot), umbellifers (e.g.
carrots, celery, etc.). We can also cite cereals such as wheat, corn, barley, triticale and rice, and oilseeds such as sunflower and soy.
The invention also relates to the seeds of transgenic plants capable of producing the rabies G glycoprotein, as well as their descendants.
The invention also relates to the use of the glycoprotein G as a therapeutic product, in particular as a vaccine against rabies infection, or by related viruses.
In particular, the invention relates to a composition pharmaceutical including.
- one or more glycoproteins) according to the invention, or - cells according to the invention, or - all or part of a plant according to the invention, in combination with an acceptable excipient from the point physiological point of view.
The pharmaceutical composition contains at least 1 ~ cg of glycoprotein. For example, it contains 1 ~ .g â
1 mg, or preferably from 10 ~ g to 200 ~ Sg, or preferably 25 to 100 mg, glycoprotein purified, per dose, for example 200 or 300 ~ g. If the glycoprotein is used in a form other than purified, for example in the form of extract of the plant, or the plant itself, the amount administered should be adjusted so that the dose of active ingredient reaches at least 100 ~ g.
The excipient used in the composition is a excipient normally used for vaccines rabies, for example alumina gel.
Normally, the vaccine is formulated for a oral or parenteral administration. I1 can also take the form of a food product made from the plant material of the invention.
The invention also includes a method to immunize mammals, including humans, against infection with this rabies virus, or with a virus related to rabies virus, including administration of a glycoprotein of the invention, or a plant material comprising this glycoprotein.
Various aspects of the invention are illustrated in the figures.
Figure 1 Western Blot of Tobacco Leaf Proteins transformed with cDNA of glycoprotein G
rabic (built pBIOC110 = PPS-RabG) 12% gel.

Track 1. Unprocessed tobacco leaves;
Track 2. Tobacco leaves processed with pBIOC110;
Track 3. Reference rabies G glycoprotein;
Track 4. Molecular weight marker.
Figure 2.
Western Blot of Tobacco Leaf Proteins processed with pBIOC110 (PPS-RabG) and seeds rapeseed transformed with pBIOC114 (PPS-RabG).
10% gel.
Track 1. Rabies G glycoprotein reconstruction reference in tobacco juice;
Track 2. Rapeseed processed with pBIOC114;
Track 3. Unprocessed rapeseed;
Track 4. Tobacco leaves processed with pBIOC110;
Track 5. Unprocessed tobacco leaves;
Track 6. Reference rabies G glycoprotein;
Track 7. Molecular weight marker.
Figure 3.
Western Corn Call Protein Blot transformed with pBI0C116 (PS-RabG).
10% gel Track 1. Reference rabies G glycoprotein;
Track 2. Molecular weight marker;
Track 3. Rabies G glycoprotein reconstruction reference in corn callus juice;
Track 4. Corn calluses processed with pBIOC116;
Track 5. Unprocessed corn calluses.
Figure 4.
8% gel Track 1. Rabies G glycoprotein reconstruction reference in tobacco leaf juice;
Track 2. Unprocessed tobacco leaf;

Track 3. Tobacco leaf processed with pBIOC110 (PPS-RabG);
Track 4. Tobacco leaf processed with pBIOC104 (PS-RabG);
Track 5. Molecular weight marker.
EXAMPLES
A. EXAMPLES OF MOLECULAR CONSTRUCTIONS
I. CONSTRUCTION OF CHEMICAL GENES ENCODING FOR
RECOMBINANT PROTEIN OF G RABIC GLYCOPROTEIN AND
ALLOWING EXPRESSION IN SHEETS AND
TOBACCO SEEDS.
The expression in tobacco leaves of the gene viral encoding rabies G glycoprotein (RabG) a need the following regulatory sequences.
1. a constituent promoter.
- or the double constitutive promoter 35S (pd35S) CaMV (cauliflower mosaic virus). II
corresponds to a duplication of sequences activating the transcript located upstream of the TATA element of natural 35S promoter (Kay et al., 1987).
- either the chimeric super-promoter (pSP; Ni et al., 1995). I1 consists of the merger of the triple repetition of an activating element transcription of the octopine gene promoter synthase of Agrobacterium tumefaciens, an element transcriptional activator of the gene promoter mannopine synthase and the mannopine synthase promoter Agrobacterium tumefaciens;
2. the transcription terminator sequence, polyA 355 terminator, which corresponds to the 3 'region non-coding for the double-stranded DNA virus sequence circular of the cauliflower mosaic producing the transcribed 35S (Franck et al., 1980).
The constructions of the different plasmids via the use of recombinant DNA techniques (Sambrook et al., 1989) derive from pHIOC4. This binary plasmid derived from pGA492 (An, 1986) which contains between the right and left borders, from plasmid pTiT37 Agrobacterium tumefaciens, on its transfer DNA, the following sequences.
- the constitutive promoter of the gene nos coding for nopaline synthase (Depicker et al., 1982), coding sequence for the nptII gene coding for neomycin phosphotransferase II (Berg and Berg, 1983) deleted from the region of the first 8 amino acids including the codon ATG methionine initiator and fused to the sequence of the first 14 amino acids in the coding sequence for gene nos (Depicker et al., 1982), the coding sequence of the nos gene devoid of the region of the first 14 amino acids, the terminator nos (Depicker et al., 1982), a polylinker (HindIII-XbaI-SacI-HpaI-KpnI-ClaI-BgIII) preceding the cat gene coding for chloramphenicol acetyltransferase (Close and Rodriguez, 1982) and the terminating sequences of gene 6 of plasmid pTiA6 from Agrobacterium tumefaciens (Liu et al., 1993).
To eliminate almost all of the coding sequence for the cat gene, the plasmid pGA492 was doubly digested with SacI (restriction site of polylinker) and by ScaI (restriction site present in the sequence of the cat gene) then subjected to the action of the T4 DNA polymerase enzyme (Biolabs) according to manufacturer's recommendations. Plasmid ligation modified (20 ng) was performed in a medium 10 ~ reaction, 1 containing 1 ~ 1 of T4 DNA buffer ligase x 10 (Amersham); 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours.

Bacteria, Escherichia coli DHSa rendered previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 ~ .g / ml tetracycline, was extracted according to the alkaline lysis method (Birnboim and Dôly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
Then, the HindIII restriction site of DNA
plasmid of the retained clone was modified in a site of EcoRI restriction using a HindIII adapter -Phosphorylated EcoRI (Stratagene Cloning Systems). For make this modification, 500 ng of plasmid DNA
of the clone retained were digested with HindIII, dephosphorylated by the enzyme alkaline phosphatase calf intestine (Boehringer Mannheim) according to manufacturer's recommendations and co-precipitated in presence of 1500 ng of HindIII-EcoRI adapter DNA, 1/10 volume of 3M sodium acetate pH 4.8 and 2.5 volumes absolute ethanol at -80 ° C for 30 min. After centrifugation at 1200og for 30 min., DNA
precipitate was washed with 70% ethanol, dried, taken up in 8 ~ 1 of water, brought to 65 ° C for 10 min., then ligated in the presence of 1 ~ 1 of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) to 14 ° C for 16 hours. After inactivation of T4 DNA
ligase at 65 ° C for 10 min., the reaction mixture of ligation was digested with EcoRI, purified by 0.8% agarose gel electrophoresis, 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 120008 for 30 min., washed with ethanol 70%, dried, then ligated as described above.
Bacteria, Escherichia coli DHSa rendered previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 ~ cg / ml tetracycline, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with HindIII
and EcoRI in particular. The resulting binary plasmid, which only have the last 9 amino acids of the coding sequences of the cat gene and whose EcoRI site is single, was called pBIOC4.
The expression cassette, consisting of the promoter pd35S and the polyA 35S terminator, was isolated at from plasmid pJIT163A. Plasmid pJIT163A
derived from plasmid pJIT163 which itself is derived from plasmid pJIT60 (Guerineau and Mullineaux, 1993). The plasmid pJIT163 has an ATG codon between the sites HindIII and Sali from the polylinker. To delete this ATG
and obtain the plasmid pJIT163 ~, the plasmid DNA
pJIT163 was doubly digested with HindIII and Sali, purified by electrophoresis on 0.8% agarose gel, electro-eluted (Sambrook et al., 1989), precipitated in presence of 1/10 of 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 Klenow enzyme (Biolabs). according to the recommendations of manufacturer, deproteinized by extraction with 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) then 1 chloroform volume: isoamyl alcohol (24: 1), precipitated in the presence of 1/10 volume of 3M acetate sodium pH4.8 and 2.5 volumes of absolute ethanol at -80 ° C for 3o min., Centrifuged at 12000g for 30 min., washed with 70% ethanal, dried, and finally, ligated in presence of 1 ~ cl of T4 DNA ligase buffer x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) to 14 ° C for 16 hours. Bacteria, Escherichia coli DH5a previously made competent, have been transformed (Hanahan, 1983). The plasmid DNA of clones obtained, selected from 50 ~ cg / ml ampicillin, was extracted using the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by digestion enzymatic by restriction enzymes. To isolate the expression cassette consisting of the pd35S promoter and of the polyA 35S terminator (SacI-XhoI fragment), the plasmid DNA of the retained clone pJIT1630 was digested by SacI and XhoI. The SacI-XhoI 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 3M volume sodium acetate pH4.8 and 2.5 volumes of ethanol absolute at -80 ° C for 30 min., centrifuged at 12000g for 30 min., washed with 70% ethanol, dried, then subject to the action of the enzyme Mung Bean Nuclease (Biolabs) according to the manufacturer's recommendations. This purified insert (200 ng) was cloned into DNA
pBIOC4 plasmid (20 ng) digested with EcoRI, treated by the enzyme Mung Bean Nuclease and dephosphorylated by the calf intestine alkaline phosphatase enzyme (Boehringer Mannheim) according to the recommendations of maker. The ligation reaction was carried out in 20 ~ tl in the presence of 2 ~ 1 of T4 DNA ligase x 10 buffer (Amersham), 2 ~ cl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DH5a returned previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 ~, g / ml tetracycline, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pHIOC21.

To obtain a binary plasmid similar to pBIOC21 but whose promoter pd35S has been replaced by the pSP promoter, the PwII - SalI fragment subjected to the action of Klénow containing the promoter pSP, was isolated from the plasmid pBISN1 (Ni et al., 1995), purified by electrophoresis on 1% agarose gel, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried and ligated to DNA
plasmid of pBIOC81 doubly digested with KpnI and EcoRI subjected to the action of T4 DNA polymerase and dephosphorylated by the enzyme alkaline phosphatase calf intestine (Boehringer Mannheim) according to manufacturer's recommendations. Plasmid pBIOC81 corresponds to pB10C21 from which the Xbal site has been deleted.
To do this, the plasmid pB1oC21 was digested with XbaI then submitted to the action of Klenow and bound by action of T4 DNA ligase.
Ligation was carried out with 20 ng of the vector dephosphorylated described above and 200 ng of fragments of DNA carrying pSP described above in a medium 20 μl reaction in the presence of 2 ~ cl of T4 buffer DNA ligase x 10 (Amersham) and 2 ~ Cl of 50% polyethylene glycol 8000, and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa previously rendered have been transformed (Hanahan, ~ 1983).
The plasmid DNA of the clones obtained, selected on 12 ~ g / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes of restriction. The resulting plasmid was called pBIOC82.
Rabies G glycoprotein (RabG), isolated from of the ERA strain, is naturally synthesized under form of a precursor. The mature protein RabG is made up of 505 amino acids. Its signal peptide is composed of 19 amino acids.
CDNA encoding the envelope glycoprotein rabies virus, strain ERA (A. Anilionis et al., Nature, 1981, 294, 275-278 and MP Rieny et al., Nature, 1984, 312, 163-166) was amplified by PCR
according to the usual methods, using the two following oligonucleotides Oligo 1 (30 mer) 5 'AAA GGA TCC ATG GTT CCT CAG GCT CTC
CTG 3 ' Oligo 2 (30 mer) 5 'AAA CTG CAG TCA CAG TCT GGT CTC ACC
CCC 3 ' The 1.69 kb PCR fragment was digested with BamHI
and Pstl then cloned into the vector pBlueScript previously digested with BamHI and Pstl, thus giving the plasmid pPB010.
The complementary DNA of the RabG precursor is contained in the plasmid pPBO10. I1 was used for construction of the binary plasmids pBIOC104 and pBIOC105 containing the sequence coding for PS-RabG, pBIOC107 and pBIOC108 containing the sequence coding for BL-RabG and pBIOC110 and pBIOCÜi containing the sequence encoding PPS-RabG o ~ 1 the sequence encoding RabG
is preceded by the sequence coding for a peptide signal, PS and BL, and for an N-terminal prepropeptide (PPS i.e. a signal peptide followed by sequences N-terminal vacuolar addressing) of plant origin respectively. The PS and PPS sequences, made up 23 and 37 amino acids respectively, are those a reserve protein from the tuberous roots of Yam . 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).
In order to obtain a sequence coding for the rabies G glycoprotein with only the first stop codon, the cDNA RabG has been modified at its end 3 'by PCR directed mutagenesis using 2 oligodeoxynucleotides, 5 'CTC AGG AGT TGA CTT GGG 3' (containing the unique HincII site in the plasmid pPB010) and 5 'CCG GAT CCT CAC AGT CTG GTC TCA C 3' (containing the unique BamHI site in the plasmid pPB010).
PCR amplification of the HincII-BamHI fragment a been performed in 100 ~ 1 of dream medium including 10 ~ C1 of Taq DNA polymerase buffer x10 (500 mM KC1, 100 mM Tris-HC1, pH9.0 and 1% Triton x100), 6 ~ 1 of 25 mM MgCl2, 3 ass of 10 mM dNTP (dATP, dCTP, dGTP and dTTP), 100 pM of each of the 2 oligodeoxynucleotides described above, 5 ng DNA
matrix (vector pPB010), 2.5 U of Taq DNA polymerase (Promega) and 2 drops of petroleum jelly oil. DNA has 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 extension to 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 were precipitated in the presence of 1/10 of 3M volume sodium acetate pH4.8 and 2.5 volumes of ethanol absolute at -80 ° C for 30 min., centrifuged at 12000g for 30 min., washed with 70% ethanol, dried and digested by the 2 restriction enzymes HincII and BamHI. The digested ADrt fragments from PCR amplification were purified by 2% agarose gel electrophoresis, 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 12000g for 30 min., washed at 70% ethanol, dried, then ligated to plasmid DNA
pPB010 doubly digested with HincII and BamHI, purified by electrophoresis on 0.8% agarose gel, electroeluted, subjected to alcoholic precipitation, dried. The ligation was carried out with 100 ng ~ of vector and 50 ng of digested DNA fragments from PCR amplification, described above, in a medium reaction of 10 ass in the presence of 1 bel of T4 buffer DNA ligase x 10 (Amersham) and 2.5 U of T4 DNA enzyme ligase (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5oc previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 50 ~ g / ml ampicillin, was extracted according to the method alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes from restriction. Some of the clones retained were verified by sequencing using the sequencing kit T7TM marketed by Pharmacia according to the dideoxynucleotides (Sanger et al., 1977)) The plasmid resulting was called pHIOCIO2.
at. CONSTRUCTION OF BINARY PHASMIDES pHIOCi04 AND
pBIOC105 CONTAINER PS-RabG.
The plasmid pBIOC102 was digested doubly by BglII and HindIII in order to delete the coding sequence for the natural signal peptide of glycoprotein G
rabies and the first 9 amino acids of the protein Mature RabG (KFPIYTIPDKL). This sequence was 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 ("first PS-9 codons of mature RabG "). The sequence" PS-9 prime mature RabG codons "was amplified by PCR from of the plasmid pMAT103 (Matuoka and Nakamura, 1991) to using the 2 oligodeoxynucleotides, 5 ' cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3 ' (containing the unique BglII and EcoRI sites additional in plasmid pPB010) 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 IündIII site in plasmid pPB010) following the protocol PCR amplification described above. Temperature hybridization was 40 ° C. After double digestion enzymatic by BglII and HindIII, the DNA fragments from PCR amplification were purified by 2% agarose gel electrophoresis, 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 12000g for 30 min., washed with ethanol 70%, dried, then ligated to the plasmid DNA of pBIOC101 doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried. The ligation was performed with 100 ng of the vector and 50 ng of fragments of digested DNA from PCR amplification, described below above, in a reaction medium of 10 ~ cl in the presence 1 ~ C1 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. Bacteria, Escherichia coli DHS ~ c rendered previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 ~, g / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones selected have been verified by sequencing using the T7TM sequencing marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
The sequences coding for PS and mature RabG were cloned while keeping their reading phases open (i.e., such that they constitute a open single reading phase). The sequence of cleavage between PS and mature RabG sequences is Ser-Lily. The resulting plasmid was called pHIOC103.
From pBIOC103, the EcoRI fragment carrying the PS-RabG sequence was isolated by enzymatic digestion by EcoRI, purified by agarose gel electrophoresis 0.8%, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried, and ligated to DNA
plasmid of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase intestine of calf (Hoehringer Mannheim) according to manufacturer's recommendations. The ligation was performed with 100 ng of dephosphorylated pBIOC21 vector and 50 ng of DNA fragments containing PS-RabG, described above, in a reaction medium of 10 presence of 1 μl of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 12 ~ cg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes of restriction. The resulting clone was named pBIOC104.
The nucleic acid sequence of the fragment coding for the PS-RabG recombinant protein has been verified by sequencing using the T7TM sequencing kit marketed by Pharmacia using the dideoxynucleotides (Sanger et al., 1977). DNA

binary vector plasmid pBIOC104 has been introduced by direct transformation in the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the selected clone has has been verified by enzymatic digestion of DNA
plasmid introduced.
Obtaining pBIOCIOS is similar to obtaining pBIOC104 except that the EcoRI fragment carrying the PS-RabG sequence was cloned to the XbaI site of pBIOC82.
The EcoRI sites of the insert and XbaI of the vector were subject to the action of Klenow. Usual methods clones have been applied.
b. CONSTRUCTION OF BINARY PLASMIDS pBIOC107 AND
pBIOC108 CONTAINER BL-RabG.
The plasmid pBIOC103 was digested doubly by BglII and HindIII in order to delete the coding sequence for the signal peptide PS and the first 9 acids amines of the mature RabG protein (KFPIYTIPDKL). This sequence has been replaced by that coding for BL 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 RabG protein mature ("BL-9 first codons of mature RabG" j. La sequence "BL-9 first codons of mature RabG" has been amplified by PCR from the plasmid BLc3 (Lerner and Raikhel, 1989) using the 2 oligodeoxynucleotides,

5 'ggagatctgaattcaacaATG AAG ATG ATG AGC ACC AGG 3' (containing the unique BglII and EcoRI sites additional in 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 pBIOC102) following the protocol PCR amplification described above. Temperature hybridization was 65 ° C. After double digestion enzymatic by BglII and HindIII, the DNA fragments from PCR amplification were purified by 2% agarose gel electrophoresis, 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 12000g for 30 min., washed with ethanol 70%, dried, then ligated to the plasmid DNA of pBIOCl02 doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried. The ligation was performed with 100 ng of the vector and 50 ng of fragments of digested DNA from PCR amplification, described below above, in a reaction medium of l0 ~ cl in the presence 1 ~, 1 of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Hscherichia coli DHSa rendered previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 ~ cg / ml ampicillin, has been extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones selected have been verified by sequencing using the T7TM sequencing marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
The sequences encoding BL and mature RabG were cloned while keeping their reading phases open (i.e., such that they constitute a open single reading phase). The sequence of cleavage between the BL and mature RabG sequences is Ala-Lily. The resulting plasmid was called pHIOCIO6.
From pBIOC106, the EcoRI fragment carrying the BL-RabG sequence was isolated by enzymatic digestion by EcoRI, purified by agarose gel electrophoresis 0.8%, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried, and ligated to DNA
plasmid of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase calf intestine (Boehringer Mannheim) according to manufacturer's recommendations. The ligation was performed with 100 ng of dephosphorylated pBIOC21 vector and 50 ng of DNA fragments containing BL-RabG, described above, in a reaction medium of 10 μl in presence of 1 ~ 1 of T4 DNA ligase buffer x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia soli DHSa previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 12 ~ cg / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes of restriction. The resulting clone was named pBIOCIO7.
The nucleic acid sequence of the fragment coding for the BL-RabG recombinant protein has been verified by sequencing using the T7TM sequencing kit marketed by Pharmacia using the dideoxynucleotides (Sanger et al., 1977). DNA
binary vector plasmid pBIOC107 has been introduced by direct transformation in the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the selected clone has been verified by enzymatic digestion of DNA
plasmid introduced.
Obtaining pHIOCIOB is similar to that of pBIOC107 except that the EcoRI fragment carrying the BL-RabG sequence was cloned at the XbaI site of pBIOC82.
The EcoRI sites of the insert and XbaI of the vector were subject to the action of Klenow. Usual methods clones have been applied.
vs. CONSTRUCTION OF BINARY PLASMIDS pBI0C110 AND
pHIOClll CONTAINING PPS-RabG.
The plasmid pBIOC102 was digested doubly by BglII and HindIII to suppress the coding sequence for the natural signal peptide of glycoprotein G
rabies and the first 9 amino acids of the protein Mature RabG (KFPIYTIPDKL). This sequence has been replaced by that coding for the signal peptide PPS
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 this code for the first 9 codons of the mature RabG protein ("first PS-9 mature RabG codons "). The sequence" Prime PPS-9 mature RabG codons "was amplified by PCR from of the plasmid pMAT103 (Matuoka and Nakamura, 1991) to using the 2 oligodeoxynucleotides, 5 ' cgagatctgaattcaacaATG AAA GCC TTC ACA CTC GC 3 ' (containing the unique HglII and EcoRI sites additional in plasmid pPB010) 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 plasmid pPH010) following the protocol PCR amplification described above. Temperature hybridization was 40 ° C. After double digestion enzymatic by BglII and HindIII, the DNA fragments from 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 12000g for 30 min., washed with ethanol 70%, dried, then ligated to the plasmid DNA of pBIOC101 doubly digested with BglII and HindIII, purified by electrophoresis on 0.8% agarose gel, electroêluê (Sambrook et al., 1989), subject to alcoholic precipitation, dried. The ligation has been performed with 100 ng of the vector and 50 ng of fragments of digested DNA from PCR amplification, described below above, in a reaction medium of 10 ~, 1 in the presence 1 ~ C1 of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa rendered previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 ug / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. Some of the clones selected have been verified by sequencing using the T7TM sequencing marketed by Pharmacia according to the dideoxynucleotide method (Sanger et al., 1977).
The sequences encoding PPS and mature RabG were cloned while keeping their reading phases open (i.e., such that they constitute a open single reading phase). The sequence of cleavage between the mature PPS and RabG sequences is Ala-Lily. The resulting plasmid was called pBIOCIO9.
From pBIOC109, the EcoRI fragment carrying the PPS-RabG sequence was isolated by enzymatic digestion by EcoRI, purified by agarose gel electrophoresis 0.8%, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried, and ligated to DNA
plasmid of pBIOC21 digested at the EcoRI site and dephosphorylated by the enzyme alkaline phosphatase calf intestine (Boehringer Mannheim) according to manufacturer's recommendations. The ligation was performed with 100 ng of dephosphorylated pBIOC21 vector and 50 ng of DNA fragments containing PPS-RabG, described above, in a reaction medium of 10 ~ C1 in presence of 1 ~ 1 of T4 DNA ligase buffer x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSoc previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 12 ~, g / ml tetracycline, was extracted according to the method of alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes of restriction. The resulting clone was called pBIOC110.
The nucleic acid sequence of the fragment coding for the PPS-RabG recombinant protein has been verified by sequencing using the T7TM sequencing kit marketed by Pharmacia using the dideoxynucleotides (Sanger et al., 1977). DNA
binary vector plasmid pBIOCllo has been introduced by direct transformation in the LBA4404 strain of Agrobacterium tumefaciens according to the method of Holsters et al. (1978). The validity of the selected clone has been verified by enzymatic digestion of DNA
plasmid introduced.
Obtaining pBIOCili is similar to that of pBIOC110 except that the EcoRI fragment carrying the PS-RabG sequence was cloned at the XbaI site of pBIOC82.
The EcoRI sites of the insert and XbaI of the vector were subject to the action of Klenow. Usual methods cloning has been applied.

II. CONSTR ~ CTION OF CHEMICAL GENES ENCODING PO
RECOMBINANT PROTEIN OF GLYCOPROTEIN G RABIQüE AND
ALLOWING AN EBPRESSION IN THE COLZA SEEDS.
The expression in the rapeseed of the gene viral encoding rabies G glycoprotein (RabG) a need the following regulatory sequences 1. one of the promoters described below.
- the pCRU promoter corresponding to the 5 'region non-coding gene for the reserve protein seeds, radish CRUCIFERIN A (Depigny-This and al., 1992), and allowing a specific expression in seeds;
- the pGEAI promoter corresponding to the 5 'region non-coding gene for the reserve protein seeds, GEA1 of Arabidopsis thaliana (Gaubier et al., 1993), and allowing a specific expression in the seeds;
- the pGEA6 promoter corresponding to the 5 'region non-coding gene for the reserve protein of seeds, GEA6 of Arabidopsis thaliana (Gaubier et al., 1993), and allowing a specific expression in the seeds;
2. one of the terminator sequences described above below.
- the transcription terminator sequence, polyA 355 terminator, which corresponds to the 3 'region non-coding for the double-stranded DNA virus sequence circular of the cauliflower mosaic producing the transcribed 35S (Franck et al., 1980);
- the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 'region non-coding of the plasmid nopaline synthase gene Ti of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).

WO 97! 43428 PCT / FR97 / 00827 To obtain a binary plasmid similar to pBIOC21 but whose promoter pd35S has been replaced by the pCRU promoter, the "EcoRI fragment treated with Klenow - BamHI ", containing the pCRU promoter, was isolated from the plasmid pBI221-CRURSP. The plasmid pBI221-CRURSP is derived from pBI221 (marketed by Clontech) by replacing the promoter 35S with the pCRU promoter.
The fragment "EcoRI treated with Klenow - BamHI"
carrying the pCRU promoter was purified by 0.8% agarose gel electrophoresis, electroeluted (Sambrook et al., 1989), subject to precipitation alcoholic, dried and ligated to the plasmid DNA of pJITl63 (described in paragraph I.) digested by KpnI, treated with T4 DNA Polymerase (Biolabs) according to manufacturer's recommendations, then digested with BamHI, purified by electrophoresis on 0.8% agarose gel, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried and dephosphorylated by the calf intestine alkaline phosphatase enzyme (Boehringer Mannheim) according to the recommendations of maker. The ligation was carried out with 20 ng of dephosphorylated vector described above and 200 ng of DNA fragments "EcoRI treated with Klenow - BamHI"
described above in a reaction medium of 20 ~ cl in the presence of 2 ~ C1 of T4 DNA ligase x 10 buffer (Amersham), 2 ~ 1 of 50% polyethylene glycol 8000-and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSoc rendered previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 ~ cg / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. The resulting plasmid was called pHIOC27.

The expression cassette, consisting of the promoter pCRU and the polyA35S terminator was isolated from of pBIOC27 by total digestion XhoI followed by partial EcoRI digestion. It was purified by 0.8% agarose gel electrophoresis, electro-eruated (Sambrook et al., 1989), subject to precipitation alcoholic, dried, Klenow-treated (Biolabs) according to manufacturer's recommendations and ligated to DNA
plasmid of pBIOC4 at the EcoRI site treated with IClenow and dephosphorylated by the enzyme alkaline phosphatase calf intestine (Boehringer Mannheim) according to manufacturer's recommendations. The ligation was performed with 20 ng of the dephosphorylated vector described above above and 200 ng of XhoI-EcoRI DNA fragments described above in a reaction medium of 20 ~ l in presence of 2 ~ Cl of T4 DNA ligase buffer x 10 (Amersham), 2 ~, 1 of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DH5a returned previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 12 ~ cg / ml tetracycline, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes. ~ The resulting plasmid has been called pBIOC28.
To obtain the binary plasmid pBIOC91, -la "pGEAID - tNOS" expression cassette, isolated from of pHSII-pGEAID, was introduced into the plasmid binary pSCVl.2 itself obtained by cloning the fragment HindIII carrying the expression cassette "p35S - nptII
- tNOS "described by Fromm et al. (1986) at the HindIII site of pSCV1 built by Edwards GA in 1990 following the usual cloning methods.
The plasmid pBSII-pGEAID was obtained in two steps .

- on the one hand, the SacI-EcoRI fragment carrying tNOS
(terminator of the nopaline synthase gene) of Agrobacterium tumefaciens, treated with the T4 DNA enzyme polymerase (Biolabs) according to the recommendations of manufacturer and purified, was cloned at EcoRV ~ site pBSIISK + marketed by Stratagene, dephosphorylated by the enzyme alkaline phosphatase from calf intestine (Boehringer Mannheim) according to the recommendations of maker. The ligation was carried out with 20 ng of dephosphorylated vector and 200 ng of DNA fragments containing tNOS described above in a medium 20 ~ cl reaction in the presence of 2 μl of T4 buffer DNA ligase x 10 (Amersham), 2 ~, 1 50%
polyethylene glycol 8000 and 5 U of T4 DNA enzyme ligase (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DHSa previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 50 ~ cg / ml ampicillin, was extracted according to the method alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes from restriction. Some of the clones selected were verified by sequencing using the sequencing kit T7TM marketed by Pharmacy according to the method of dideoxynucleotides (Sanger et al., 1977). The plasmid resulting was called pBSII-tNOS.
- on the other hand, the fragment carrying pGEAID digested doubled with HindIII treated with the Klenow enzyme and BamHI, purified, was cloned at "XbaI treated sites Klenow and BamHZ "of the plasmid pBSII-tNOS.
ligation was. performed with 100 ng of the vector described above and 50 ng of DNA fragments described above above in a 10 μl reaction medium in the presence 1 ~ 1 of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa previously made competent, have been transformed (Hanahan, 1983). The plasmid DNA of clones obtained, selected on 50 ~ cg / ml ampicillin, was extracted using the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by digestion enzymatic by restriction enzymes. The The resulting plasmid was called pBSII-pGEAlD.
Then, the expression cassette "pGEAlD - tNOS"
carried by the XbaI-HindIII fragment treated with Klenow, was cloned at the SmaI site of pSCVl.2 dephosphorylated by the calf intestine alkaline phosphatase enzyme (Boehringer Mannheim) according to the recommendations of maker. The ligation was carried out with 20 ng of dephosphorylated pSCVl.2 and 200 ng of fragments carrying the expression cassette "pGEAID - tNOS", in a medium reaction of 20 ~ 1 in the presence of 2 here of T4 buffer DNA ligase x 10 (Amersham), 2 ~ 1 of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa previously made competent, have been transformed (Hanahan, 1983). The plasmid DNA of clones obtained, selected on 50 ~ g / ml ampicillin, was extracted using the alkaline lysis method (Birnboim and Doly, 1979} and analyzed by digestion enzymatic by restriction enzymes. The plasmid resulting was called pHIOC91.
To obtain the binary plasmid pBIOC92 similar to pBIOC21 but whose promoter pd35S has been replaced by the promoter pGEA6D, the EcoRI fragment -BamHI treated with Klenow, containing the promoter pGEA6, was isolated from the plasmid pGUS2-pGEA6.
The pGUS-2-pGEA6 clone derived from pBI221 by replacement of p35S with the promoter pGEA6. he has 2 ATGs in phase. GEA6 (Em6) gene ATG and ATG of the gus gene. The GEA6 gene ATG has been destroyed. 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 '. Hybridization temperature has been adapted.
The PCR amplified fragment was digested with AccI and BamHI, purified by gel electrophoresis 2% agarose, electroeluted (Sambrook et al., 1989), precipitated in the presence of 1/10 volume of 3M acetate sodium pH4.8 and 2.5 volumes of absolute ethanol â -80 ° C for 30 min., Centrifuged at 1200og for 30 min., washed with 70% ethanol, dried, then ligated to DNA
pGUS-2-GEA6 plasmid doubly digested with AccI
and BamHI, purified by agarose gel electrophoresis 0.8%, electroeluted (Sambrook et al., 1989), subject to the alcoholic precipitation, dried. The ligation was performed with 100 ng of the vector described above and 50 ng of digested DNA fragments from amplification PCR described above in a reaction medium of 10 µl in the presence of 1 ~ 1 T4 DNA ligase x 10 buffer (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected for safety ~ g / ml ampicillin, was extracted according to the method alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes from restriction. Some of the clones retained were verified by sequencing using the sequencing kit T7TM marketed by Pharmacia according to the dideoxynucleotides (Sanger et al., 1977). The clone resulting was called pGUS-2-pGEA6D.

WO 97/43428 PCT / F'R97 / 00827 The EcoRI - BamHI fragment carrying the promoter pGEA6D isolated from pGUS-2-pGEA6D, treated with Klenow according to manufacturer's recommendations (Biolabs), was purified by gel electrophoresis 0.8% agarose, electroeluted (Sambrook et al., 1989), subjected to alcoholic precipitation, dried and ligated to Klhoow-treated XhoI site of DNA
modified pBIOC21 plasmid. Plasmid pBIOC21 modified was obtained by double digestion with HindIII
treated with Klenow and KpnI from pHIOC21 to delete the fragment carrying the pd35S promoter and replace it with the KpnI-EcoRV fragment carrying the compound polylinker KpnI-XhoI-Sali-ClaI-HindIII-BamHI-SmaI-EcoRI- sites EcoRV from pBSIISK +.
The ligation was carried out with 20 ng of pBIOC21 modified dephosphorylated and 200 ng of DNA fragments EcoRI-BamHI, described above, in a medium reaction of 20 ~ 1 in the presence of 2 ~ 1 of T4 buffer DNA ligase x 10 (Amersham), 2 ~ cl of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa previously made competent, have been transformed (Hanahan, 1983). The plasmid DNA of clones obtained, selected from 12, ~ g / ml tetracycline, was extracted using the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by digestion enzymatic by restriction enzymes. The plasmid resulting was called pBIOC92.
Then, the expression cassette "pGEA6D - t35S" was was isolated from pBIOC92. This cassette of expression carried by the KpnI-EcoRV fragment treated with the T4 DNA polymerase enzyme (Biolabs) according to manufacturer's recommendations and purified, has been cloned at the SmaI site of pSCVl.2 dephosphorylated by the enzyme calf intestine alkaline phosphatase (Boehringer Mannheim) according to the manufacturer's recommendations. The ligation was carried out with 20 ng of pSCVl.2 dephosphorylated and 20o ng of fragments carrying the expression cassette "pGEAID - tNOS", in a medium reaction of 20 ~ 1 in the presence of 2 ~ 1 of T4 buffer DNA ligase x 10 (Amersham), 2 ~ 1 of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, ~ scherichia co 'DH5oc previously made competent, have been transformed (Hanahan, 1983). The plasmid DNA of clones obtained, selected on 50 ~ .g / ml ampicillin, was extracted using the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by digestion enzymatic by restriction enzymes. The plasmid resulting was called pBIOCiI2.
CONSTR ~ CTION OF BINARY PLASMIDS pHIOC113, pBIOC114 AND pBIOC115 CONTAINING PPS-RANG UNDER CONTROL
PROMOTER B ALLOWING EXPRESSION IN
BEMENCEB.
The plasmid pBIOC109, described previously in Ic, contains the EcoRI fragment carrying the sequence PPS-RabG.
This fragment was isolated by enzymatic digestion by EcoRI, purified by gel electrophoresis 0.8% agarose, electroeluted, subject to precipitation alcoholic, dried and ligated to pBIOC28, pBIOC91 et pBIOC112 digested with EcoRI and dephosphorylated to result in pBIOC113, pBIOCiI4 and pBIOC115 respectively.

III. CONSTRUCTION OF CHEMICAL GENES ENCODING POÜR TA
RECOMBINANT PROTEIN OF G RABIC GLYCOPROTEIN AND
ALLOWING EXPRESSION IN CORN SEEDS.
at. CONSTRUCTION OF PhABMIDEB pBIOC116 AND

CONSTITOTIVE IN CORN SEEDS.
The constitutive expression in the seeds of but of the viral gene coding for the glycoprotein G
rabies (RabG) required regulatory sequences following 1. one of the two promoters allowing a constitutive expression - active rice promoter followed by active intron of rice (PAR-IAR) ~ contained in the plasmid pActi-F4 described by McElroy et al. (1991);
- double constitutive promoter 35S (pd35S) of CaMV
(cauliflower mosaic virus). I1 corresponds to a duplication of the sequences activating the transcription located upstream of the TATA element of the 35S promoter natural (Kay et al., 1987);
2. one of the two terminators.
- the transcription terminator sequence, polyA 35S terminator, which corresponds to the 3 'region non-coding sequence of double-stranded DNA virus circular of the cauliflower mosaic producing the transcribed 35S (Franck et al., 1980);
- the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 'region non-coding of the plasmid nopaline synthase gene Ti of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
The plasmid pBIOC116 where the sequence coding for PS-RabG is under the control of PAR-IAR has been obtained by cloning of the EcoRI fragment carrying the sequence coding for PS-RabG at the "NcoI and Sali" sites from 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, then treated with the Klenow enzyme. The plasmid pBSII-pAR-IAR-tNOS has been doubly digested by Sali and NcoI, purified, treated with the enzyme Mung Bean Nuclease (Biolabs) and dephosphorylated by the enzyme phosphatase veal alkaline (Boehringer Mannheim) according to manufacturers' recommendations. The ligation was performed with 20 ng of the dephosphorylated vector and 200 ng DNA fragments containing the sequence coding for RS-LGC, described above, in a reaction medium of 20 ~, 1 in the presence of 2 ~ 1 of T4 DNA ligase x buffer lo (Amersham), 2 ~ 1 of 50% polyethylene glycol 8000 and 5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C
for 16 hours. Bacteria, Escherichia coli DHSa. previously made competent, have been transformed (Hanahan, 1983). The plasmid DNA of clones obtained, selected from 50 ~, g / ml ampicillin, was extracted using the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by digestion enzymatic by restriction enzymes. The resulting plasmid was called pBIOC116.
The plasmid pBSII-pAR-IAR-tNOS results from cloning to the sites "Eco0109I treated with KlEnow and KpnI" from pBSII-tNOS of the SnaBI-KpnI fragment carrying the sequence corresponding to "pAR-IAR-start of coding sequence for the gus "gene 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 10 ~ reaction medium, 1 digested in the presence of 1 ~ 1 of T4 DNA ligase x 10 buffer (Amersham) and 2.5 U
T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa rendered previously competent, have been transformed (Hanahan, 1983). The plasmid DNA of the clones obtained, selected on 50 ~ g / ml ampicillin, was extracted according to the alkaline lysis method (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with restriction enzymes.
Plasmid pBSII-tNOS was obtained by cloning to the dephosphorylated EcoRV site of pBSIISK + marketed by Stratagene, of the SacI-EcoRI fragment carrying the tNOS sequence isolated from pBI121 marketed by Clontech by double enzymatic digestion by SacI
and EcoRV, subjected to purification by electrophoresis on 2% agarose gel, and treated with the T4 DNA enzyme polymerase. Ligation was carried out with 2 ng of dephosphorylated vector and 200 ng of DNA fragments containing the sequence tNOS, described above, in a reaction medium of 20 ~, 1 in the presence of 2 ~ 1 of T4 DNA ligase x 10 buffer (Amersham), 2 ~ C1 50%
polyethylene glycol 8000 and 5 U of T4 DNA enzyme ligase (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DHSa previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 50 ~ .g / ml ampicillin, was extracted according to the method alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes from restriction.
The plasmid pHIOCiI7 where the sequence coding for PS-RabG is placed under control of pd35S has been obtained by cloning at the "KpnI and XbaI" sites of the plasmid pBSIISK + marketed by Stratagene, from the fragment KpnI-XbaI carrying the sequence corresponding to "pd35S-PS-RabG "isolated from pBIOC103. The ligation was performed with 100 ng of vector and 50 ng of fragments of DNA, described above, in a reaction medium of 10 ~ cl digested in the presence of 1 ~ cl of T4 DNA buffer ligase x 10 (Amersham) and 2.5 U of T4 DNA enzyme ligase (Amersham) at 14 ° C for 16 hours. The bacteria, Escherichia coli DH5a, previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 50 ~ .g / ml ampicillin, was extracted according to the method alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes from restriction.
b. CONSTRUCTION OF pBIOC118 AND PLASMIDS
pBIOC119 CONTAINING PS-RabG AND pS-RabG-RDEL
RESPECTIVELY AND ALLOWING ONE EXPRESSION IN
THE CORN SEED ALBUMEN.
The expression in the albumen of corn seeds of the viral gene coding for rabies glycoprotein G
(RabG) required the following regulatory sequences 1. the promoter of the corn yzein gene (pyzein) contained in the plasmid py63 (Reina et al., 1990, NAR, ~, 6426). Plasmid pY63 results from cloning of pyzeine at the HindIII and XbaI sites of a plasmid pUClB
containing, between its HindIII and EcoRI sites, the pBI221 "p35S-gus-tNOS" expression cassette marketed by Clontech. I1 allows an expression in the albumen of corn seeds.
2. the transcription terminator sequence, polyA NOS terminator, which corresponds to the 3 'region non-coding of the plasmid nopaline synthase gene Ti of Agrobacterium tumefaciens nopaline strain (Depicker et al., 1982).
The plasmid pBIOC118 or the sequence coding for PS-RabG is under the control of pyzeine, has been obtained by cloning at the "SacI sites treated with the enzyme T4 DNA polymerase and BamHI "from plasmid py63, from fragment "BamHI treated with Klenow - BglII" isolated at 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 ~ C1 in presence of 1 ~ 1 of T4 DNA ligase buffer x 10 (Amersham) and 2.5 U of T4 DNA ligase enzyme (Amersham) at 14 ° C for 16 hours. Bacteria, Escherichia coli DHSa previously rendered have been transformed (Hanahan, 1983).
The plasmid DNA of the clones obtained, selected from 50 ~, g / ml ampicillin, was extracted according to the method alkaline lysis (Birnboim and Doly, 1979) and analyzed by enzymatic digestion with enzymes from restriction. The resulting clone was named pBIOC118.
Plasmid pBIOC119 results from substitution of the BamHI-HincII fragment of pBIOCiI8 by the fragment BamHI-HincII carrying the sequence coding for KDEL
placed before the stop codon obtained by amplification PCR according to the methods described above. The 2 oligodeoxynucleotides used in this reaction have been . 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. Cloning has was performed as described above.

WO 97/43428 PCT / F'R97 / 00827 B. EXAMPLES OF TOBACCO GENETIC TRANSFORMATION, COLZA AND DUE BUT
I. OBTAINING TRANSGENIC TOBACCO PLANTS
Tobacco plants used for transformation experiences (Nicotiana tabacum var.
Xanthi NC and PDB6) are cultured in vitro on the base medium of Murashige and Skoog (1962) added vitamins from 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 potash solution before autoclaving at 120 ° C for 20 min. Tobacco seedlings are transplanted by internode cuttings every 30 days on this multiplication medium MS20.
All in vitro cultures are carried out in air-conditioned enclosure, under the conditions defined above below.
- Light intensity of 30; CE.m-2.S-1;
4 p.m. photoperiod;
- Thermoperiod of 26 ° C during the day, 24 ° C at night.
The processing technique used is derived from that of Horsch et al. (1985).
A preculture of Aarobacterium tumefaciens strain LBA4404 containing binary plasmids, is performed for 48 hours at 28 ° C with stirring, in LB medium supplemented with adequate antibiotics (rifampicin and têtracycline). The preculture is then diluted with 50th in the same environment and cultivated in memes conditions. After one night, the culture is centrifuged (10 min, 3000 rpm), the bacteria are taken up in an equivalent volume of MS30 medium (30g / L sucrose) liquid and this suspension is diluted to l0th.

Explants of approximately lcm2 are cut out from the leaves of the seedlings described above They are then brought into contact with the bacterial suspension for 1 hour, then dried quickly on filter paper and placed on a medium coculture (solid MS30).
After 2 days, the explants are transferred to Petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200mg / L), bacteriostatic, augmentin (40omg / L) and hormones necessary for the induction of buds (BAP, lmg / L and ANA, O, lmg / L). A transplant of the explants is performed on the same medium after 2 weeks of culture. After 2 more weeks, the buds are transplanted into petri dishes on the middle of development composed of MS20 medium supplemented with kanamycin and augmentin. After 15 days, buds are transplanted into pots on the same medium whose kanamycin concentration has been reduced by half. Rooting takes about 2o days, at the end from which the seedlings can be cloned by internode cuttings or greenhouse outings.
II. OBTAINING TRANSGENIC COLZA PLANTS
Seeds. spring rapeseed (Brassiaa napus cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a Domestos solution at 15%. After 4 rinses with sterile water, the seeds are put to germinate, at the rate of 20 seeds per pots 7 cm in diameter, 10 cm high, in the middle Murashige and Skoog mineral (Sigma, reference M
5519) with 30 g / 1 of sucrose and solidified with the agargel at 5 g / 1. These pots are placed in a culture chamber at 26 ° C with a photoperiod of 4 p.m. / 8 a.m. and at a light intensity of the order of 80 ACE m-2 S'1. After 5 days of germination, the cotyledons are removed sterilely by cutting each petiole about 1 mm above the cotyledon node.
At the same time a culture of Aarobacterium tumefaciens strain LBA4404, containing the plasmid pBI0C114, is grown in a 50 ml Erlenmeyer flask, for 36 h at 28 ° C in 10 ml of YT bacterial medium supplemented with antibiotics useful for selection of the strain used. This preculture is used to seed a new bacterial culture at 1%
performed under the same conditions. After 2 p.m.
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 5 cm petri dishes diameter at the rate of 5 ml / box.
The severed end of the petiole is submerged a few seconds in the agrobacteria solution thus prepared, then the petiole is pushed in a few millimeters in the middle of regeneration.
This medium has the same basic composition as the medium germination with an additional 4 mg / 1 of benzylamino-purine, phytohormone favoring the neoformation of buds. Twelve explants (cotylêdon avec pëtiole) are grown in a 9 cm bunch of petri dishes diameter (Greiner, reference 664.102).
After 2 days of coculture in the same environmental conditions that germinations, explants are transplanted into phytatray boxes (Sigma, reference P1552) containing the above medium supplemented with a selective agent: 45 mg / 1 of the sulfate kanamycin (Sigma, reference K4000) and a bacteriostatic: mixture of 1/6 (by weight) of salt potassium clavulanic acid and 5/6 salt ammonium amoxicillin (Augmentin injectable) to 600 mg / l.

Twice in a row, 3 weeks apart, the explants are sterile transplanted onto medium new under the same conditions.
The green buds appeared at the end of the second or the third transplant are separated from the explant and individually grown in pots transparencies 5 cm in diameter and 10 cm high containing a medium identical to the above but BAP-free. After 3 weeks of culture the stem of the transformed bud is cut and the bud is transplanted into a jar of fresh medium. At the end of three to four weeks the roots are pretty developed to allow acclimatization of the seedling with phytotron. The buds that are not green or rooted are eliminated. These seedlings are then transplanted into 7 cm side cups filled with potting soil (standard NF U44551: 40% peat brown, 30% sifted heather and 30% sand) saturated in water. After two weeks of phytotron acclimatization (temperature 21 ° C, photoperiod 16h / 8h and 84%
relative humidity), the seedlings are repotted in 12 cm diameter pots filled with the same potting soil enriched with late fertilizer (Osmocote, 4g / 1 potting soil) then transported to the greenhouse (class S2) regulated at 18 ° C, with two daily waterings of 2 minutes in the water.
As soon as the flowers appear, these are bagged (Crispac, reference SM 570y 300 mm * 700 mm) so as to prevent cross-fertilization.
When the pods have reached maturity, they are harvested, dried and then beaten. The seeds obtained are used to measure the activity biochemical. Selection of descendants transgenic is done by germination on a medium containing kanamycin sulfate in an amount of 100 to 150 mg / 1 (depending on genotypes). Conditions are identical to those described at the beginning of this document except that germinations are made in glass tubes with a single seed per tube. Only seedlings developing roots secondary the first three weeks are acclimatized in a phytotron before going into tight.
III. OBTAINING PLANTEB FROM TRANSGENIC BUT
a) Obtaining and using corn callus as target for Qenetic transformation.
Genetic transformation of the mat, whatever either the method used (electroporation, Aarobacterium, microfibers, particle gun), usually requires the use of cells undifferentiated into rapid divisions having retained an ability to regenerate whole plants. This type of cells composing the embryogenic friable callus (called type II) corn.
These calluses are obtained from embryos immature of genotype HI, II or (A188 x 873) depending on the method and on the media described by Armstrong (Malte Handbook, 1994, M. Freeling, V. Walbot Eds., pages 665-671). The calluses thus obtained are multiplied and maintained by successive subcultures every fifteen days on the initiation medium.
Seedlings are then regenerated from these calluses by altering the hormonal balance and osmatic of cells according to the method described by Vain et al. (Plant Cell Tissue and Organ Culture, 1989, 18. 143-151). These plants are then acclimated in the greenhouse where they can be crossed or self-fertilized.

b) Use of the particle gun for the genetic transformation of corn.
The preceding paragraph describes how to obtain and regeneration of cell lines necessary for the transformation; we describe here a method of genetic transformation leading to integration stable modified genes in the plant genome.
This method is based on the use of a particles identical to that described by J. Finer (Plant Cell Report, 1992, 11. 323-328); cells targets are fragments of calluses described in the paragraph 1. These fragments with an area of 10 to 20 mm2 were placed, 4 hours before bombing, at due to 16 fragments per box in the center of a box of petri dish containing a culture medium identical to initiation medium, supplemented with 0.2M mannitol +
0.2 M sorbitol. Plasmids carrying the genes to 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, 3 ~,: 70-73). The particles as well coated are projected towards the target cells at using the cannon and according to the protocol described by J.
Finer (Plant Cell Report, 1992, 11. 323-328).
The boxes of calluses thus bombarded are then sealed with Scellofrais ~ and then cultivated $
darkness at 27 ° C. The first transplant takes place 24 hours after, then every fortnight for 3 month on medium identical to the initiation medium added with a selective agent whose nature and concentration may vary depending on the gene used (see paragraph 3). Selective agents that can be used generally consist of active compounds of certain herbicides (Basta ~, Round up ~) or some antibiotics (Hygromycin, Kanamycin ...) After 3 months, or sometimes sooner, calluses whose growth is not inhibited by the agent selection, usually and mostly composed of cells resulting from the division of a cell having integrated in its genetic heritage one or multiple copies of the selection gene. Frequency obtaining such calluses is about 0.8 cal per bombed box.
These calluses are identified, individualized, amplified then cultivated so as to regenerate. of seedlings (see paragraph a). In order to avoid any interference with unprocessed cells, all these operations are carried out on culture containing the selective agent.
The plants thus regenerated are acclimatized then grown in a greenhouse where they can be crossed or self-fertilized.
C. EVIDENCE OF GLYCOPROTEIN G RABIQOE
I. ANALYSIS OF GLYCOPROTEIN G EXPRESSION
RABIQüE IN LEAVES, TOBACCO SEEDS AND
RASP SEEDS.
The glycoprotein G extraction protocol rabies from the leaves is as follows:
- 1 g of tobacco leaves (fresh weight) is ground in liquid nitrogen and then suspended, at 4 ° C, in 4 ml of 100 mM Tris-HC1 buffer pH 7.5, added 1 mM EDTA, 0.2% Triton X100 and 250 mM NaCl.
The homogenate thus obtained is immediately centrifuged at 4 ° C for 10 min at 10,000 rpm.
For tobacco or rapeseed seeds, the extraction is carried out at the rate of 100 mg of seeds for 4 ml of buffer. For corn calluses, the extraction is carried out at a rate of 0.5 g in 1 ml of buffer.
After centrifugation, the supernatant is recovered then filtered on MIRACLOTH (CALBIOCHEM). A dosage of soluble protein is then produced by the method of Bradford (1976).
Rabies G glycoprotein highlighted by "Western" type immunodetection ("Western blots ") (Renart and Sandoval, 1984) and dosed by a ELISA test.
II. IMMUNODETECTION BY RESTERN BLOT OF THE
GLYCOPROTEIN G RABIQUE EXPRESSED IN SHEETS OF
TOBACCO, IN TOBACCO AND CANNED SEEDS
TRANSGENIQOES AND IN THE CALIS DE MAIS PROCESSES.
Proteins extracted according to the protocol below above are denatured by heating at 95 ° C for 5 min, in the presence of Tris HC1 50 mM ph 6.8 buffer; SDS
4%; BSH 1%; 20% sucrose and bromophenol blue 0.01%.
The proteins are then separated by electrophoresis, on a polyacrylamide gel in denaturing conditions at 8%, 10% or 12.5%, depending on the technique by Laemmli UK (1970) at a rate of 50 ug of total protein per sample.
After migration the proteins are transferred on a nitrocellulose membrane. An anti-antibody glycoprotein G of rabies) obtained in sheep is used as a probe and the revelation is carried out at using a sheep-labeled anti-IgG antibody alkaline phosphatase.
The tests were carried out on plants transformed with the following constructs. pBIOC104, pBIOC110, pBIOC114, pBIOC116.

Control protein migrates as 2 apparent molecular weight bands 66 and 61 KDa.
The lightest form corresponds to a protein, truncated at the terminal C end (Wunner et al, 1983), more soluble than the complete protein:
Analysis of leaf and seed extracts tobacco, as well as in rapeseed and corn callus, reveals a molecular weight band about 66 KDa, (Fig 1, 2, 3 and 4) which corresponds to that of the virus’s glycoprotein G.
No band corresponding to glycoprotein G
rabies is only detected in protein extracts from tobacco and rapeseed leaves and seeds not transformed.
III. GLYCOPROTEIN G ELISA TEST ASSAY
RABIQUE EXPRESSED IN TOBACCO LEAVES AND
TRANSGENIC TOBACCO AND CANNED SEEDS.
For the determination of the expressed glycoprotein G
in the leaves and in tobacco seeds, the following protocol has been implemented.
- extraction of glycoprotein. the leaf or seed samples taken from ice the day of the test, are crushed at the rate of ig for 4m1 extraction buffer for leaves and 10omg for 4m1 of extraction buffer for seeds.
The amount of total protein is then determined by the Bradford method and all extracts are brought to a concentration of 5 ~ cg / ~, 1.
These extracts are diluted extemporaneously to 1/1000.
The reference range (glycoprotein G. 0 to 20 mg / ml) is prepared in an unprocessed plant extract ground, standardized and diluted to 1/1000 of the same way.
Seeds. ELISA sandwich technique WO 97! 43428 PCT / FR97 / 00827 Rabies glycoprotein, after being retained by a monoclonal anti-glycoprotein antibody G of rabies adsorbed on a polychloride plate vinyl, is contacted with an antibody polyclonal anti-glycoprotein G of rabies. This one is revealed using IgG, labeled with peroxidase or with alkaline phosphatase and directed against fragment F (ab ') 2 of the preceding polyclonal antibody.
The dosage is then carried out by direct reading on a standard curve performed in the same conditions, with purified glyco-protein G.
Leaves. Direct ELISA technique Each of these samples is absorbed on the plate (150 ~ cl / well) for 2 hours at 37 ° C, then marked, without prior saturation step, by a purified polyclonal horse antibody (100 ~, 1 / well, 1 hour, room temperature), and finally revealed by an antibody labeled with alkaline phosphatase (SIGMA
ref. A6063), directed against the previous one and diluted in 1/1000 (100 ~ Ci / well, 30 minutes, temperature ambient). Between each step, washes are carried out with PBS added with 0.1% Tween 20 (P / V). The results are reduced to a quantity of glycoprotein per mg of total protein.
The amount of G glycoprotein varies from 1.5 to 0.4 ~ g / ml for the different extracts tested.

WO 97/43428 PCTIF'R97 / 00827 D. EVIDENCE OF G RABIC GLYCOPROTEIN
IN CORN GRAINS.
The protocol for extracting G glycoprotein from rabies from corn kernels is as follows - 200 mg of grains are ground in liquid nitrogen, then suspended in 1 ml of 100 mM Tris-HCl buffer pH 7.5, supplemented with lmM EDTA, 0.2% Triton X100 and 250 mM NaCl.
The homogenate thus obtained is immediately centrifuged at 4 ° C
for 10 min at 13000 g. Then, the supernatant is removed and filtered on Miracloth (CALBIOCHEM). A dosage of soluble proteins is then produced by the method of Bradford (1976).
Rabies G glycoprotein is demonstrated by "Western blot" type immunodetection (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 Tris-HCl buffer 50mM pH 6.8, SDS 4%, BSH 1%, Sucrose 20% and bromophenol blue 0.01%.
The proteins are then separated by electrophoresis on a gel.
polyacrylamide under denaturing conditions at 8% or 10%, depending on the Laemmli technique (1970) at a rate of 30 ~ g of soluble proteins per sample.
After migration, the proteins are transferred to a membrane PVDF (polyvinylidene difluoride). One year anti-polyclonal serum rabies glycoprotein G, obtained in sheep, is used as a probe and the revelation is carried out by means of a sheep anH-IgG antibody labeled with alkaline phosphatase.
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 truncated protein at the C end terminal (Wunner et al., 1983), more soluble than the complete protein.
No band corresponding to rabies G glycoprotein is detected in unprocessed corn extracts.
Analysis of extracts of corn kernels transformed with pBIOC118 highlights specific bands characteristic of the glycoprotein G. One of the bands has a molecular weight of approximately 66 KDa.
E. VEGETATION OF THE SEQUENCE CODING THE
GLYCOPROTEIN G RABIQUE
I. MODIFICATION OF THE SEQUENCE ENCODING GLYCOPROTEIN
G RABIQUE. CREATION OF SYNTHETIC SEQUENCES
FOR USE IN GENETIC TRANSFORMATION OF MAòS.
To increase the level of expression, the sequence encoding the glycoprotein G
rabic has been subject to several changes - use of plant genetic code (Murray EE Nucleic Acids Res., 1989, 17, 477) - modification of content in GC
- removal of cryptic introns - suppression of internal polyadenylation signals - removal of RNA destabilizing sequences - suppression of Polymerase II termination signals The modification of the cDNA encoding the rabies glycoprotein G was carried out according to the LbPCR technique by modifying the 5 'parts [fragment BgIII (agatct) - XhoI
(ctcgag)], central [fragment XhoI - AatII (gacgtc)] and 3 '[fragment AatII -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 at using "sense" oligodeoxynucleotides and a PCR step to obtain DNA
double strand. Each of the modified parts has been verified by sequencing at help from Amersham Sequenase PCR product sequencing kit as recommended from the manufacturer. Then either the modified part S 'or the parts 5' and modified central, either the modified S ', central and 3' parts, were introduced in pBIOC
109 by enzymatic digestions (restriction enzymes delimiting the fragments).
The 5 ′ part was constituted using the following oligodeoxynucleotides - "sense" oligodeoxynucleotides to form continuous single strand DNA
cgagatctgaattcaacaATGAAAGCCTTCACACTCGCTCTCTTCTTAGCTCTTTC
CCTCTATCT
2.
CCTGCCCAATCCAGCCcattccaggttcaatcccatccgcctccccaccacacacgaacccgccAA
3.
gTTCCCaATcTACACcATCCCAGACaAGCTcGGcCCaTGGAGCCCaATcGAC
ATcCAcCACCTCA
4.
GCTGCCCAAACAAcTTGGTcGTcGAGGACGAgGGcTGCACCAACCTcTCcG
GcTTCTCCTACATG
5.
GAgCTcAAgGTcGGcTACATCTTgGCCATcAAgATGAACGGcTTCACcTGCA
CcGGCGTcGTcAC

WO 97! 43428 PCT / FR97 / 00827

6.
cGAGGCcGAgACCTACACcAACTTCGTcGGcTAcGTCACcACCACcTTCAAg AGgAAGCAcTTCCG

7.
gCCAACcCCAGAcGCcTGcAGgGCCGCcTACAACTGGAAGATGGCCGGcG
ACCCaAGgTAcGAg

8.
GAGTCcCTcCACAAcCCaTACCCaGACTACCGCTGGCTcCGcACcGTcAAgA
CCACCAAGGAGTC

9.
cCTCGTcATCATcTCcCCAAGcGTcGCcGAcTTGGACCCATAcGACAGgTCC
CTcCACTCGAGGGTCTT
- "guide" oligodeoxynucleotides to link the oligodeoxynucleotides "meaning":
1. CTGGATTGGGCAGGagatagagggaaagagctaag 2. GATgGTGTAGATTGGGAACttggcgggttcg 3. AGTTGTTTGGGCAGCtgaggtggtggatgtc 4. GCCGACCTTGAGCTCcatgtaggagaagccg 5. GGTCTCGGCCTCGgtgacgacgccg 6. GTCTGGGGTTGGCcggaagtgcttcctct 7. GGTTGTGGAGGGACTCctcgtaccttgggtcg 8. GGAGATGATGACGAGGgactccttggtggtctt - oligodeoxynucleotides to carry out PCR amplification of simple DNA
strand obtained cgagatctgaattcaacaATGAAAGCC
and AAGACCCTCGAGTGGAGGgacc The 5 ′ part amplified by PCR was doubly digested with BgIII and XhoI, then, cloned to replace the BgIII - XhoI fragment of pBIOC 109 according to the processes already described above. The resulting plasmid was called pBIOC 109-5'M. This plasmid was used in genetic transformation of corn.
The central part was formed using oligodeoxynucleotides following - "sense" oligodeoxynucleotides 1.
CCCaAGCGGcAAGTGCTCcGGcGTcGCcGTcTCcTCcACCTACTGCTCCACc AACCACGAc 2.
TACACCATcTGGATGCCaGAGAAcCCaAGgCTcGGcATGTCcTGcGACATcT
TcACCAAcA
3.
GcAGgGGcAAGAGgGCcTCCAAgGGcAGcGAGACcTGCGGCTTcGTcGAcG
AgAGgGGCCT
4.
cTAcAAGTCcTTgAAgGGcGCcTGCAAgCTCAAGTTgTGcGGcGTcCTcGGcC
TcAGgCTc 5.
ATGGAcGGcACcTGGGTCGCcATGCAAACcTCcAAcGAgACCAAgTGGTGC
CCaCCaGAcC

6.
AaTTGGTcAACCTcCACGACTTcCGCTCcGACGAgATcGAGCACCTcGTcGT
cGAGGAGTT
7.
GGTCAGGAAGAGgGAGGAGTGcCTcGAcGCcCTtGAGTCCATCATGACcA
CCAAGTCcGTcA
- "guide" oligodeoxynucleotides 1. GCATCCAGATGGTGTAgtcgtggttggtgga 2. CTCTTGCCCCTGCtgttggtgaagatgtcg 3. CCCTTCAAGGACTTGTAGaggcccctctcgt 4. CAGGTGCCGTCCATgagcctgaggccg 5. GTGGAGGTTGACCAATTggtctggtgggcac 6. CTCCCTCTTCCTGACCaactcctcgacgacg - oligodeoxynucleotides to carry out PCR amplification tccgctcgagCCCAAGCGGCAAGTGCTCCGgcg and actgacgtcTGACGGACTTGGTGGTCATGATGGACtcaagggcgtcgag The central part amplified by PCR was doubly digested with XhoI and AatII, then) cloned to replace the XhoI - AatII fragment of pBIOC 109-5'M according to the methods already described above. The resulting plasmid was called pBIOC 109-5'CM. This plasmid was used in genetic transformation of corn.
Part 3 'was formed using the following oligodeoxynucleotides - "sense" oligodeoxynucleotides GcTTCAGACGTCTCAGcCAcTTgAGgAAgCTcGTCCCaGGcTTcGGcAAgGC
cTAcACCATcTTCAACA
2.
AGACCTTGATGGAgGCCGAcGCcCACTACAAGTCcGTCAGgACcTGGAAc GAGATCCTCCCaTCc 3.
AAgGGcTGcTTgAGgGTcGGcGGcAGGTGcCAcCCaCAcGTcAACGGcGTcTT
cTTCAAcGGcAT
4.
cATcTTgGGcCCaGACGGCAAcGTCTTgATCCCAGAGATGCAATCcTCCCT
CCTCCAaCAACAcAT
5.
GGAGTTGTTGGAgTCCTCcGTcATCCCaCTcGTcCACCCaCTcGCcGACCCa TCcACCGTcTTC
6.
AAGGACGGcGACGAGGCcGAGGAcTTcGTcGAgGTcCACCTcCCaGATGTc CACAAcCAaGTCT
7.
CcGGcGTcGACTTGGGcCTCCCaAACTGGGGcAAGTAcGTcTTgCTcAGcGC
cGGcGCCCTcAC
8.
cGCCTTGATGTTGATcATcTTCCTcATGACcTGcTGcAGgAGgGTCAAcCGc TCcGAgCCaACc WO 97/43428 PCTJF'R97 / 00827 9.
CAACACAAcCTCAGgGGcACcGGcAGGGAGGTcTCcGTCACcCCaCAAAGC
GGcAAGATCATcT

10.
CcTCcTGGGAgTCcCACAAGAGcGGcGGcGAGACCAGgCTcTGAggatccgggg aattctgtga - "guide" oligodeoxynucleotides I. GCCTCCATCAAGGTCTtgttgaagatggtgtagg 2. CTCAAGCAGCCCTTggatgggaggatctc 3. TGGGCCCAAGATGatgccgttgaagaagac 4. AGGACTCCAACAACTCCatgtgttgttggaggag 5. CGTCGCCGTCCTTgaagacggtggatgg 6. AGTCGACGCCGGagacttggttgtggacat 7. GATCAACATCAAGGCGgtgagggcgccg 8. CCCTGAGGTTGTGTTGggttggctcggag 9. GGACTCCCAGGAGGagatgatcttgccgct - oligodeoxynucleotides to carry out PCR amplification GCTTCAGACGTCTCAGCCACTTga and TCACAGAATTCCCCGGATCCtc The 3 ′ part amplified by PCR was doubly digested with AatII and BamHI, then, cloned to replace the AatII - BamHI fragment of pBIOC 109-5'CM according to the processes already described above. The resulting plasmid was called pBIOC I 09-5'C3'M. This plasmid was used in genetic transformation of But.

II. CONSTRUCTION OF BINARY PLASMIDS CONTAINING
DIFFERENT MODIFIED SEQUENCES OF THE cDNA ENCODING THE
G RABIC GLYCOPROTEIN FOR USE IN TRANSGENESIS
VIA AGROBACTERIUM TUMEFACIENS
EcoRI fragments carrying the different modified sequences of the coding cDNA
rabies G glycoprotein were isolated from plasmids pBIOC 109-5'M, pBIOC 109-5'CM and pBIOC 109-5'C3'M. The processes used have already been described previously.
The isolated fragments were ligated to the plasmid DNA of pBIOC21, pBIOC28, pBIOC82, pBIOC91 and pBIOCII2 digested with EcoRI and dephosphorylated. The usual cloning methods were applied. The plasmid DNA of 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 processes, glycoproteins and their uses, nucleic acids, transformed plant cells, plants chimeric or transgenic, with seeds of transgenic plants, antibody monoclonal or polyclonal, to pharmaceutical compositions and to products foods according to the invention characterized in that the coding sequence, allowing expression in the cell of protein G, has been subjected to modifications to increase the expression for example was subjected to a "revegetation" according to Example E above.

REFERENCES
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Koprowski, Eds), 3rd ed., Pages 354-357, Geneva, Switzeland.

2254115.seq LIST OF SEQUENCES
(1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: BIOCEM - Cezeaux University Campus (B) STREET: 24 avenue des Landais (C) CITY: AUBIERE
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 63170 (A) NAME: MERIAL
(B) STREET: 17 rue Bourgelat (C) CITY: LYON
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 69002 (ii) TITLE OF THE INVENTION: TRANSGENIC PLANTS EXPRESSING THE
GLYCOPROTEIN G IN RABIES, AND
GLYCOPROTEINS SO OBTAINED
(iii) NUMBER OF SEQUENCES: 72 (iv) ADDRESS OF CORRESPONDENCE:
(A) RECIPIENT: Robic (B) STREET: 55 St-Jacques (C) VILE: Montreal (D) PROVINCE: QC
(E) COUNTRY: CANADA
(F) POSTAL CODE: H2Y 3X2 (G) TELEPHONE: 514-987-6242 (H) FAX: 514-845-7874 (v) COMPUTER-READABLE FORM:
(A) TYPE OF MEDIA: 3.5 "/ 1.44 MB floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: ASCII text (vi) CURRENT DEMAND DATA:
(A) APPLICATION NUMBER: 2,254,115 (B) DEPOSIT DATE: May 07, 1997 (vii) DATA RELATING TO PRIOR APPLICATION:
(A) APPLICATION NUMBER: PCT / FR97 / 00827 (B) DEPOSIT DATE: May 07, 1997 (A) REQUEST NUMBER: FR 96/06029 (B) DEPOSIT DATE: May 09, 1996 2254115.seq (2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1.30 (D) OTHER INFORMATION: / note = "oligo 1 page 29"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1 .18 (D) OTHER INFORMATION: / note = "oligo containing the site HincII page 30 "
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
CTCAGGAGTT GACTTGGG lg (2) INFORMATION FOR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(ix) CHARACTERISTIC:
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

(2) INFORMATION FOR SEQ ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1.69 (D) OTHER INFORMATION: / note = "signal peptide page 31"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

2254115.seq GCCCATTCC 6g (2) INFORMATION FOR SEQ ID NO: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 38 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: l .38 (D) OTHER INFORMATION: / note = "oligo containing the sites BglII and EcoRI page 32 "
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:

2254115.seq (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 78 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
GGAGATCTGA ATTCAACAAT GA.AGATGATG AGCACCAGG 39 (2) INFORMATION FOR SEQ ID NO: 10:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 55 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA

2254115.seq (ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1.55 (D) OTHER INFORMATION: / note = "Oligo containing HindIII
page 34 "
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 111 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1,111 (D) OTHER INFORMATION: / note = "Peptide signal PPS page 37"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:

(2) INFORMATION FOR SEQ ID NO: 12:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(ix) CHARACTERISTIC:
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2254115.seq (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:
CGAGATCTGA ATTCAACAAT GAAAGCCTTC ACACTCGC 3g (2) INFORMATION FOR SEQ ID NO: 13:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1.60 (D) OTHER INFORMATION: / note = "oligo containing HindIII
page 37 "
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:

(2) INFORMATION FOR SEQ ID NO: 14:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: l .19 (D) OTHER INFORMATION: / note = "oligo 1 page 45"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:

(2) INFORMATION FOR SEQ ID NO: 15:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15:

(2) INFORMATION FOR SEQ ID NO: 16:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 35 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1.35 (D) OTHER INFORMATION: / note = "oligo containing the site HincII page 52 "
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16:

(2) INFORMATION FOR SEQ ID NO: 17:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 46 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1 .46 2254115.seq (D) OTHER INFORMATION: / note = "oligo page 52 (KDEL and site BamHI) "
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17:

(2) INFORMATION FOR SEQ ID NO: 18:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 65 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: l .65 (D) OTHER INFORMATION: / note = "oligo sens 1 page 65"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18:

(2) INFORMATION FOR SEQ ID NO: 19:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 66 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: misc_feature (B) LOCATION: 1.66 (D) OTHER INFORMATION: / note = "OLIGO SENS 2 page 65"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19:

2254115.seq (2) INFORMATION FOR SEQ ID N0: 20:
(i) CHARACTERISTICS OF THE SEQUENCE:
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Claims (36)

1 - Process for the production of rabies virus glycoprotein G, or a virus related to the rabies virus, characterized by:
i) the introduction into a plant cell of a nucleic acid molecule comprising a sequence chimeric coding, comprising on the one hand a sequence encoding said mature viral protein G, or for an analogous protein, and on the other hand, a sequence encoding an N-terminal signal peptide other than the one naturally associated with viral protein G, the introduction of the chimeric coding sequence allowing expression in the cell of the protein G;
ii) multiplication, in the form of culture cell, transformed cells, or the regeneration of whole transgenic plants or chimeric from these cells;
iii) possibly the extraction and purification of G-glycoprotein from cells or tissues plants thus obtained. 2 - process according to claim 1 characterized in that the sequence encoding the signal peptide N-terminal code for an original signal peptide vegetable. 3 - Method according to claim 1 or 2 characterized in that the nucleic acid introduced into the cell further comprises sequences transcription regulators recognized by the plant cell. 4 - Method according to claim 3 characterized in that the regulatory sequences include at minus a transcription initiation signal and a transcription termination signal. 5 - Method according to claim 3 characterized in that the initiation and termination signals of transcription are of plant origin, or come from a plant virus, or Agrobacterium. 6 - Method according to any one of previous claims characterized in that the chimeric coding sequence further comprises a sequence coding for a retention signal endoplasmic of plant origin, or for a signal vacuolar addressing. 7 - Method according to any one of previous claims characterized in that sequence encoding an agent for selection transformed cells is also introduced in the plant cell. 8 - Method according to any one of previous claims characterized in that the glycoprotein G extraction step comprises the use of one or more detergent (s), non-ionic preference (s). 9 - Glycoprotein, characterized in that:
- it is recognized by antibodies specific to the rabies virus glycoprotein or virus related to rabies virus;
- it has a molecular weight of 66 kDa approximately;
- it is insoluble;
- It is N-glycosylated by at least one glycan of polymannosidic type, and / or by at least one glycan complex type including within its structure a or more residues of xylose .beta.-1,2, and / or one or several residues of fucose linked in .alpha.-1,3, and being free of sialic acid residues. 76 - Glycoprotein according to claim 9 characterized in that it further comprises a signal of plant endoplasmic retention. 11 - Glycoprotein according to any one of claims 9 or 10 characterized in that it is capable of inducing the formation of protective antibodies against the rabies virus or against a virus related to rabies virus. 12 - Glycoprotein according to any one of claims 9 to 11 characterized in that they are of rabies virus glycoprotein G, having a weight molecular of about 66 kDa. 13 - Glycoprotein according to claim 9 characterized in that it is an analogue of the rabies virus G glycoprotein with approximately 90% to 95% homology with protein G from rabies virus. 14 - Glycoprotein capable of being produced by the method according to any one of claims 1 to 8. - Nucleic acid comprising a sequence chimeric coding comprising on the one hand a sequence coding for mature rabies virus G protein or a virus related to the rabies virus, or for an analogous protein, and on the other hand, a sequence encoding an N-terminal signal peptide other than the one naturally associated with the virus, or the sequences complementary to the above coding sequences. 16 - Nucleic acid according to claim 15 characterized in that the sequence encoding the signal peptide N-terminal code for a signal peptide of plant origin. 17 - Nucleic acid according to claim 16 characterized in that it is DNA. 18 - Nucleic acid according to claim 16 characterized in that it is RNA. 19 - Nucleic acid according to any one of claims 16 to 18, characterized in that it further includes regulatory sequences of transcription recognized by a plant cell. 20 - Vector comprising a nucleic acid according to claims 16 to 19. 21 - Plant cells transformed in a way stable by nucleic acid according to any one of claims 16 to 19 and capable of producing the rabies virus glycoprotein or virus related to rabies virus, or a glycoprotein similar. 22 - Plant cells according to claim 21 characterized in that it is a culture of plant cells, for example in a liquid medium or immobilized. 23 - Plant cells according to claim 21 characterized in that they are cells making part of a whole transformed plant. 24 - Chimeric or transgenic plant capable of produce the rabies virus glycoprotein G or a virus related to the rabies virus, or a analog glycoprotein, characterized in that it has cells according to claim 21. - Transgenic plant seeds according to the claim 24. 26 - Monoclonal or polyclonal antibodies capable to specifically recognize the glycoprotein according to claims 9 to 14. 27 - Pharmaceutical composition comprising:
- one or more glycoprotein (s) according to one any of claims 9 to 14, or - cells according to one of claims 21 to 23, or - all or part of a plant according to claim 24, in combination with an acceptable excipient from the point physiological point of view. 28 - Pharmaceutical composition according to claim 27 characterized in that it is a vaccine. 29 - Pharmaceutical composition according to claim 28 characterized in that it is a vaccine for oral administration or injectable, possibly in combination with one or several adjuvant (s). - Glycoprotein according to any one of Claims 9 to 14 for use in therapy and in particular as a human or veterinary vaccine against rabies virus infection, or a virus related to the rabies virus. 31 - Plant cells according to any one of claims 21 to 23 for use in therapy and especially as a human vaccine or veterinarian against infection with the rabies, or by a virus related to the rabies virus. 32 - Transgenic or chimeric plant depending on the claim 24, or extract of a plant according to the claim 24, for use in therapy and especially as a human or veterinary vaccine against rabies virus infection, or a virus related to the rabies virus. 33 - Use of a glycoprotein according to one any of claims 9 to 14, cells according to any one of claims 21 to 23, plants according to claim 24, or an extract of said plant, for the preparation of a medicament against rabies virus, or a virus related to rabies virus. 34 - Food product characterized in that it includes a glycoprotein which has the characteristics following:

- it is recognized by antibodies specific to the rabies virus glycoprotein or virus related to rabies virus;
- it has a molecular weight of 66 kDa approximately;
- it is insoluble;
- It is N-glycosylated by at least one glycan of polymannosidic type, and / or by at least one glycan complex type including within its structure a or more residues of xylose .beta.-1,2, and / or one or several residues of fucose linked in .alpha.-1,3, and being free of sialic acid residues. 35 - Food product characterized in that it includes transformed plant cells from stably with a nucleic acid, said acid nucleic acid comprising a chimeric coding sequence comprising on the one hand a sequence coding for the mature G protein of rabies virus or virus related to rabies virus, or for a protein analog, and on the other hand, a sequence coding for a N-terminal signal peptide other than that naturally associated with the virus, or the sequences complementary to the above coding sequences. 36 - Food product characterized in that it includes a transgenic or chimeric plant, or a extract from a transgenic or chimeric plant, said plant being capable of producing the rabies virus glycoprotein or virus related to rabies virus, or a glycoprotein analog, the plant cells being transformed with a nucleic acid comprising a coding sequence chimeric comprising on the one hand a sequence coding for mature protein G of rabies virus or a virus related to the rabies virus, or for a protein analog, and on the other hand, a sequence encoding an N-terminal signal peptide other than the one naturally associated with the virus, or the sequences complementary to the above coding sequences.