WO2004050692A2

WO2004050692A2 - Fusion of the e2 protein of csfv with kdel , vts and/or ubiquitin for expression in transgenic plants for vaccine production

Info

Publication number: WO2004050692A2
Application number: PCT/PL2003/000136
Authority: WO
Inventors: Andrzej B. Legocki; Jozef Kapusta; Tomasz Pniewski; Olesia Lisowa; Katarzyna Miedzinska; Magdalena Czaplinska; Jacek Wojciechowicz; Femiak Iowona; Andrzej Plucienniczak; Malgorzata Kesik; Anna Porebska; Boguslaw Szewczyk; Monika Gut-Winiarska; Jolanta Tyborowska; Jolanta Ficinska; Krystyna Bienkowska-Szewczyk
Original assignee: Institute Of Bioorganic Chemistry; Institute Of Biotechnology Antibiotics
Priority date: 2002-12-04
Filing date: 2003-12-04
Publication date: 2004-06-17
Also published as: PL201420B1; AU2003287108A8; PL357518A1; AU2003287108A1; WO2004050692A3

Abstract

The present invention relates to a chimaeric protein, a sequence, a construct, a plant cell, a method for the production of a chimaeric protein and a transgenic plant, the transgenic plant and the application of transgenic plants in the manufacture of oral vaccines, a pharmaceutical composition, a vaccine and a method of inducing immunity to infections. Generally, the invention relates to a method of inducing an immune response, encompassing the oral administration of plant material containing a protein with immunogenic properties. The topics of the present invention serve to produce oral vaccines against the Classical Swine Fever Virus (CSFV).

Description

G imaeric protein, sequence, construct, plant cell, transgenic plantj application of the transgenic plants, vaccine and a method of induction

This invention relates to chimaeric proteins, sequence construct, plant cell, the method . of manufacturing of the chimaeric protein and the transgenic plant, the transgenic plant, the application of transgenic plants in the manufacture of oral vaccines, a pharmaceutical composition, vaccine and a method of induction of immunity in mammals. In general the invention relates to a method of obtaming an immune response, encompassing the oral administration of plant material containing a protein with i-mmunogenic properties. The subjects of this invention serve to produce oral vaccines against the Classical Swine Fever Virus (CSFN).

Classical Swine Fever is a disease whose etiological factors are pestiviruses, belonging to the family Flaviviridae. Pestiviruses attack animals from the family Suidae (pigs), some species of ruminants (cows, sheep and goats) as well as a series of wild animals. Classical swine fever was first described in 1833 in Ohio. From that time it has drastically increased its range throughout the world, becoming one of the most limiting economical factors in the production of swine. The global organization Office International des Epizooties lists this disease on its A list, as one of the fifteen most dangerous diseases of animals. This disease has been not been found in only 16 countries in the world.

Depending on the virulence of the virus, the disease may be manifested in one of three forms: acute, moderate and mild. An acute attack of the disease manifests itself in animals with a high fever, diarrhoea, leukopenia (leukocyte apoptosis), skin lesions, strong immunosuppression, and in most cases the animals' death.

A mild case may be borne without symptoms. Moreover, the disease has a rather long incubation stage, from 2 to 14 days, which complicates the control of an epidemic. The classic swine fever virus is very resistant to environmental conditions, it can remain viable outside the organism for as much as a month. It is most often transmitted through contact between a sick animal and a healthy one, but may also be physically borne by humans, birds and insects, as well as agricultural implements, vehicles, clothing, etc. To date no cure exists for this disease. Following the discovery of the pathogen, all herds within 25km of the epidemic must be destroyed. Many attempts have been made to produce a vaccine. Presently, three vaccines exist: one based on the attenuated live strain C, one based on a modified infective clone of cDNA [Meyers 1996, J.Virol. 70, 1588-1595], as well as on glycoprotein E2 [van Rijn 1999, Vaccine 17:433-440]. In no European countries is massive vaccination used, chiefly due to the relatively high cost per dose of the vaccine. The E2 viral protein is the chief neutralizing antigen in the course of a swine fever infection. This means that antibodies directed against epitopes of this protein most effectively neutralize the virulence of the pathogen.

The possibilities of application of genetically modified plants are very broad and encompass many facets of the economy. Most often, transgenic plants find uses in agriculture as varieties resistant to herbicides, diseases and pests or various stressful conditions such as drought, cold or hyperhalination. During the last few years, more and more importance has been attached to the usage of transgenic plants as bioreactors producing useful proteins, useful in therapy, diagnostics and medical analysis, as well as in industry.

In comparison to industrial fermenters or bioreactors, the plant system is much more economical in terms of exploitation costs or transport. Such costs may be reduced even tenfold. [Kusnadi 1997, Biotechnol. Bioeng. 56, 473-484]. For comparison, the cost of producing an IgG antibody in alfalfa growing in a hothouse is some 10 times lower than the cost of producing it in hybridoma cells. The technology of collecting and processing plants on a large scale has been known and applied for a long time, and in the case of the usage of transgenic plant tissues for direct consumption (i.e. as an oral vaccine), the processing of this material is superfluous. In plants it is possible to direct the production of the produced protein into cellular compartments in which it is more stable. At the same time, the amount of protein produced in plants may be extremely large, and it may even reach an industrial scale. Such plants as tobacco, soya or alfalfa may produce over 20 kg of heterologous protein per hectare [Khoudi 1999, Biotechnol. Bioeng. 64, 135-143]. In contrast to bacteria, plants produce multi- subunit proteins (i.e. antibodies), and they possess a system of protein folding and glycosylation, through which structural proteins may reach the appropriate spatial conformation. At the same time, the risk of contamination of the product with technological process side products is not great. In contrast to animal systems, plants cannot be hosts to human pathogens such as HIV, prions, viruses, hepatitis, etc. They also do not contain oncogenic sequences. [Fischer 2000, J. Biol. Regul. Homeost. Agents 14, 421-477; Fischer 2000, Transgenic Res. 9, 279-299].

The level of synthesis of the useful protein in plants can be increased through the application of organ specific promoters controlling the expression of the transgenes. Promoters active in developing storage organs of plants such as seeds or bulbs facilitate synthesis during a strictly defined stage of the plant's development. Genes coding heterologous proteins can be equipped with translocation sequences for particular cellular compartments, which on one hand isolate the protein from the rest of the cell, and on the other facilitate their greater accumulation. An example of a translocation sequence is the KDEL amino-acid motif which causes the retention of proteins in the endoplasmic reticulum [Doran P. M. 2000, Curr. Opin. Biotechnol. 11, 199-204].

The CSFN genome is comprised of a single strand of RΝA, 12,5 thousand bases long, with positive polarity. The RΝA molecule contains one reading frame coding one 4 thousand amino-acid polyprotein, which becomes post-translationally divided into the following structural proteins: C, E0, El, E2, and non-structural i.e. viral proteases. The virion's structure is not yet known in detail. It is known that the virions are likely icosahedral particles with a diameter of around 50nm and are composed of a spherical nucleocapsid and coat. The nucleocapsid is composed of many moleculs of protein C, whereas the coat contains membrane phospholipids and the E0, El and E2 surface glycoproteins. The coat proteins form homo- (E0 and E2) and heterodimers (E1-E2) with the aid of disulphide bridges. Only such dimers comprise the viral coat. The El and E2 proteins contain transmembrane domains, ensuring their anchoring in the membrane [Meyers G. et al. 1996, J.Nirol. 70, 1588-1595; van Rijn P. A. et al. 1993, Vaccine 17, 433-440]. All of the coat glycoproteins have immunogenic properties. It is thought that the E2 protein is the strongest immunogen, stronger than E0 and El.

In the manufacture of vectors the most common promoter sequence used is 35S CaMV, due to the expression of the gene in plants. It is of a constitutive character. Genes under its control are expressed in all plant tissues. It is believed that this promoter possesses optimal characteristics in terms of the production of heterologous proteins. A stronger promoter, as is thought, would very probably cause the post-transcriptional silencing of the gene [Weising 1988, Annu. Rev. Genet. 22, 421-477], while a weaker one would not facilitate the production of a high enough level of the heterologous protein.

Seed specific promoters have been primarily used in research of the expression of heterologous proteins to increase the nutritional value of the seeds. The sequences used usually originated from plants other than peas. Earlier, the phaseoline promoter from the bean and the β-conglycinine promoter from soya were used in the regulation of the expression of a lysine-rich protein in tobacco seeds [Keeler 1997, Plant Mol. Biol., 34: 15-29]. Transformation efforts in plants to date (chiefly tobacco) using seed-specific promoters of peas: legumines and other storage proteins, lectins and other proteins; have concentrated mainly on the discernment of the functions and mechanisms of promoter sequences [Bδttcher B. et al. 1997, Nature, 386:88-91; Brennan F. et al. 1999, Journal of Viroly, 73:930-938; Calgene Pacyfic PTY Ltd 1994] .

For the selection of transgenic plants, genes coding for antibiotic resistance are most often used. The Food and Drug Administration (FDA) of the USA studied the application of kanamycin resistance in three transgenic plants: the tomato, cotton and rape-seed [Daniell 1999, Trends Plant Sci. 4, 467-469]. Kanamycin and neomycin are toxic antibiotics, whose clinical utility is limited. These drugs are used solely in the situation when the patient takes no food, and when the food does not contain a sufficient concentration of the ATP cofactor for the degradation of a significant portion of the antibiotic. The FDA stresses that to date no mechanism is known of transfer of genes from the plant chromosome to microorganisms. In the construction of vectors for the transformation of plants, the bar gene is often used, which codes for a resistance factor to the Basta herbicide [D'Halluin 1990, Crop Sci. 30, 866-871]. This is a gene derived from the Streptomyces hygroscopicus bacterium, coding the phosphinotricine acetylotransferase enzyme. This enzyme carries out the acetylation of the active ingredient of the herbicide, glucosulphonate (a symthetic phosphinotricine). Phosphinotricine is a structural analogue of glutamate and inhibits glutamate synthase, which results in the accumulation of ammonia, which is toxic to plant cells. The experiments described in literature show that phosphinotricine and its acetylate derivative lack toxic effects in animal cells, and that this substance undergoes rapid degradation in soil and surface water. An additional benefit of this selection markeris the fact that in comparison to the kanamycin resistance gene, the application of the bar gene increases the efficiency of the transformation [Tabe 1995, J. Animal Sci. 73, 2752-2759]. The Basta herbicide is also a very effective selection factor. It has been proven that resistance to the herbicide was retained after 2 months of growing of an alfalfa callus, without selection.

Patent description number US6084156 (published 2000.07.04) describes a solution relating to plants producing lytic peptides. Except for description US6084156, patent descriptions US 6448391 (published 2002.09.10) and US6018102 (published 2000.01.25) and US5955573 (published 1999.09.21) also describe solutions relating to the stabilizing combination of polypeptides with ubiquitin lytic peptide, as well as a method of manufacturing through the subcloning of a nucleic acid coding the sequence of a lytic peptide into a plasmid vector containing a promoter and a sequence coding the ubiquitin polypeptide, in which the ubiquitin polypeptide is bound with the 5' end of the sequence of the nucleic acid sequence of the lytic peptide undergoing translation as a fusion polypeptide. Descriptions US6018102 and US5955573 further describe solutions regarding gene constructs combining the ubiquitin lytic peptide, their protein products and their production and application.

Patent description US6306663 (published 2001.10.23) describes a solution relating to new compounds containing ubiquitin recognition elements and a protein binding factor. This invention also relates to the application of said compounds in order to modulate the level and/or level of activity of the target protein. These compounds are useful in application against infection, inflammation states, cancer and genetic diseases, as well as herbicides and insecticides. Patent description US6068994 (published 2000.05.30) describes an invention relating to the expression of ubiquitin. The solution describes a method for conjugating ubiquitin to the expression of a gene, facilitating a controllable high level of production of heterologous proteins possessing destabilizing amino-acid residues in a terminal position. Ubiquitin fusion proteins undergoing expression in yeast are precisely cleaved in vivo through endogenous hydrolases specific for ubiquitin into heterologous yeast proteins such as human alfa-1- antitrypsin, human gamma-interferon and human HIV virus protein, which are initiated with destabilising residues. A synthetic vector containing a synthetic gene of monomeric yeast ubiquitin was constructed and underwent expression under the control of the yeast regulatory glucose promoter. This system may be used to increase the level of expression for proteins showing a low level of expression and for the production of proteins possessing destabilizing amino-acid residues in a terminal position.

In patent descriptions US 5847097 (published 1998.12.08) and US5646017 (published 1997.07.08) a method is presented for the production or modification of protein structure at the level of protein or gene in order to produce specific amino-termini in vivo or in vitro. These methods are based on the unnatural introduction of protein-ubiquitin conjugates and the determination that the half-life of the protein in vivo is a function of the N-terminal amino- acid of the protein.

Patent description US5620923 (published 1997.04.15) describes a method of manufacturing synthetic protein products containing approximately forty amino-acid residues lengthened by ubiquitin at the carboxyl end, undergoing expression in prokaryotic cells such as E. coli. This process may be applied in the production of peptides containing from 2 to around 40 amino-acid residues and is particularly applicable in the production of peptides containing from 5 to 40 amino-acid residues.

Patent description US 5494818 (published 1996.02.27) relates to a generic class of ubiquitin-specific proteases which specifically cleave groups near the C-end residue of ubiquitin in a ubiquitin fusion protein, regardless of size. More specifically, the invention relates to ubiquitin-specific proteases from a class isolated from a cell and isolated DNA sequences coding this class of protein.

The stability of a heterologous protein and the arrangement of multi-subunit structures depend on the biochemical surroundings of the plant cell, and what this entails, the localization of the sub-cellular expression. The structures, where the localization of heterologous protein expression seems to be optimal are: the cell surface, the endoplasmic reticulum, and the Golgi body [Haq 1995, Science 268, 714-716].

KDEL is a sequence of four amino-acids (lysine (K), asparagine (D), glutaminic acid (E), and leucine (L)), which is found at the C-end of the protein and causes it to be retained in the endoplasmic reticulum. The studies of Wandelt et al. have shown that the attachment of the KDEL sequence causes a significant increase in the level of expression of the heterologous protein in transgenic plants [Wandelt 1992, The Plant Journal, 2:181-182]. It turned out that the highest level of expression was observed when the protein was directed towards the ER. The retention of the protein in the can increase the level of expression to a large degree, from 10 to 100 fold. The expressed level can amount to from 0,35% to 2% of the total soluble protein and sometimes may even reach a higher level [Fischer 2000, J. Biol. Regul. Homeost. Agents 14, 421-477; Fischer 2000, Transgenic Res. 9, 279-299].

Patent description US6207165 (published 2001.03.27) describes a solution relating to a vaccine protecting swine against pathological states connected to respiratory and reproductive tract diseases. In this solution, an immunogenic composition is presented inducing an immunological response against CSVF, containing a plasmid coding the E2 protein (earlier called El) and undergoing expression in host cells in vivo.

In patent applications US6136320 (published 2000.10.24), US6034298 (published 2000.03.07), US5914123 (published 1999.07.22), US5484719 (published 1996.01.16) pa vaccine is presented, undergoing expression in plants. An anti-virus vaccine is described, produced in transgenic plants and introduced into the organism in two ways, either through a standard introduction of the vaccine or through the consumption of a portion of such aplant, particularly in the form of a vegetable or fruit juice. The DNA sequence responsible for the coding of an antigen of a pathogenic virus and its expression is isolated and fused to a promoter, which can regulate the level of antigen produced in a transgenic plant. This gene is then transferred into a plant cell using procedures enabling its inclusion in the plant's genome, using Agrobacterium tumenfaciens. Here, the viral immunogen is a protein from a virus selected from a group containing the gastroenteritis or hepatitis virus. Solution US6034298 related to potatoes and tomatoes. Patent description US5965134 (published 1999.10.12) describes immunogenic polipeptides of pestiviruses, particularly CSFV, and particularly its non-structural protein plO and vaccines and diagnosis using the described polypeptides or nucleic acid.

Despite the above described studies concerning the delivery of a vaccine against CSFV a need still exists for the manufacture of effective tools facilitating the easy production of effective vaccines immunising against this virus.

The goal of the present invention is to produce the means which may be used to obtain a vaccine against CSFV. A particular goal of the present invention is the production of chimaeric proteins which would contain the selected antigens and which could be used in vaccination against CSFV, as well as tools facilitating their expression, particularly in plant systems. A particular goal of the present invention is the production of means for the production of a transgenic plant which could be used to produce edible vaccines against CSFV.

Unexpectedly, the realization of such a determined goal and the solution of the described problems in the techniques connected with proteins arising in cells on the basis of exogenous DNA were achieved in the present invention.

The topic of the invention is a chimaeric protein characterized in that it contains the amino-acid sequence of the E2 protein of the Brescia strain of CSFV or its fragment, fused to at least one from among the following sequences: the VTS sequence, the KDEL sequence, or the ubiquitin sequence. Preferentially, it contains one of the following sequences: the amino- acid sequence of the E2 protein of the Brescia strain of CSFV fused to the VTS sequence and the KDEL sequence, or the amino-acid sequence of the E2 protein of the Brescia strain of CSFV with the KDEL sequence, or a shortened amino-acid sequence of the E2 protein of the Brescia strain of CSFV fused to a ubiquitin sequence. The topic of the invention is sequence coding a chimaeric protein characterized in that it contains the sequence coding the E2 protein of the Brescia strain of CSFV or its fragment, fused to at least one from among the following sequences: the sequence coding the VTS, the sequence coding the KDEL sequence, the sequence coding the bar marker sequence, or the sequence coding ubiquitin. Preferentially it contains one of the following sequences: the sequence coding the E2 protein of the Brescia strain of CSFV fused to the sequence coding the VTS, the sequence coding the KDEL sequence, and the sequence coding the bar marker sequence, or the sequence coding the E2 protein of the Brescia strain of CSFV fused to the sequence coding the KDEL sequence and the sequence coding the bar marker sequence, or the sequence coding the E2 protein of the Brescia strain of CSFV fused to the sequence coding the bar marker sequence or a shortened sequence coding the E2 protein of the Brescia strain of CSFV fused to the sequence coding ubiquitin. Preferentially, the sequence according to the invention contains a sequence selected from among sequences presented in Fig. 18 to Fig. 20. The next topic of the present invention is a construct containing a sequence coding a chimaeric protein according to the invention, as mentioned above. Preferentially, in the construct according to the present invention, the sequence coding the chimaeric protein is fused to the sequence of the 35SCaMN promoter or the seed-specific promoter leg A. Preferential constructs are shown in Figs. 1 to 4.

The next topic of the present invention is a plant cell characterized in that it contains the above defined construct according to the present invention. The next topic of the present invention is a method for the production of the chimaeric protein characterized in that it encompassed the introduction into cells of Agrobacterium tumefaciens using the electroporation method, the transformation of plants with a bacterial strain containing this vector, and the obtainment of the expression of the chimaeric protein, where the vector contains the construct defined above according to the present invention. The next topic of the present invention is a method for the production of a transgenic plant characterized in that it encompassed the introduction into cells of Agrobacterium tumefaciens using the electroporation method, the transformation of plants with a bacterial strain containing this vector, and regeneration of the plants, where the vector contains the construct defined above according to the present invention. The next topic of the present invention is a transgenic plant characterized in that it possesses cells transformed with the construct according to the present invention as defined above.

The next topic of the present invention is the application of the transgenic plant possessing cells transformed with the construct according to the present invention as defined above.

The next topic of the present invention is the application of the protein containing the amino-acid sequence of the E2 protein of the Brescia strain of CSFV or its fragment preferentially fused to at least one sequence selected from among the following sequences: the VTS sequence, the KDEL sequence, or the ubiquitin sequence in order to obtain a vaccine against the Classical Swine Fever Virus (CSFV). Preferentially the protein applied contains one of the following sequences: the amino-acid sequence of the E2 protein of the Brescia strain of CSFV fused to the VTS sequence and the KDEL sequence, or the amino-acid sequence of the E2 protein of the Brescia strain of CSFV with the KDEL sequence, or a shortened amino-acid sequence of the E2 protein of the Brescia strain of CSFV fused to the ubiquitin sequence. The next topic of the invention is a pharmaceutical composition characterized in that it contains a chimaeric protein containing the amino-acid sequence of the E2 protein of the Brescia strain of CSFV or its fragment fused to at least one sequence selected from among the following sequences: the VTS sequence, the KDEL sequence, or the ubiquitin sequence, or a plant cell containing a construct containing a plant cell containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein, or a transgenic plant possessing cells transformed with a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein, or said plant's tissues or products of their processing and possibly a pharmaceutically acceptable delivery agent. It is preferential that the amino-acid sequence of the said chimaeric protein contains one of the following sequences: the amino-acid sequence of the E2 protein of the Brescia strain of CSFV fused to the VTS sequence and the KDEL sequence, or the amino-acid sequence of the E2 protein of the Brescia strain of CSFV with the KDEL sequence, or a shortened amino-acid sequence of the E2 protein of the Brescia strain of CSFV fused to the ubiquitin sequence. It is preferential that the pharmaceutical composition takes the form of a lyophilysate.

The next topic of the present invention relates to a vaccine against the Classical Swine Fever Virus (CSFV), the amino-acid sequence of the E2 protein of the Brescia strain of CSFV or its fragment fused to at least one sequence selected from among the following sequences: the VTS sequence, the KDEL sequence, or the ubiquitin sequence, or a plant cell containing a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein, or a transgenic plant possessing cells transformed with a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein, or said plant's tissues or products of their processing and possibly a pharmaceutically acceptable delivery agent. It is preferential that the amino-acid sequence of the said protein contains a sequence selected from among the following sequences: the amino-acid sequence of the E2 protein of the Brescia strain of CSFV fused to the VTS sequence and the KDEL sequence, or the amino- acid sequence of the E2 protein of the Brescia strain of CSFV with the KDEL sequence, or a shortened amino-acid sequence of the E2 protein of the Brescia strain of CSFV fused to the ubiquitin sequence.

It is preferential that the vaccine takes the form of a lyophilysate. It is preferential that the vaccine is an oral vaccine.

The next topic of the present invention is a method for the induction of immunity against infections with the Classical Swine Fever Virus (CSFV) in mammals, characterized in that the said mammal has administered to it a composition containing a chimaeric protein containing the amino-acid sequence of the E2 protein of the Brescia strain of CSFV or its fragment fused to at least one sequence selected from among the following sequences: the VTS sequence, the KDEL sequence, or the ubiquitin sequence, or a plant cell containing a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein, or a transgenic plant possessing cells transformed with a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein, or said plant's tissues or products of their processing at such a dose as contains a sufficient quantity of antigen to induce immunity. It is preferential that the composition is administered orally.

The attached figures facilitate a better description of the nature of the present invention.

Figure 1 represents the preparation of the pP35SBE2BAR vector for the transformation of plants, where the foundation stok were the plasmids: pCSFVE2, containing the sequence coding E2 Brescia, pGEM-T, pMG2A, and pP35SGIB.

Figure 2 represents a binary vector, pP35SBE2RBAR, containing the sequence coding E2 with an ER retention signal, under the control of the 35SCaMV promoter, and a bar marker sequence conferring phosphinotricin resistance.

Figure 3 represents the ROK2 plant expression plasmid, where: NosPRO is the Nos promoter, NPT II (Kanamycyna R) is the canamycin resistance gene, NosTER is the Nos terminator, CaMV 35S is the cauliflower mosaic virus promoter, XX is the cloned in nucleotide sequence being the shortened amino-acid sequence of the E2 protein of the

Brescia strain of CSFV fused to the ubiquitin sequence.

Figure 4a and 4b represent the construction of pP35SVBE2RBAR and pPLAVBE2RBAR vectors.

Figure 5 represents a schematic of a VTS molecule. Construction of a two-strand oligomolecule of DNA encompassed the chemical synthesis of single complementary oligodeoxynucleotides called VTS1 and VTS2, and then their hybridization into the VTS molecule. The two single-stranded oligodeoxynucleotides were designed in such a way that following hybridization they made a two-stranded molecule with such "sticky" ends which would arise from digestion with- b and Pst I endonucleases. Figure 6a represents a comparison of the sequence of the legumine A promoter from the Gene Bank, No. X02982 (legA) and the sequence of the isolated promoter of legumine A. Figure 6b represents the entire sequence of the synthesized, two-stranded VTS. Figure 7 represents an analysis of the genomic PCR of lettuce, in order to discover the presence of the sequence coding E2 Brescia, where: M is the DNA mass marker of λ/EcoRI, Hind III, W is the negative control with water as the template, 1-7 are the PCR reactions with transformed plant DNA as templates, K+ is the PCR with plasmid p35SBΕ2BAR as a template, K- is the negative control, a PCR with genomic DNA from a non-transgenic plant as a template.

Figure 8 represents a confirmation of the presence of the transgene in lettuce plants from the TI generation using PCR where: M is the DNA mass marker of λ/EcoRI, Hind III, W is the negative control with water as the template, K- is the negative control PCR with genomic DNA from a non-transgenic plant as a template, 1-8 are the PCR reactions with transformed plant DNA as templates, K+ is the PCR with plasmid p35SBΕ2BAR as a template.

Figure 9 represents an antigen analysis using Western blotting of protein extracts from lettuce of the TO generation transformed with the pP35SBE2BAR vector, where: K+ is the positive control with purified E2 Brescia protein produced in a baculovirus system, 1-6 are protein extracts from transgenic plants, and K- is a protein extract from control plants.

Figure 10 represents an antigen analysis using Western blotting of protein extracts from lettuce of the TI generation transformed with the pP35SBE2BAR vector, where: K+ is the positive control with purified E2 Brescia protein produced in a baculovirus system, 1-8 are protein extracts from transgenic plants, and K- is a protein extract from control plants.

Figure 11 represents a PCR analysis of Genomic DNA from lettuce in order to confirm the presence of the sequence coding E2 Brescia, wherein: W- negative control, water as a template; K- -negative control, PCR using genomic DNA of a non-transgenic plant as a template; 1-10 - PCRs using genomic DNA of transformed plants as templates; K+ - PCR using the plasmid p35SBE2RBAR as a template; M- DNA mass marker λ/EcoRI, Hind III.

PCR analysis of the genomic DNA of lettuce in order to determine the presence of the sequence coding Ε2 Brescia where: W is the negative control with water as the template, K- is the negative control PCR with genomic DNA from a non-transgenic plant as a template, 1 - 10 are the PCR reactions with transformed plant genomic DNA as templates, K+ is the PCR with plasmid p35SBE2BAR as a template, and M is the DNA mass marker of λ/EcoRI, H d

III.

Figure 12 represents an antigen analysis using Western blotting of protein extracts from lettuce transformed with the pP35SBΕ2BAR vector, where: K+ is the positive control with purified E2 Brescia protein produced in a baculovirus system, 1-8 are protein extracts from transgenic plants, and K- is a protein extract from control plants.

Figure 13 represents the confirmation of the presence of the transgene in alfalfa plants where: W is the negative control with water as the template, K- is the negative control PCR with genomic DNA from a non-transgenic plant as a template, and 1-10 are the PCR reactions with transformed plant genomic DNA as templates, K+ is the PCR with plasmid p35SBE2BAR as a template, and M is the DNA mass marker of λ/EcoRI, H d III. Figure 14 represents an ELISA immunoenzymatic assay to determine the expression of the E2 gene in RR lucerne, where: (1, 5,10 ng) + K- are negative controls with the addition of the E2 antigen, 6 to 19 are transgenic plants transformed with the p35SBE2RBAR vector, „-"K is a negative control, untransformed plant. Figure 14a represents an ELISA immunoenzymatic assay to determine the content of antigen E2 in lyophilised lucerne where: (1, 5,10 ng) + K- are negative controls with the addition of the E2 antigen, „-"K is a negative control of lyophilised untransformed plant, - E2 is a system without added E2 antigen being lyophilised plants containing lOμg of E2 antigen per gram of material Figure 15 represents a confirmation of the presence of the transgene in lettuce plants from the TO generation using PCR where : M is a molecular mass marker (lkb Sigma), 1 - 5 are PCR products, K+ is a positive control containing the pP35SE2BUBIROK plasmid, K- is a negative control of untransformed plant

Figure 15a represents an ELISA immunoenzymatic assay to determine the content of antigen E2 in lyophilised lettuce where: -K+ (30, 50, 70, lOOng) is a negative control of untransformed plant with E2 antigen added, -K is a negative control, untransformed plant, E2Ubi are lyophilised lettuce plants transformed with the pP35SE2BUBIROK vector.

Figure 16 represents a PCR analysis of genomic DNA to confirm the presence of the E2 Brescia transgene, where: Kn is untransformed tobacco (negative control), M is a molecular mass DNA marker, Tt6-1 etc. are plants transformed with the pPLAVBE2RBAR vector, Tt7-1 etc. are plants transformed with the pP35SVBE2RBAR vector, W is water (negative control), Kp is the pPLAVBE2RBAR plasmid (positive control).

Figure 17 represents an ELISA immunoenzymatic assay to confirm the expression of the E2 glycoprotein gene in tobacco leaves, where: K-lng, 5ng, lOng are control plants with the addition E2 antigen at the rate of 1, 5 and lOng, 7/2 - 7/20 are tobacco plants transformed with the pP35SVBE2RBAR vector, K- control plant, non transgenic, „-" E2 is a negative control without the addition of purified E2 antigen, „-" II antibody, negative control a system without the addition of the II Mab anti-E2 antibody.

Figure 17a represents an ELISA immunoenzymatic assay to confirm the expression of the E2 glycoprotein gene in tobacco seeds, where: K-lng, 5ng, lOng are control plants with the addition E2 antigen at the rate of 1, 5 and lOng, 6/1 - 6/20 are tobacco seeds transformed with the pPLAVBE2RBAR vector, K- is a protein extract from an untransformed plant, „-" E2 is a negative control without the addition of purified E2 antigen, „-" II antibody, negative control a system without the addition of the II Mab anti-E2 antibody. Figure 17b represents a comparative ELISA analysis for the confiromation of the expression of the E2 glycoprotein gene in tobacco leaves, where: K-lng, 5ng, lOng are control plants with the addition of the E2 antigen at a rate of 1, 5, and lOng, 7/2 - 7/20 are tobacco plants transformed with the pP35SVBE2RBAR vector, 6/1 - 6/20 are tobacco seeds transformed with the pPLAVBE2RBAR vector, K- is a protein extract from an untransformed plant, „-" E2 is a negative control without the addition of purified E2 antigen, „-" II antibody, negative control a system without the addition of the II Mab anti-E2 antibody.

Figure 17c represents a comparative ELISA analysis for the confirmation of the expression of the E2 glycoprotein gene in tobacco leaves, where: K-lng, 5ng, lOng is a protein extract from the seeds of a control plant with the of the E2 antigen at a rate of 1, 5, and lOng, 7/1 - 7/9 are protein extracts from the seeds of tobacco transformed with the pP35SVBE2RBAR vector, 6/4 - 6/17 are protein extracts from the seeds of tobacco transformed with the pPLAVBE2RBAR vector, K- is a protein extract from an untransformed plant.

Figure 18 represents a sequence coding the ubiquitin:: swine fever virus E2 antigen fragment hybrid protein. The fragment was included into a PBS vector into cleavage sites following digestion with the EcoRI and HindUI restriction nucleases.

Figure 19 represents a sequence coding the ubiquitin:: swine fever virus Ε2 antigen fragment hybrid protein. The fragment was included into a pIGCmT7 vector into cleavage sites following digestion with the Ndel and HindUI restriction nucleases.

Figure 20 represents a sequence coding the ubiquitin:: swine fever virus E2 antigen fragment hybrid protein. The fragment was included into a ROK2 vector into cleavage sites following digestion with ϋxeXbal and BamHl restriction nucleases.

Figure 21 represents an ELISA immunoenzymatic assay of a - serum and b -stool of laboratory animals (mice) for the presence of IgG (serum) and IgA (stool) specific for the administered E2 antigen, where: - m E are mice fed transgenic lucerne

- m G are mice fed non-transgenic lucerne m H are mice which were administered phosphate buffer

Example embodiments of the present invention defined above are presented below.

Embodiment 1. Construction of vectors for plant transformation

The first variant related to the construction of the binary vector pP35SBE2BAR for the transformation of plants, containing the sequence coding the E2 protein of the Brescia strain of CSFV. The initial material were the plasmids: pCSFNE2, containing the E2 coding sequence of

Brescia, pGEM-T, pMG2A, and pP35SGIB. The preparation of the vector encompasses the following stages [fig. 1]:

An E2 coding region was amplified on the basis of the pCSFNE2 template using PCR and through specially designed oligonucleotide starters, with a length of 1.2 kb, with the simultaneous introduction of restriction sites for Xbal and Pstl on the 5' and 3' ends.

Additionally, to ensure a proper reading frame, the ATG codon was introduced at the 5' end of the E2 sequence.

The amplified fragment was introduced into the pGEM-T plasmid, a universal plasmid for the cloning of PCR products. The pGTBE2 plasmid was the result. This plasmid was used as a template for the accurate sequencing of the region coding Brescia E2.

The pGTE2B plasmid was cleaved with Xbal and Pstl enzymes, and thus a fragment was obtained containing the E2 sequence flanked by Xbal and Pstl sites. The region coding E2 was introduced into the pMG2A plasmid. The pMG2ABE2 plasmid was the result.. From the pMG2ABE2 plasmid, the sequence coding E2 along with the Νt terminator was cleaved out using Xbal and H dIII enzymes.

Vector pP35SGIB was cleaved with the Xbal and H dIII enzymes in order to remove the gus gene with intron, and the Νt terminator. Next, into the cleaved pP35SGIB vector, the sequence coding E2 was introduced along with the Nt terminator. The result was the plant transformation plasmid pP35SBE2BAR, containing the sequence coding Brescia E2 under the control of the 35SCaMV promoter and a bar marker sequence conferring phosphinotricine resistance.

The second variant related to the construction of the binary vector pP35SBE2BAR, containing the sequence coding the Brescia E2 with the KDEL sequence directing the E2 protein into the ER [fig. 2].

The initial material were the plasmids: pCSFVE2, containing the E2 coding sequence of Brescia, pGEM-T, and pP35SBE2RBAR. The preparation of the vector encompasses the following stages. An N-terminal section of the E2 coding region was amplified on the basis of the pCSFVE2 template using PCR, with a length of around 670 kb. To ensure the correct readingto ensure a proper reading frame, the ATG codon was introduced at the 5' end of the E2 sequence. At the 3 '-end the AAA GAT GAA CTT sequence was introduced, coding the four amino acids, KDEL. This sequence causes proteins to be retained in the ER. The whole was outfitted with appropriate cleavage sites, Xbal at the 5' end and Xhόl at the 3 '-end. All of these elements were introduced using pecially designed oligonucleotide starters. Next, the amplified fragment was inserted into pGEM-T, a universal plasmid for the cloning of PCR products. The pGTBE2KR plasmid was the result. This plasmid served as a template for the sequencing of the E2R coding region. The sequence coding E2KR was cleaved out of the pGTBE2KR plasmid using the Xbal and Xhόl enzymes. Simultaneously, the pP35SBE2KRBAR plasmid was also cleaved using the Xbal and Xhόl enzymes. Thus, after purification, the E2 coding sequence was eliminated. Using ligation, the E2R sequence was inserted in place of the E2 region. This resulted in the pP35SBE2RBAR plasmid, containing the E2 coding sequence with an ER retention signal under the control of the 35SCaMV promoter, and a bar marker sequence conferring phosphinotricine resistance. The third variant was the construction of the pP35SE2BUBIROK vector containing a shortened sequence of the E2 glycoprotein fused with a ubiquitin sequence. Transfer of the binary pP35SE2BUBIROK vector into cells of A. tumefaciens using the electroporation method.

Schematic of the production of ihe Ubi_BresciaE2_SR_w_PBS fragment included in the PBS(-) vector- Stage I and cloning of ubiquitin in the PBS(+) and pBuescript SK(-) vectors.

The ubiquitin-coding gene was amplified by PCR using Saccharomyces cerevisiae yeast genomic DNA as a template using the forward UBl and reverse UBKl primers. The primers were designed on the basis of a DNA sequence coding the ubiquitin protein from position 767 to 995. The leading primer lengthens the amplified DNA molecule at the 5' end by introducing EcoRI and Ndel restriction endonuclease recognition sites, as well as a nucleotide substitution, while maintaining the amino-acid sequence. Guanine 785 is replaced by adenine. The reverse primer introduces changes to the nucleotide sequence of the 3' end of the DNA molecule, while maintaining the amino-acid sequence. The changes are:

• guanine 977 is replaced by cytosine,

• cytosine 978 is replaced by thymine,

• guanine 983 is replaced by adenine, • adenine 986 is replaced by cytosine,

• adenine 987 is replaced by cytosine,

• adenine 989 is replaced by cytosine.

As a result of changes in the nucleotide sequence of the amplified gene stemming from the use of the UBKl primer, recognition sites appear for the restriction nucleases -4/711 and SαcII. The mixture was separated electrophoretically after PCR in a polyacrylamide gel. The amplified fragment, 240 bp long, was eluted from the polyacrylamide gel³. The resultant DNA fragment was amplified using PCR using the forward UBl primer and the reverse UBK2 primer. The UBK2 primer lengthens the elongated fragment of DNA at the 3 ' end, introducing the TAA stop codon, and a BamRl restriction nuclease recognition site. The mixture was separated electrophoretically after PCR on a polyacrylamide gel. The amplified fragment, 259 bp long, was eluted from the polyacrylamide gel. The eluted fragment was digested with the restriction nucleases EcoRI and BamHl, and de-proteinated . The resulting DNA fragment was ligated with the plasmids:

1. PBS(+) digested with the same restriction nucleases 2. pBluescript SK(-) digested with the same restriction nucleases.

The ligation product used to transform E. coli cells of the NM522 strain. Following the isolation of plasmid DNA from the transformed E. coli colonies, restriction analysis was used to confirm the presence of the DNA insert coding ubiquitin. The accuracy of the gene sequence was verified by sequencing. The SΕQBLT3 and Ml 3 primers were used for sequencing. The PBS(+) vector with the cloned gene coding ubiquitin was digested with the restriction nucleases Aflϊl and Hindlϊl. Following digestion, the mixture was separated electrophoretically in an agarose gel, from which linear plasmid DNA was excised and then eluted. Stage II Cloning of the E2 antigen

From the plasmid containing the full sequence of antigen E2 of CSFV, a 660 bp fragment, from position 2428 to 3087, was amplified using PCR and primers UBIBRES1 and UBIBREKl . Primers UBIBRES1 and UBIBREKl were designed based on the sequence of of the gene coding antigen E2 of CSFV. The forward UBIBRES1 primer introduces changes to the nucleotide sequence of the 5' end of the DNA molecule, while maintaining the amino-acid sequence. The changes are:

• guanine 2430 is replaced by thymine,

• adenine 2452 is replaced by cytosine,

• guanine 2454 is replaced by cytosine. The reverse (UBIBREKl) primer lengthens the amplified fragment of DNA at the 3' end, introducing a stop codon, and Xhόl and Hindlϊl restriction nuclease recognition sites. The mixture was separated electrophoretically after PCR on a polyacrylamide gel. The amplified fragment, 680 bp long, was eluted from the polyacrylamide gel³. The resulting DNA fragment was used as a template in a PCR reaction using the forward primer UBIBRE2 and the reverse primer UBIBREKl . The forward primer UBIBRE2 lengthens the 5' end of the DNA molecule introducing a complementary sequence of 16 bp to the 3' fragment coding the C-end region of ubiquitin. The mixture was separated electrophoretically after PCR on a polyacrylamide gel. The amplified fragment, 700 bp long, was eluted from the polyacrylamide gel³, The eluted fragment was digested with the restriction nucleases 4 711 and HindUI, and de-proteinated The resulting DNA fragment was ligated with the vector resulting from the completion of Stage I. The ligation product was used to transform E. coli cells of the NM522 strain. Following the isolation of plasmid DNA from the transformed E. coli colonies, restriction analysis was used to confirm the presence of the Ubi_Rresc.αΕ2_SR_wPBS insert. The accuracy of the Ubi_Rre-.cz^' E2_SR_wPBS fragment cloned in the PBS(+) vector was verified by sequencing using SEQBLT3 and Ml 3 primers.

A schematic for the production of the Ubi_BresciaE2_SR w_PIG fragment isereted into the pIGCmTl vector.

The PBS(+) vector with the Ubi_Rresc.^'αE2_SR_wPBS insert was digested with NJel and Hindϊll restriction nucleases. The mixture was separated electrophoretically after PCR on a polyacrylamide gel. The digested DΝA fragment 906 bp long was eluted from the polyacrylamide gel³. The reulting fragment was ligated with the pIGCmT7 vector digested with the restriction nucleases Ndel and Hindlϊl, deproteinated. The ligation product was used to transform E. coli cells of the ΝM522 strain. Following the isolation of plasmid DNA from the transformed E. coli colonies, restriction analysis was used to confirm the presence of the Ubi_Rre-.cz^'αE2_SR_w_PIG insert. The accuracy of the Ubi_--.re-.c/αE2_SR_w_PIG fragment cloned in the pIGCmT7 plasmid was verified by sequencing.

A schematic for the production of the Ubi_BresciaE2_SR_w_ROK2 fragment inserted into the ROK2 vector

From the pIGCmT7 plasmid containing the Ubi_Rresc-αE2_SR_w_PIG insert, a fragment of DNA was amplified using PCR with the forward UBIROCK primer and the reverse BREROCK primer. The UBIROCK and BREROCK primers were designed based on the sequence of the amplified fragment. The UBIROCK primer introduces changes in the sequence of the 5' end of the amplified DNA fragment, removing the Ndel restrictase recognition site, and introducing a recognition site for the Xbal restriction nuclease. The BREROCK primer introduces changes to the 3' end of the amplified DNA fragment, by removing recognition sites for the Xhol and HindUI restriction nucleases, and introducing a recognition site for the Bamlll restriction nuclease. The mixture was separated electrophoretically after PCR on a polyacrylamide gel. The digested DNA fragment 911 bp long was eluted from the polyacrylamide gel³, and then treated with the BamRl and Xbal restrictases, and deproteinated. The resulting DNA fragment was ligated with the pBluescript SK(-) vector digested with the same enzymes and deproteinated. The ligation product was used to transform cells of E. coli of the NM522 strain. Following the isolation of plasmid DNA from the transformed E. coli colonies, restriction analysis was used to confirm the presence of the Ubi_Rre-.czαΕ2_SR_w_Blue insert. The accuracy of the Ubi_Rre-?cz^'αE2_SR_w_Blue fragment was verified by sequencing. The SEQBLT3, Ml 3, UBIROCK, CGLYNK2 primers were used in the sequencing. The pBluescript SK(-) vector, containing the cloned insert Ubi_Rre-? cz αE2_SR_w_Blue was digested with Xbal and BamHl restriction nucleases. The mixture was separated electrophoretically after PCR on a polyacrylamide gel. The digested DΝA fragment 911 bp long was eluted from the polyacrylamide gel . The resulting DΝA fragment was ligated with the ROK2 vector digested with Xbal and BamHl restriction nucleases, and deproteinated following digestion. The ligation product was used to transform cells of E. coli of the ΝM522 strain. Following the isolation of plasmid DNA from the transformed E. coli colonies, restriction analysis was used to confirm the presence of the Ubi_Rre-.cz^'αE2_SR_w_ROK2 insert, [fig.3].

The fourth and fifth variants of the experiment encompassed the construction of vectors: pP35SNBE2RBAR containing the 35S promoter, the VTS sequence and the KDEL sequence directing the E2 protein to the ER and pPLAVBE2RBAR containing the legA promoter, the VTS sequence and the KDEL sequence directing the E2 protein to the ER [fig.

4a and b].

All of the vectors used during the work were based on the binary plasmid pGPTF- BAR. The vectors prepared thus contained the bar gene as a selection marker for transgenic plants. The bar gene codes a specific phosphofransferase which deactivates phosphinotricine, the active ingredient in such herbicides as Basta or Glyfosat. In the pGPTV-BAR vector and its derivatives, the mentioned marker gene is found under the control of the constitutive nopaline synthase promoter (ΝOS).The pGPTV-BAR plasmid also contains a uid gene coding β-glucuronidase (GUS) along with the nopaline synthase terminator (NOSt) within the restriction sites for EcoR I, Xba I, Sma and Hind III. Following the removal of the uid sequence and NOSt, arbitrary expression cassettes can be inserted this spot.

The construction of binary vectors encompassed several stages: a) preparation of pea seed-specific storage protein promoters Fragments of DNA encompassing promoter sequences along with fragments of sequences coding storage proteins of the pea were isolated from the pool of total pea DNA through amplification on the template of genomic DNA using appropriate starters. Based on sequences available from the GeneBank (GB), specific starters were designed for the promoter of the storage protein legumine A (legA), (GB ID X02982, for the DNA fragment, 1388bp). The PCR reaction was performed on a template of genomic DNA of the pea isolated from the Feltham First variety (legA) and the Greenfeast variety. These varieties were the primary source of sequences placed in the Gene Bank. Seeds of these genotypes were acquired from the English collection at Norwich and the Polish collection from the Horticultural Station in Wiatrόw. The amplified reaction product obtained was separated on an agarose gel and then purified. Using restriction sites inserted during the PCR, the DNA fragment was cloned into the plasmid vector pBluscriptSK, producing the pBSKlegA vector. The subsequent sequence analysis showed a nearly complete identity, 99,7% of the cloned DNA fragment with the sequence of the legA promoter available in Gene Bank. The resulting legA sequence contained TATA-box motifs typical for eukaryotic promoters and a characteristic legumin-box sequence responsible for the specific activity of the promoter in developing seeds. The cloned sequence also contained a portion of the coding sequence, encompassing initiation codons and signal peptides (SP_ of storage proteins. b) preparation of a two-stranded oligomer of DNA for the construction of binary vectors

Construction of a two-stranded oligomer of DNA encompassed the chemical synthesis of single, complementary oligodeoxynucleotides called VTSl and VTS2, and their subsequent hybridization into the VTS particle.

The synthesis of individual oligomers was performed at the Laboratory of Bioconjugate Chemistry, IChB PAN.

Two single stranded oligodeoxynucleotides designed in such a way that following hybridization they comprised a two-stranded molecule with such s"sticky" ends that would arise during digestion with the Xba and Pst restriction nucleases [fig. 5] . Thanks to this, the resultant two-stranded fragment was suitable for direct cloning into a plasmid digested with the mentioned enzymes, following the phosphorylation of the 5' ends of the molecule. c) preparation of vectors:

1. Insertion of restriction sites for EcoR and Xba via PCR into the sequence encompassing the legA promoter (PlegA) and the signal peptide (SP) of legumine A and the cloning of the modified promoter into pUC18, resulting in the plδPlegAEX plasmid, and then sequence analysis. 2. Using PCR, the insertion of restriction sites for Spe and Xho the TAA (STOP) codon into the E2 sequence, and additionally the signal sequence of the KDEL amino-acid motif. The modified E2 region was cloned into the pGEM-T plasmid, resulting in pGTE2 and pGTBE2R. The accuracy of the E2 sequence was verified by sequencing.

3. Using PCR, the insertion of restriction sites for Pst, Xho and HindUI into the sequence of the NOS terminator (NOSt). The terminator was then cloned into pUC18, resulting in p 18NOSt, and then sequenced.

4. A two-stranded oligomer of DNA was prepared containing a signal sequence directing proteins into vacuole (vacuolar targeting sequence, VTS). It was then cloned into pUC18, resulting in plδVTS and its accuracy was verified through sequencing.

5. The previously cloned 35S CaMV promoter was adapted in the pBluescriptKS vector by removing the Pst restriction site, resulting in pKSP35SGI.

Following the preparation of the VTS sequence, the construction of expression cassettes (boxes) and binary vectors was undertaken.

6. NOSt was cloned into pi 8VTS, resulting in pi 8VNt.

7. The ΕlAlfort sequence was inserted into the p 18 VNt plasmid. 8. The prepared expression cassette, VTS-E2Alfort-NOSt, was moved onto the pGPTV-

BAR vector replacing GUS-NOSt, resulting in pVE2BAR

9. The legA promoter was cloned into pVE2BAR, along with the SP sequence, resulting in the binary vector pPLANE2B AR

The remaining vectors bearing the sequence coding the E2 antigen were prepared through modifications of the pPLANE2BAR vector:

10. Replacing P/eg-4, the 35S promoter was inserted, resulting in the pP35SNE2BAR vector.

11. In the pPLANE2BAR plasmid, in place of the ΕlAlfort sequence, the modified ElBrescia was inserted, resulting in pPLANBE2RBAR. 12. In the pP35SNE2BAR vector, in place of the ΕlAlfort sequence, the modified

HlBrescia was inserted, resulting in pP35SNBE2RBAR.

Embodiment 2. Transformation and preparation of transgenic plants

The first experimental variant encompassed the transfer of the binary vector pP35SBE2BAR into cells of A. tumefaciens using electroporation [fig. 2].

Bacteria suspended in glicerol and stored in an ultrafreezer (temperature of -70° C) were defrosted in an icebath. To bacteria prepared in such fashion 1 to 20 ng of plasmid DΝA was added, gently stirred and incubated on ice for 2 - 6 min. Next, the bacteria were transferred to a chilled cuvette for electroporation. The cuvette was placed in the electroporator and current was applied. The electroporation was performed at 25 μF and 2500 V. Following electroporation, the bacteria were suspended in 1ml of SOC medium and transferred into flasks. The bacteria were shaken for 1 - 2 hrs. at 28°C, 50 - 60 rpm. Following incubation, the suspension was inoculated into YEB media with antibiotics and cultured for 2 days at 28°C. The grown agrobacteria were inoculated into 10ml of liquid YEB medium with selection factors: canamycin 50mg/l, rifampicine lOOmg/1 ' in Erlenmeyer flasks and cultured at 28°C. Following 16 - 20 hours of culturing the suspension of agrobacteria was inoculated onto fresh YEB ^K50'^R10° medium 1:100 and further cultured at 28°C until the bacteria attained the logarithmic stage of growth. The bacterial growth phase was estimated based on OD of the suspension at a 600 nm wavelength. The optimal OD value was around 1. In further stages of the experiment, the cultures were centrifuged for 10 min. at 6 000 rpm and 4°C. The supernatant was removed, and the bacteria were suspended in fresh MSGA medium, in a volume equivalent to that of the culture. The prepared suspension was poured into Petrie dishes. At the same time plant material was prepared for transformation. The next stage of the first variant was the transformation of plants by A. tumefaciens, strain EHA105 containing the binary vector pP35SBE2BAR.

The initial material for the transformation were seeds of lettuce, of the Aramir variety. The material was collected into flasks, and then bathed for 2 min in 70 % ethanol. Next, the ethanol was removed, and the seeds were rinsed in sterile water. In a further stage , the seeds were incubated for 20 min. in a 25 % solution of the commercial bleach, Clorox with an addition of Tween 20 (at a concentration of 0,01 %). Following the required time for sterilisation, the material was washed 4 - 5 times for 2-3 minutes with sterile distilled water, until the Clorox was completely washed out. Sterilised lettuce seeds were placed on dishes with 0,8% agar. The seeds germinated at a temperature of 22 - 24°C, under weak lighting. After 2 to 3 days the cotyls were cut off, and placed in the suspension of A. tumefaciens and incubated for 10 min., with gentle mixing. After the inoculation, the cotyls were placed with the ventral side onto LR3A medium. The co-culture was maintained for 2 - 4 days, in the dark at 28°C. Following the end of co-culture the cotyls were transferred onto selective LR3 A medium, containing the Basta selective factor at a concentration of 2,5 mg/1 and carbencilin at a concentration of 500 mg/1.

The cotyls on which a callus formed were transferred every 5 days onto fresh LR3A medium containing the same concentration of selection factors. The regenerating plants were excised from the callus, and grafted onto LR2A medium with a constant concentration of selection factors. When the plants reached a size of around 2 - 3 cm, they were transferred into jars containing 1/2SH medium with the Basta selection factor at a concentration of 2,5 mg/1 and carbencilin at a concentration of 500 mg/1. Rooted plants were transplanted into pots containing sterile sand mixed 1:1 with pearlite and they were adapted to in vivo conditions. Culture of lettuce seedlings, TI generation.

Seeds from the primary transformants were sterilised and placed in Petri dishes containing 0,8%) agar with added Basta herbicide at 2,5 mg/1. The seeds germinated for 1-2 days at a temperature of 28°C in the dark. The saplings developing from seeds were transferred to pots and in vivo conditions.

The second variant of the experiment encompassed the transfer of the binary vector pP35SBE2RBAR into _J4. tumefaciens cells using electroporation.

Bacteria suspended in glicerol and stored in an ultrafreezer (temperature of -70°C) were defrosted in an icebath. To bacteria prepared in such fashion 1 to 20 ng of plasmid DNA was added, gently stirred and incubated on ice for 2 - 6 min. Next, the bacteria were transferred to a chilled cuvette for electroporation. The cuvette was placed in the electroporator and current was applied. The electroporation was performed at 25 μF and 2500 V.

Following electroporation, the bacteria were suspended in 1ml of SOC medium and transferred into flasks. The bacteria were shaken for 1 - 2 hrs. at 28°C, 50 - 60 rpm. Following incubation, the suspension was inoculated into YEB media with antibiotics and cultured for 2 days at 28°C. The grown agrobacteria were inoculated into 10ml of liquid YEB medium with selection factors: canamycin 50 mg/1, rifampicine 100 mg/1 ' in Erlenmayer flasks and cultured at 28°C. Following 16 - 20 hours of culturing the suspension of agrobacteria was inoculated onto fresh YEB ^K50'^R10° medium 1 :100 and further cultured at 28°C until the bacteria attained the logarithmic stage of growth. The bacterial growth phase was estimated based on OD of the suspension at a 600 nm wavelength. The optimal OD value was around 1. In further stages of the experiment, the cultures were centrifuged for 10 min. at 6 000 rpm and 4°C. The supernatant was removed, and the bacteria were suspended in fresh MSGA medium, in a volume equivalent to that of the culture. The prepared suspension was poured into Petrie dishes. At the same time plant material was prepared for transformation. The next stage of the first variant was the transformation of plants by A. tumefaciens, strain EHA105 containing the binary vector pP35SBE2RBAR.

The initial material for the transformation were seeds of lettuce, of the Bibb Burple strain. The material was collected into flasks, and then bathed for 2 min in 70 % ethanol. Next, the ethanol was removed, and the seeds were rinsed in sterile water. In a further stage , the seeds were incubated for 20 min. in a 25 % solution of the commercial bleach, Clorox with an addition of Tween 20 (at a concentration of 0,01 %). Following the required time for sterilisation, the material was washed 4 - 5 times for 2-3 minutes with sterile distilled water, until the Clorox was completely washed out!

Sterilised lettuce seeds were placed on dishes with 0,8% agar. The seeds germinated at a temperature of 22 - 24°C, under weak lighting. After 2 to 3 days the cotyls were cut off, and placed in the suspension of A. tumefaciens and incubated for 10 min., with gentle mixing. After the inoculation, the cotyls were placed with the ventral side onto LR3A medium. The co-culture was maintained for 2 - 4 days, in the dark at 28 °C. Following the end of co-culture the cotyls were transferred onto selective LR3A medium, containing the Basta selective factor at a concentration of 2,5 mg/1 and carbencilin at a concentration of 500 mg/1.

The cotyls on which a callus formed were transferred every 5 days onto fresh LR3A medium containing the same concentration of selection factors. The regenerating plants were excised from the callus, and grafted onto LR2A medium with a constant concentration of selection factors. When the plants reached a size of around 2 - 3 cm, they were transferred into jars containing 1/2SH medium with the Basta selection factor at a concentration of 2,5 mg/1 and carbencilin at a concentration of 500 mg/1.

Rooted plants were transplanted into pots containing sterile sand mixed 1 : 1 with pearlite and they were adapted to in vivo conditions. The total number of explanations used in the transformation was 148. As a result of regeneration, 25 plants were obtained on the selective media, so the efficiency of the transformation was 16,9%.

The third variant of the experiment was identical procedure- wise to the stage of the transfer of the binary vector pP35SBE2RBAR into A. tumefaciens cells using electroporation, inclusive. It further encompassed the transformation of plants with A. tumefaciens strain EHA105 containing the binary vector pP35SBE2RBAR and the adaptation of the method of the regeneration of seeding lucerne, varieties Rangelander and A2.

The initial materials were leaves, cotyls or leaf stems of commercial varieties of lucerne, Rangelander and A2. The Rangelander variety was made available by the Department of Crop Science, University of Guelph, Canada. The A2 variety was made available by the lab of prof. A. Kondorosi, Institute des Sciences Vegetales CNRS, Gif-sur-Yvette, France. The material was collected into flasks, and then bathed for 2 min in 70 % ethanol. Next, the ethanol was removed, and the seeds were rinsed in sterile water. In a further stage , the seeds were incubated for 20 min. in a 25 % solution of the commercial bleach, Clorox with an addition of Tween 20 (at a concentration of 0,01 %). Following the required time for sterilisation, the material was washed 4 - 5 times for 2-3 minutes with sterile distilled water, until the Clorox was completely washed out.

The sterilized material was damaged through cutting its edges with a scalpel, and then placed in a suspension of A. tumefaciens and incubated for an hour, in the dark with gentle agitation. Following inoculation, the explanted tissues were dried on sterile tissue and put onto medium for co-culture. The co-culture was maintained for 1-2 days in the dark, at a temperature of 26°C. Following the end of the co-culture, the explants were rinsed free of the bacteria in sterile water and placed on selection medium. For the first ten days the explants were cultured in a phytotron, at a temperature of +24°C, in the dark, and then brought into the light in the culture room, and onto a medium with a different composition of plant growth regulators.

Regenerating explants were transferred onto fresh medium every 14 days. The forming somatic embryos were separated from the callus. When the somatic embryos reached the stadium of advanced torpedo, they were transferred onto a medium with a different composition of growth regulators. Rooted plants were planted in pots containing a sterilised mixture of sand and pearlite (1:1). They were then inoculated with Rhizobium melilotti and adapted to in vivo conditions.

Adaptation of the method of regeneration of commercial lucerne varieties Rangelander and A2 Based on the experiments of the Department of Crop Science, University of Guelph, Canada, where work has been carried out for years on the transformation of the Rangelander commercial lucerne variety and where the media based on the SH composition [Shenk 1972] have been used, and for this variety an optimal procedure has been set up (Tab.l). The method of regeneration of the A2 variety, however, has been adapted from the Institute des Sciences Vegetales at Gif-sur-Yvette. However, when applied in its original form, this method was not as effective, as it was after some modifications (Tab.2). The changes pertained to the stimulation of morphogenesis. Somatic embryos developed into complete plants only after the reduction of macroelements and saccharose by half in the medium. The activity of the medium was also slightly elevated (by 0.1 units), when it was noticed that a slightly higher activity caused the medium to clarify a little. Furthermore, it was noticed that the application of iron in the form of EDFS, as recommended by the French researchers gave worse results than the application of iron chelate according to the MS medium. Tab. 1 Schematic ofthe regeneration ofthe Rangelander variety.

Tab. 2 Schematic ofthe regeneration ofthe A2 variety.

The fourth variant was the transformation of plants with A. tumefaciens variety LBA4404 containing the binary vector pP35SE2BUBIROK.

The initial material for the transformation were seeds of lettuce, ofthe Aramir variety. The material was collected into flasks, and then bathed for 2 min in 70 % ethanol. Next, the ethanol was removed, and the seeds were rinsed in sterile water. In a further stage , the seeds were incubated for 20 min.^" in a 25 % solution of the commercial bleach, Clorox with an addition of Tween 20 (at a concentration of 0,01 %). Following the required time for sterilisation, the material was washed 4 - 5 times for 2-3 minutes with sterile distilled water, until the Clorox was completely washed out.

Sterilised lettuce seeds were placed on dishes with 0,8% agar. The seeds germinated at a temperature of 22 - 24 °C, under weak lighting. After 2 to 3 days the cotyls were cut off, and placed in the suspension of A. tumefaciens and incubated for 10 min., with gentle mixing. After the inoculation, the cotyls were placed with the ventral side onto LR3A medium. The co-culture was maintained for 2 - 4 days, in the dark at 28 °C. Following the end of co-culture the cotyls were transferred onto selective LR3A medium, containing the canamycin selective factor at a concentration of 200mg/l and timentine at a concentration of 300mg/l. The cotyls on which a callus formed were transferred every 5 days onto fresh LR3A medium containing the same concentration of selection factors. The regenerating plants were excised from the callus, and grafted onto LR2A medium with a constant concentration of selection factors. When the plants reached a size of around 2 - 3 cm, they were transferred into jars containing 1/2SH medium with the canamycin selection factor at a concentration of 200mg/l and timentine at a concentration of 300mg/l.

The fifth variant encompassed the transfer of the binary vectors pP35SVBE2RBAR and ρPLAVBE2RBAR into cells of A. tumefaciens using electroporation.

Bacteria suspended in glicerol and stored in an ultrafreezer (temperature of -70° C) were defrosted in an icebath. To bacteria prepared in such fashion 1 to 20 ng of plasmid DNA was added, gently stirred and incubated on ice for 2 - 6 min. Next, the bacteria were transferred to a chilled cuvette for electroporation. The cuvette was placed in the electroporator and current was applied. The electroporation was performed at 25 μF and 2500 V. Following electroporation, the bacteria were suspended in 1ml of SOC medium and transferred into flasks. The bacteria were shaken for 1 - 2 hrs. at 28°C, 50 - 60 rpm. Following incubation, the suspension was inoculated into YEB media with antibiotics and cultured for 2 days at 28°C.

The grown agrobacteria were inoculated into 10ml of liquid YEB medium with selection factors: canamycin 50mg/l, rifampicine lOOmg/1 ' in Erlenmayer flasks and cultured at 28°C. Following 16 - 20 hours of culturing the suspension of agrobacteria was inoculated onto fresh YEB ^K50>R10° medium 1:100 and further cultured at 28°C until the bacteria attained the logarithmic stage of growth. The bacterial growth phase was estimated based on OD of the suspension at a 600 nm wavelength. The optimal OD value was around 1. In further stages of the experiment, the cultures were centrifuged for 10 min. at 6 000 rpm and 4°C. The supernatant was removed, and the bacteria were suspended in fresh MSGA medium, in a volume equivalent to that of the culture. The prepared suspension was poured into Petrie dishes. At the same time plant material was prepared for transformation with A. tumefaciens LBA4404 containing vectors ρP35SVBE2RBAR and pPLAVBE2RBAR. The initial material were leaves of tobacco plants of the Wisconsin 38 variety, originating from the in vitro culture at the ICHB PAN in Poznan. From the in vitro culture, from 3-4 week old plants, explants were taken, pieces of leaves of about 1-2 cm . They were placed in the suspension of agrobacteria and inoculated for 10-20 min., with intermittent, gentle mixing. Following inoculation, the leaf fragments were lightly dried on tissue, and then placed bottom side down on MS medium. The co-culture was maintained for 2-3 days, in the dark at a temperature of 28°C. Following the end of the co- culture, the agrobacteria were rinsed off in sterile water, the explants were then gently dried on tissue and placed bottom side down onto the regenerative-selective medium MST with the selective factors Basta 20mg/l and carbencilin 500mg/l..

The in vitro tobacco culture was maintained at 22-24°C and a photoperiod of 16/8 (16 hrs. light, 8 hrs. darkness). Following 4-5 weeks of culture, the developing grains ofthe transgenic callus were excised from transformed fragments of leaves and placed singly onto MST medium, with the same concentration of selection factor as before. The tobacco plants regenerating over the next 3-5 weeks were excised from the callus and again transferred onto MST medium (Basta 20 mg/1, carbencilin 250 mg/1). Plants of about 2cm were transferred into jars containing 1/2MS medium (Basta 20 mg/1, carbencilin 250 mg/1). Developed and rooted tobacco plants were transferred into in vivo conditions. In vivo tobacco cultures were maintained in glasshouses. Rooted tobacco transformants were placed in pots containing potting soil with pearlite (1:1), and were then tempered by nightly cloche removal for extended periods of time. The adaptation of the plants to in vivo conditions was carried out over 3-7 days. The plants were watered with aged tap-water depending on their needs.

Embodiment 3. Expression and analysis

The resulting plant material from the first variant was examined using molecular analysis: a) PCR analysis of genomic DNA of lettuce to determine the presence of the sequence coding E2 Brescia [fig. 7]. The material for the isolation of genomic DNA were young leaves of both the TO and TI generations. The PCR showed that not all of the plants growing on a selective concentration of phosphinotricin also possessed the transgene sequence. The result obtained may suggest the chimaeric character of TO generation plants. Analysis of the TI generation confirmed the presence of the transgene E2 Brescia in the genomes of 97% of lettuce plants [fig. 8] .

b) analysis of antigens using Western blotting of protein extracts from lettuce of the TO generation transformed with the pP35SBE2BAR vector [fig. 9, 10]. The results of this analysis suggest that the level of expression of the antigen protein E2 in lettuce leaves is relatively low. It is also noticeable that the level of expression of the antigen in TO generation plants seems to be very variable.

The resulting plant material from the second variant was examined using molecular analysis: a) PCR analysis of genomic DNA of lettuce to determine the presence ofthe sequence coding E2 Brescia [fig. 11]

The material for the isolation of genomic DNA were young leaves ofthe TO generation. b) analysis of antigens using Western blotting of protein extracts from lettuce of the TO generation transformed with the pP35SBE2BAR vector [fig.12]. The material for protein isolation were young lettuce leaves.

In the third variant, for both varieties of commercial lucerne, the transformation wash characterized by unusually high efficacy. For the purposes of analysis, 50 plants were regenerated (separate genotypes) of each variety. The resulting plants were examined using molecular analysis: a) PCR analysis of genomic DNA of lettuce to determine the presence of the sequence coding E2 Brescia [fig. 13].

The material for the isolation of genomic DNA were the leaves of transformed lucerne plants. b ) immunoenzymatic ELISA test analysis The analysis material was protein isolated from transgenic plants. For this 12 of 50 plants were selected of the commercial lucerne variety Rangelander transformed with A. tumefaciens containing the binary vector pP35SBE2RBAR [fig. 14].

Plate immunoenzymatic test ELISA

The analysis material was protein isolated from the leaves of lucerne. In a majority of the plants analysed, the expression of the protein takes place at a very high level. The protein content per gram of fresh plant mass hovers between 1300 - 1700 ng. Thanks to such a high expression of the E2 glycoprotein in plant material it may be used in research on lab animals in order to provoke an immune response.

The Maxisorp microwell plate was coated with monoclonal MabV8 anti-E2 antibody suspended in a carbonate buffer pH=9,6, diluted to 1:5000, and incubated at 37^°C for 3 hours. Next, they were rinced three times with phosphate buffer PBS pH=7,4 with 0,05% Tween 20. At a further stage, the plate was blocked with 5% skim milk dissolved in PBS, 390μl per well. After 30 min., the plate was rinsed as described earlier, and coated with the E2 antigen. This stage ofthe experiment was carried out overnight at 4°C. Next, the plate was rinsed again, and coated with monoclonal MabV3 anti-E2 antibody suspended in PBS buffer, lOOμl/well at a dilution of 1 :5000. The plate was incubated at a temperature of 37°C at lOOrpm for 1 hour, and then rinsed, and coated with monoclonal anti-mouse IgG (whole molecule) conjugated with alkaline phosphatase (Sigma) suspended in PBS buffer, 100 μl/well at a dilution of 1 :20000. The stage was performed at 37°C over 1 hour. Next, the colour-forming reaction was performed by the addition of substrate (INC): 2-amino-2metyl-l,3 propanediol (10-times concentrated) and an aqueous solution of p-nitrophenylphosphate (50-times concentrated) and topped to the desired volume with MQ water. The volume of substrate per well was 100 μl. It was incubated in the dark for 30min. The readings ofthe reaction was made at λ=405nm on a BioRad Model 550 plate reader. For further immunoenzymatic analysis, those plants were selected which contained the highest level of antigen per gram of fresh mass. These plants were lyophilised, which allowed for the maximum homogenization of the green material and condensation of the antigen. To estimate the concentration of antigen the same immunoenzymatic ELISA test was used which was used in the analysis of the plants. Finally, in 1 gram of lyophilised lucerne, 1 Oμg of antigen E2 were found[Fig. 14a]. This material was used to immunise the lab animals. In the fourth variant the resulting plants were examined using molecular methods: a ) PCR analysis of genomic DNA of lettuce to determine the presence of the transgene in lettuce plants transformed with A.tumefaciens containing the pP35SE2BUBIROK [fig. 15]. b) immunoenzymatic ELISA test analysis

The analysis material were lyophilised lettuce plants of the Aramir variety containing the E2 glycoprotein sequence fused to the ubiquitin sequence. For this purpose, protein was isolated from lyophilised green material, and analysed immunoenzimatically. The ELISA test protocol determined earlier was used. The experiments carried out showed the correctness of the application of the ubiquitin sequence as a fusion protein to increase the expression of the E2 glycoprotein in plants. A perliminary analysis of the protein content showed that the lyophilised lettuce samples contained around 200μg [Fig.15a].

In the fifth variant, the resulting transgenic tobacco plants from the TO generation were analysed with regards to the presence of the sequences coding the E2 CSFV protein in their genomes.

Molecular methods were used for this purpose: a) PCR analysis of genomic DNA of lettuce to determine the presence ofthe sequence coding

El Brescia [fig. 16].

The analysis material was genomic DNA isolated from leaf fragments from transformed plants.

Using the pP35SVBE2RBAR for the transformation, 100% transformation efficiency was obtained. For the pPLAVBE2RBAR vector, the efficiency reached 89.5%. b) immunoenzymatic ELISA analysis to determine the expression ofthe E2 protein.

The analysis material was protein isolated from leaves and seeds of tobacco. For this purpose, a selection was made of 18 out of 20 plants transformed with A. tumefaciens containing the vector pP35SVBE2RBAR [Fig.17] and 10 out of 20 plants transformed with A. tumefaciens containing the pPLAVBE2RBAR vector. [Fig. 17a]. In both cases, the immunoenzymatic analysis confirmed the expression of glycoprotein E2 in the plants, both in the leaves and in the seeds. An experiment was made to compare the level of E2 antigen between the leaves and seeds, which were transformed with a vector, where the E2 gene was controlled by the constitutive promoter and the legumine promoter. It was observed that the more efficient expression of the protein occurs in those plants, where the E2 glycoprotein sequence is under the control of the constitutive 35SCaMV promoter [Fig.17b], as compared to the legumine promoter [Fig. 17c]. The effect of this may be the presence of storage lipids in the tobacco seeds, and the problems they entail in the isolation and marking of protein in the immunoenzymatic assay. It is known that lipids obstruct interactions between proteins, including antigen and antibody in ELISA assays.. It is possible, then, that the obtained values ofthe level of E2 antigen are somewhat underestimated.

Embodiment 4. Immunization of mice with transgenic material.

Groups of mice: In the experiment, 22 week-old female Balb/C mice were used. Two experimental groups of 8 mice were set up, and one of six mice. E - the group receiving the lyophilisate containing the antigen: the E2 protein of the Brescia strain of CSFV fused with the KDEL sequence

G - the group receiving non-transgenic lucerne lyophilisate

H- the group receiving PBS buffer

Plant material used in the procedure. The lucerne lyophilisate from the commercial variety Rangelander containing the sequence of the E2 glycoprotein fused with the amino-acid motif KDEL, which causes the expression ofthe protein in the endoplasmic reticulum. As a control, lyophilisate from non-transgenic lucerne ofthe same strain as the transgenic one was used.

Method of immunization. The lyophilisate was administered orally, using a gastric tube. A schematic ofthe immunization: a) The immunised group E received the transgenic lucerne lyophilisate. The control group G received the non-transgenic lucerne lyophilisate. Group H received PBS buffer. b) Double immunization at a pre-determined interval. c) The dose of each respective group of mice was : Antigen dose: lg of lyophilisate contains: 10 μg of antigen

I immunization. The mice were given 500 ng of antigen, suspended in 200 μl of PBS. After administration ofthe antigen, the mice were starved 20 hours.

1. Collection of animal material.

Blood collection took place from periocular blood vessels using a capillary tube of 150 μl before the experiment, 2 and 4 weeks post immunization I and 2 weeks post immunization II. Stool sample collection took place at the same time as the blood collection

2. Preparation ofthe material for analysis. BLOOD: a) centrifugation immediately following collection in Eppendorf tubes, at 4°C, 3000 rpm, 20 min. b) the separated serum was kept at -20 °C until time of determination STOOL: a) the material collected was homogenised in PBS at a ratio of 1 :5 with a polypropylene pestle b) it was centrifuged for 10 min, at 4 °C, at 12rpm. c) the supernatant was separated and stored for further analysis at - 20°C. 3. Immunological analysis.

The samples collected were used after defrosting to carry out assays using a highly sensitive ELISA.

Microwell plate immunoenzymatic ELISA assay a) A polisorp (Nunc) plate was coated with antigen E2 0.5μg/ml in PBS, at 50μl/well b) The plate was incubated overnight at 4 °C. c) It was rinsed three times in PBS with Tween (0,05%), pH 7,4. d) Blocking was performed with 5% skim milk in PBS for 30 min. at room temperature, 390 μl/well e) Rinsing as in point c f) Serum samples were applied in appropriate gradient dilutions, 50 μl/well g) Incubation took place over 2 hrs. at 37°C, and 60 rpm rotation h) Rinsing as in point c i) Incubation with antibodies anti-mouse IgG and IgA conjugated with alkaline phosphatase or peroxidase, 100 μl/well. The antibody dilution was 1:2000., and the incubation time was

1.5 hours at 37°C, and 60 rpm rotation j) Rinsing as in point c k) Incubation with substrates for peroxidase or alkaline phosphatase lOOμl/well, 1 hour in the dark, at room temperature. 1) The reaction was stopped with 1M sulphuric acid m) The reaction was read with a BioRad, model 550 plate reader, at λ=405nm.

Analysis of the content of IgG antibody in the serum and IgA antibody content in the stool of animals immunised with the lyophilisate of lucerne containing the E2 antigen of the

Classical Swine Fever Virus has indicated the presence of antibodies specific for the antigen administered. These results seem to indicate that the administration of plant material containing immunogenic proteins causes an immune response. The administration pathway, along with feed, does not lead to the inactivation or destruction of the immunogenic molecules, which may simplify future vaccination of animals.

Claims

Patent Claims

1. A chimaeric protein, characterized in that it contains the amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus, or its fragment fused with at least one of the following sequences: the VTS sequence, the KDEL sequence, the ubiquitin sequence.

2. A protein according to Claim 1, characterized in that it contains one of the following sequences:

- amino-acid sequence of protein E2 ofthe Brescia strain of the CSFV virus fused with the VTS sequence and the KDEL sequence;

- amino-acid sequence of protein E2 ofthe Brescia strain of the CSFV virus fused with the KDEL sequence; - a shortened amino-acid sequence of protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence of ubiquitin.

3. A sequence coding a chimaeric protein, characterized in that it contains a sequence coding protein E2 ofthe Brescia strain of the CSFV virus or its fragment, fused with at least one sequence selected from among the following sequences: the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence, the sequence coding ubiquitin.

4. A sequence according to Claim 3, characterized in that it contains one of the following sequences: the sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding VTS, the sequence coding KDEL and the sequence coding the bar marker sequence;

- the sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding KDEL and the sequence coding the bar marker sequence;

- the sequence coding protein E2 of the Brescia strain ofthe CSFV virus fused with the sequence coding the bar marker sequence; a shortened sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding ubiquitin.

5. A sequence according to Claim 3, characterized in that it contains one of the sequences selected from among the sequences presented in Fig. 18 - Fig. 20.

6. A construct containing a sequence coding a chimaeric protein, characterized in that it contains the sequence coding protein E2 of the Brescia strain of the CSFV virus or its fragment, fused with at least one sequence selected from among the following: the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence, the sequence coding ubiquitin.

7. A construct according to Claim 6, characterized in that the sequence coding the chimaeric protein is bound with the 35SCaMV promoter sequence or the seed specific promoter legA.

8. A construct according to Claim 6, characterized in that it contains one of the following sequences: - the sequence coding protein E2 of the Brescia strain ofthe CSFV virus fused with the sequence coding VTS, the sequence coding KDEL and the sequence coding the bar marker sequence;

- the sequence coding protein E2 ofthe Brescia strain of the CSFV virus fused with the sequence coding KDEL and the sequence coding the bar marker sequence; - the sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding the bar marker sequence; a shortened sequence coding protein E2 ofthe Brescia strain of the CSFV virus fused with the sequence coding ubiquitin.

9. A construct according to Claim 6, characterized in that it has been selected from among the constructs represented in Fig. 1 - 4.

10. A plant cell, characterized in that it contains a construct containing the sequence coding protein E2 of the Brescia strain ofthe CSFV virus, or its fragment, fused with at least one sequence selected from among the following: the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence, the sequence coding ubiquitin.

11. A cell according to Claim 10, characterized in that it contains a construct containing one ofthe following sequences:

- the sequence coding protein E2 of the Brescia strain ofthe CSFV virus fused with the sequence coding VTS, the sequence coding KDEL and the sequence coding the bar marker sequence; - the sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding KDEL and the sequence coding the bar marker sequence;

- the sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding the bar marker sequence; - a shortened sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding ubiquitin.

12. A method for the preparation of a chimaeric protein, characterized in that it encompasses the introduction of a vector into cells of Agrobacterium tumefaciens using electroporation, the transformation of plant varieties containing this vector, and the obtainment of the expression of the chimaeric protein, where the vector contains a construct containing a sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus, or its fragment, fused with at least one sequence selected from among the following: the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence, the sequence coding ubiquitin.

13. A method according to Claim 12, characterized in that it encompasses the insertion of a construct containing one ofthe following sequences:

- the sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding VTS, the sequence coding KDEL and the sequence coding the bar marker sequence; - the sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding KDEL and the sequence coding the bar marker sequence;

- the sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding the bar marker sequence; a shortened sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding ubiquitin.

14. A method of manufacturing of transgenic plant, characterized in that it encompasses the introduction of a vector into cells of Agrobacterium tumefaciens using electroporation, the transformation of plant varieties containing this vector and the regeneration of plants, where the vector contains a construct containing the sequence coding protein E2 of the Brescia strain of the CSFV virus, or its fragment, fused with at least one sequence selected from among the following: the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence, the sequence coding ubiquitin, into a plant cell.

15. A method according to Claim 14, characterized in that it encompasses the introduction of a construct containing one ofthe following sequences: - the sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding VTS, the sequence coding KDEL and the sequence coding the bar marker sequence;

- the sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding the bar marker sequence; a shortened sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding ubiquitin.

16. A transgenic plant, characterized in that it possesses cells transformed with a construct containing the sequence coding protein E2 of the Brescia strain of the CSFV virus, or its fragment, fused with at least one sequence selected from among the following: the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence, the sequence coding ubiquitin.

17. A transgenic plant according to Claim 16, characterized in that it possesses cells transformed with a construct containing one ofthe following sequences:

- the sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding the bar marker sequence; a shortened sequence coding protein E2 of the Brescia strain ofthe CSFV virus fused . with the sequence coding ubiquitin.

18. An application of a transgenic plant possessing cells transformed with a construct containing the sequence coding protein E2 of the Brescia strain of the CSFV virus, or its fragment, fused with at least one sequence selected from among the following: the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence, the sequence coding ubiquitin. to produce a vaccine against Classical Swine

Fever Virus (CSFV).

19. An application according to Claim 18, characterized in that, it possesses cells transformed with a construct containing one ofthe following sequences:

- the sequence coding protein E2 of the Brescia strain of the CSFV virus fused with the sequence coding VTS, the sequence coding KDEL, the sequence coding the bar marker sequence; - the sequence coding protein E2 ofthe Brescia strain ofthe CSFV virus fused with the sequence coding KDEL and the sequence coding the bar marker sequence;

20. An application of the protein containing the amino-acid sequence protein E2 of the Brescia strain of the CSFV virus, or its fragment, fused with preferentially at least one sequence selected from among the following: the VTS sequence, the KDEL sequence, the ubiquitin. sequence to produce a vaccine against Classical Swine Fever Virus (CSFV).

21. An application according to Claim 20, characterized in that, the protein contains one of the following sequences:

- the amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus fused with the VTS sequence and the KDEL sequence; - the amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus fused with the KDEL sequence;

- the amino-acid sequence of protein E2 ofthe Brescia strain ofthe CSFV virus;

- the shortened amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus fused with the ubiquitin sequence.

22. A pharmaceutical composition, characterized in that it contains a chimaeric protein containing the amino-acid sequence of protein E2 ofthe Brescia strain of the CSFV virus or its fragment, fused with at least one from among the following sequences: the VTS sequence, the KDEL sequence, the ubiquitin sequence, or a plant cell containing a construct containing a nucleotide sequence coding the amino-acid sequence of such a protein or a transgenic plant possessing cells transformed with a construct containing a nucleotide sequence coding the amino-acid sequence of such a protein, or its tissues or products of their processing, and possibly a pharmaceutically permissible carrier.

23. A pharmaceutical composition according to Claim 22, characterized in that the amino-acid sequence ofthe said chimaeric protein contains one ofthe following sequences: - the amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus fused with the VTS sequence and the KDEL sequence;

- the amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus fused with the KDEL sequence;

24. A pharmaceutical composition according to Claim 22, characterized in that it takes the shape of a lyophilisate.

25. A vaccine against the Classical Swine Fever Virus (CSFV), characterized in that it contains a chimaeric protein containing the amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus or its fragment, fused with at least one from among the following sequences: the VTS sequence, the KDEL sequence, the ubiquitin sequence or a plant cell containing a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein or a transgenic plant possessing cells transformed with a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein or its tissues or products of their processing and possibly a pharmaceutically permissible carrier.

26. A vaccine according to Claim 25, characterized in that the amino-acid sequence of the said chimaeric protein is one ofthe following sequences:

- the amino-acid sequence of protein E2 of the Brescia strain of the CSFV virus fused with the VTS sequence and the KDEL sequence;

27. A vaccine according to Claim 25, characterized in that it takes the shape of a lyophilisate.

28. A vaccine according to Claim 25, characterized in that it is an oral vaccine.

29. A method for the induction of immunity against infections of the Classical Swine Fever Virus (CSFV) in mammals, characterized in that said mammal receives a preparation containing the chimaeric protein containing the amino-acid sequence of protein E2 of the

Brescia strain of the CSFV virus or its fragment, fused with at least one from among the following sequences: the VTS sequence, the KDEL sequence, the ubiquitin sequence or a plant cell containing a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein or a transgenic plant possessing cells transformed with a construct containing a nucleotide sequence coding the amino-acid sequence of such a chimaeric protein or its tissues or products of their processing at such a dosage as which contains a sufficient quantity of antigen to induce immunity.

30. A method according to Claim 29, characterized in that said preparation is administered orally.