ES2304812A1 - System to produce peptides and proteins, multimericos, and their applications. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

System to produce peptides and proteins, multimeric, and their applications. The system is based on a dna construct comprising, operatively linked, at least one nucleic acid sequence (a) containing the nucleotide sequence encoding a product of interest, and a nucleic acid sequence (b) that contains the nucleotide sequence that codes for a tetramerization domain, such as the tetramerization domain of the p53 protein; wherein the 3''-end of said nucleic acid sequence (a) is linked to the 5''-end of said nucleic acid sequence (b). The system allows the production of products of interest, for example, peptides and multimeric recombinant proteins, useful in vaccination, therapy or diagnosis, or with application in the industry. (Machine-translation by Google Translate, not legally binding)

Description

System to produce peptides and proteins, Multimerics, and their applications.

Field of the Invention

This invention relates to the production of Multimeric recombinant peptides and proteins through use of an expression system comprising the coding sequence for said peptide or protein of interest and a sequence that encodes a tetramerization domain.

Background of the invention

Currently transgenic plants they represent an alternative to the expensive expression systems of traditional proteins, since they are easily transformable, and, in addition, production costs can be reduced by growing large extensions and harvest them with the infrastructure existing.

In addition, transgenic plants have other potential advantages, since they could be managed with therapeutic or preventive purposes without purification: using the edible organs of this one. In fact, it has demonstrated the possibility of using the plants orally, as inducers of an immune response. Therefore, the plants are they present as ideal candidates to produce new vaccines: more Cheap, safer and easily manageable. However, in At present, the use of plants as biofactories presents Some inconvenients. On the one hand, the low levels of xenoproteins expression, being in the best cases around 0.1-3% of total soluble protein, and, on the other part, the instability in the expression of the transgene during the Plant development or in successive generations. So, to produce recombinant proteins for therapeutic use, industrial or plant vaccines it is necessary to develop strategies that increase the expression levels of the transgene and the accumulation of foreign protein in the plant. Although various strategies have been described to improve the expression and accumulation of foreign proteins in plants, follow There is a need to develop alternative strategies to those currently existing in order to increase the arsenal of available means to express and accumulate foreign proteins in plants.

Different strategies have been published to improve the expression and accumulation of recombinant proteins in plants, such as using different promoters and transcription enhancing sequences, using different polyadenylation signals, optimizing the use of codons for plants or directing foreign proteins to different compartments. cell phones to prevent degradation and obtain a correct conformation (endoplasmic reticulum, chloroplasts, etc.). All these strategies have given discrete improvements in the performance of plants as bio-factories. Other strategies involve the use of fusions to low degradation proteins, such as β-glucuronidase (GUS) and green fluorescent protein (GFP). Good yields have also been published in the production of recombinant proteins in plants when they have the capacity to acquire complex structures such as viral pseudoparticles (virus-like particles), for example, the Norwalk virus capsid protein, or multimerize as they are the cases of the cholera toxin and the thermolabile enterotoxin of Escherichia coli . These results, taken as a whole, seem to indicate, indirectly, the importance of the structure of the foreign protein expressed in its stability and accumulation in plant cells.

Compendium of the invention

The inventors have evaluated the importance of conformation and structure of the foreign protein in its accumulation in transgenic plants, with special emphasis on peptides of little size. To do this, they have evaluated the possibility of increasing the expression levels of a linear vaccine peptide increasing the its stability in the plant by obtaining a complex structure (multimeric) and the levels of expression and / or accumulation of the same peptide without fusing or fusing to a high stability protein (GUS). In this way, they have achieved high levels of expression of a vaccine peptide in genetically transformed plants, increasing the stability of this one in the plant by obtaining a complex structure (multimeric) For this, the domain of tetramerization of p53 protein fused to peptide 2L21 of Canine Parvovirus (CPV), a synthetic vaccine peptide.

P53 is a transcription factor, involved in different cellular functions, such as the cell cycle control, apoptosis and cell differentiation. The p53 protein is formed by 4 functional domains, in addition to a transactivation domain and a proline-rich domain (residues 1 to 43 and 61 to 94, respectively) in the region N-terminal The DNA binding domain has central location in the protein (residues 110-286). The tetramerization domain (DT) is found in the C-terminal region of the protein (residues 326 to 355) (Figure 1).

The structure of the DT has been determined previously both by X-ray crystallography as by nuclear magnetic resonance (NMR). A monomer contains a β-sheet (residues 326 to 333), attached to an α-helix (residues 335 to 355) by a single residue (glycine 334). A monomer is V-shaped and both elements in the secondary structure are an arm of the V. Three amino acids (Ile-332, Phe-338 and Phe-341) form a small hydrophobic cluster in the region of union. Estimates with respect to the surface of interaction between monomers and experimental evidence indicate that the DT is a dimer of dimers. The dimer is formed by the interaction of two monomers via the interaction of their β-sheets to form a double stranded sheet antiparallel, and, via the antiparallel association of its propellers, Create a double helix beam. The formation of the β-antiparallel sheet allows the formation of Eight hydrogen bridges. Β-sheets they are outside the tetramer and therefore are not directly involved in the association between the two dimers. The interface between the propellers is predominantly hydrophobic and envelops Met-340, Leu-344, Ala-347, Leu-348 and Leu-350

The peptide chosen for the practical embodiment of the present invention is the 2V21 epitope of CPV, which is formed by two antigenic sub-sites distant from the amino terminal region of the VP2 protein of the CPV capsid and which had previously been published as the first peptide synthetic vaccine (López de Turiso, JA, Cortés, E., Martínez, C., Ruiz de Ybánez, R., Simarro, I., Vela, C., Casal, I. (1992) J. Virol. 66: 2748 -2753). The knowledge of the three-dimensional structure and the links involved in the formation of the tetramer allows, a priori , to design a fusion strategy that does not affect the conformation of the tetramer. Therefore, the 2L21 peptide was fused in the N-terminal region of the tetramerization domain where it was expected not to prevent the formation of the tetramer. In turn, the same peptide was expressed in unbound plants using the same plasmid and under the control of the same promoter. In addition, peptide 2L21 had previously been successfully expressed in the inventors' laboratory fused to GUS (Gil F., Brun A., Wigdorovitz A., Martínez-Torrecuadra JL, Catalá R., Casal I., Salinas J., Borca MV and Escribano JM, FEBS Letters 488: 13-17, 2001), a very stable protein, with a low degradation rate and perfectly adapted to plant expression, which provided a good reference standard for comparing these strategies.

The invention is illustrated by the development of a high production system of the 2L21 peptide fused to the DT of p53 (2L21-DT) in transgenic plants of A. thaliana , based on the multimerization capacity of the fusion protein with respect to the same peptide without merging or merging to GUS. It was found that the 2L21-DT monomers expressed in A. thaliana transgenic plants are assembled into dimers and tetramers. A high percentage of transgenic plant lines with high levels of recombinant peptide expression was obtained with this strategy. Expression levels obtained with the 2L21-TD construct were even higher than those obtained in transgenic plants transformed with the 2L21-GUS construct. The recombinant peptide (2L21-DT) maintained the antigenic and immunogenic characteristics of the 2L21 peptide, inducing high specific antibody titres in groups of animals immunized both orally and intraperitoneally and this being far superior to that induced by the 2L21- fusion protein. GUS. These results seem to suggest that the tetramers formed could offer not only great stability, but also immunogenic advantages. No transgenic plants were obtained that expressed the unbound fusion peptide, which demonstrates that its stability is dependent on the protein to which it is fused, which confers a greater or lesser resistance to degradation by cellular proteases.

Brief description of the figures

Figure 1 is a schematic representation of the domain structure of the p53 protein and the domain of tetramerization of said p53 protein, including a detail of the amino acid sequence corresponding to the domain of tetramerization (DT) of the p53 protein as well as the structure of the tetramer once formed (Science, vol. 67, March 10, nineteen ninety five).

Figure 2 schematically shows the obtaining binary plasmid pBI-TEV 2L21 -DT.

Figure 3 shows the structure of the region of expression of the binary plasmid pBI-TEV 2L21-DT and the amino acid sequence of the protein of fusion 2L21-DT, the promoter CaMV35S (35S prom), which directs the expression of the corresponding sequence to the antigen 2L21 fused to the tetramerization domain of the p53 protein and the polyadenylation sequence (Nos-ter); LB and RB, delimit the T-DNA that will be integrated into the genome Nuclear plants. The amino acid sequence of the protein 2L21-DT fusion includes the peptide sequence 2L21, the tetramerization domain of p53 and 4 protein amino acid separators (GSPG). The structure is also included expected monomer 2L21-DT and tetramer predictable

Figure 4 shows the result of a Western blot analysis of the expression of the 2L21-DT fusion protein in transgenic Arabidposis thaliana plants. Figure 4A shows the result of a Western blot analysis on denaturing tricine gel loaded with 40 µg of total protein per well; an immunoreactive band close to 8 kDa is observed. Figure 4B shows the result of a Western blot analysis on a gel under native conditions loaded with 40 µg of total soluble protein extracted with a buffer without reducing agents or denaturing agents; two immunoreactive bands are observed one above 16 kDa (dimers) and a majority band of 32 kDa (tetramer). The structure of the 2L21-DT monomer and dimers and tetramers are also included
predictable

Figure 5 illustrates the transcriptional analysis of some lines expressing various amounts of the 2L21-DT fusion antigen. The results of a Northern blot analysis of the transcription of the 2L21-DT fusion gene in Arabidopsis transgenic plants are shown, on 5 µg of total and hybridized RNA using as a probe the complete DNA sequence of the 2L21-DT fusion ( 200 bp) labeled with 32 P.

Figure 6 shows the antibody titer in serum in the different groups of immunized mice. Figure 6A it is a bar chart that collects antibody titers anti-2L21 achieved in the different groups of animals immunized intraperitoneally (DT1, DT2, DT3, DT4, GUS1, GUS2, DT Pool IP, GUS Pool IP) [each DT is a group of mice, like every GUS; Pool IP means "joint intraperitonal inoculum "]. Figure 6B is a bar chart which shows the average of the title developed by the different groups of animals fed with plant leaves expressing each of the peptide fusions.

Figure 7 illustrates the possibility of obtaining multivalent vaccines containing 2 peptides or proteins vaccines according to the present invention and schematically shows the tetramer formation containing 2 vaccine proteins or peptides from a single construction that includes the domain of tetramerization of the p53 protein and 2 foreign genes.

Detailed description of the invention

In one aspect, the invention relates to a DNA construction, hereinafter DNA construction of the invention, comprising, operably linked, at least:

to)

a nucleic acid sequence (A) containing the nucleotide sequence encoding a product of interest; Y

b)

a nucleic acid sequence (B) containing the nucleotide sequence encoding a tetramerization domain;

where the 3 'end of said nucleic acid sequence (A) is attached to the 5 'end of said nucleic acid sequence (B).

The nucleic acid sequence (A) contains the nucleotide sequence that codes for a product of interest. The product of interest can be practically any peptide or protein, regardless of its origin (eukaryotic, prokaryotic, viral, etc.), which can be expressed recombinantly. In a particular embodiment, said product of interest is a peptide or protein useful in vaccination, therapy or diagnosis, for example, CPV 2L21 peptide, RHDV VP60 protein, antibiotic peptides, etc., while in another particular embodiment , said product of interest is a peptide or protein with application in any type of industry, for example, industrial enzymes, such as cellulases, glucanases, proteases, etc.

The nucleic acid sequence (B) contains the nucleotide sequence that codes for a tetramerization domain. A tetramerization domain is a peptide sequence that promotes dimerization and / or tetramerization in the proteins that contain it. Virtually any tetramerization domain can be used in the DNA construction of the invention. In a particular embodiment, said tetramerization domain comprises or is constituted by the tetramerization domain (DT) of the p53 protein, which is in the C-terminal region of the p53 protein (residues 326-355) (Chéne P. , (2001), The role of tetramerization in p53 function. Oncogene 20: 2611-2617).

In a particular embodiment, the construction DNA of the invention comprises a single acid sequence nucleic acid (A) and a single nucleic acid sequence (B). This type DNA construction can result in homotetramers of the product of interest (in the form of a fusion protein where the peptide or protein of interest is fused to DT).

In another particular embodiment, the DNA construct of the invention comprises, in addition to said nucleic acid sequences (A) and (B), a nucleic acid sequence (A ') containing the nucleotide sequence encoding a product of of interest, wherein the 5 'end of said nucleic acid sequence (A') is attached to the 3 'end of said nucleic acid sequence (B). The products of interest encoded by the nucleotide sequences contained in the nucleic acid sequences (A) and (A ') may be the same or different. When they are equal, homotetramers of the product of interest formed by 4 equal monomers each containing 2 residues of the same product of interest are obtained (in the form of fusion proteins where the products of interest are fused to both ends of the DT). However, when they are different, tetramers of the products of interest formed by 4 equal monomers are obtained each containing 2 different products of interest (in the form of fusion proteins where each of the products of interest is fused to a DT end).

In general, the nucleic acid sequence (B) that contains the nucleotide sequence that codes for the tetramerization domain does not fuse directly to the nucleic acid sequence (A) that contains the nucleotide sequence that codes for the product or products of interest but it is advantageous to introduce a spacer (flexible) peptide between the end of the nucleic acid sequence (A) and the beginning of the nucleic acid sequence (B). Thus, if desired, the DNA construct of the invention may also contain, in addition, a nucleic acid sequence (C) containing the nucleotide sequence encoding a spacer peptide (Cl) located between said nucleic acid sequences. (A) and (B), wherein the 5 'end of said nucleic acid sequence (C) is attached to the 3' end of said nucleic acid sequence (A) and the 3 'end of said nucleic acid sequence ( C) is attached to the 5 'end of said nucleic acid sequence (B). Advantageously, said spacer peptide (C1) is a peptide with structural flexibility. Virtually any peptide with structural flexibility can be used. By way of illustration, said flexible peptide may contain repetitions of amino acid residues, in particular of Gly and Ser residues or any other suitable amino acid residue repeat. In a particular embodiment, said flexible spacer peptide comprises the sequence Gly-Ser-Pro-Gly (GSPG) or the sequence (Gly-Ser) 4.

When the DNA construct of the invention contains said nucleic acid sequence (A '), in addition to said nucleic acid sequences (A) and (B), said DNA construct may also contain, if desired, a sequence of nucleic acid (C ') containing the nucleotide sequence encoding a spacer peptide (C2) located between said nucleic acid sequences (A') and (B), wherein the 5 'end of said nucleic acid sequence ( C ') is attached to the 3' end of said nucleic acid sequence (B) and the 3 'end of said nucleic acid sequence (C') is attached to the 5 'end of said nucleic acid sequence (A'). Like (C1), the spacer peptide (C2) can be virtually any structurally flexible peptide, for example, a flexible spacer peptide comprising the GSPG or (Gly-Ser) 4 sequence. The nucleic acid sequence (C ') containing the nucleotide sequence encoding a spacer peptide (C2) may be the same or different from that encoding said spacer peptide (C1) contained in said nucleic acid sequence (C) , whereby said spacer peptides (C1) and (C2) may be the same or different.

In order to facilitate the isolation and purification of the fusion peptide or protein obtained by the present invention, the DNA construct of the invention may contain, if desired, a nucleic acid sequence encoding a peptide capable of being used with isolation or purification purposes of the fusion peptide or protein. Thus, in a particular embodiment, the DNA construct of the invention includes, if desired, a nucleic acid sequence (D) containing the nucleotide sequence encoding a peptide capable of being used for isolation or purification purposes. . Said nucleic acid sequence (D) may be located in any position that does not alter the functionality of the tetramerization domain or of the product or products of interest. By way of illustration, said nucleic acid sequence (D) may be located between said nucleic acid sequences (A) and (B), wherein the 5 'end of said nucleic acid sequence (D) is attached to the 3' end of said nucleic acid sequence (A) and the 3 'end of said nucleic acid sequence (D) is attached to the 5' end of said nucleic acid sequence (B), or said nucleic acid sequence (D) is located upstream of the 5 'end of said nucleic acid sequence (A), or said nucleic acid sequence (D) is located downstream of the 3' end of said nucleic acid sequence (B).

Virtually any peptide or sequence peptide that allows the isolation or purification of the peptide or fusion protein can be used, for example, sequences of polyhistidine, peptide sequences that can be recognized by monoclonal antibodies that can serve to purify the resulting fusion protein by immunoaffinity chromatography, such as tag peptides, etc.

If desired, the DNA construct of the invention may also contain a nucleotide sequence that encodes an amino acid sequence that can be specifically cleaved by enzymatic or chemical means in order to release the product of interest once isolated. fusion protein. In this case, the DNA construct of the invention may further include a nucleic acid sequence (E) containing the nucleotide sequence encoding an amino acid sequence (E1) that can be specifically cleaved by enzymatic or chemical means. , wherein said nucleic acid sequence (E) is next to the 3 'end of said nucleic acid sequence (A). Virtually any amino acid sequence that can be specifically cleaved by enzymatic or chemical means can be used, for example, protease recognition sites (enterokinase, Arg-C endoprotease, Glu-C endoprotease, Lys-C endoprotease, Coagulation factor Xa, etc.) or sites susceptible to being specifically cleaved by chemical reagents (cyanogen bromide, etc.).

When the DNA construct of the invention contains said nucleic acid sequence (A '), in addition to said nucleic acid sequences (A) and (B), said DNA construct may also contain, if desired, a sequence of nucleic acid (E ') containing the nucleotide sequence that encodes an amino acid sequence (E2) that can be specifically cleaved by enzymatic or chemical means, wherein said nucleic acid sequence (E') is next to the end 3 'of said nucleic acid sequence (A'). Like (E1), (E2) can be virtually any amino acid sequence that can be specifically cleaved by enzymatic or chemical means. The nucleic acid sequence (E ') that contains the nucleotide sequence encoding the amino acid sequence ( E2) capable of being specifically cleaved by enzymatic or chemical means may be the same or different from said amino acid sequence (E1 capable of being specifically cleaved by enzymatic or chemical means encoded by said nucleic acid sequence (E), whereby said sequences (E1) and (E2) can be the same or different.

The DNA construct of the invention can be obtained by employing techniques widely known in the state of the art [Sambrook et al ., "Molecular cloning, a Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press , NY, 1989 Vol 1-3]. Said DNA construct of the invention may, operably linked, incorporate a sequence regulating the expression of the nucleotide sequence encoding the product or products of interest, thereby constituting an expression cassette. As used in this description, the term "operably linked" means that the product or products of interest is (are) expressed in the correct reading frame under the control of the control or regulatory expression sequences.

Therefore, in another aspect, the invention provides an expression cassette comprising the construction of DNA of the invention operably linked to a sequence of expression control of the nucleotide sequence that it encodes For the product of interest. The control sequences are sequences that control and regulate transcription and, where appropriate, the translation of the product of interest, and include sequences promoters, coding sequences for regulators transcriptional, ribosome binding sequences (RBS) and / or transcription terminator sequences. In one embodiment in particular, said expression control sequence is functional in prokaryotic cells and organisms while in another embodiment in particular, said expression control sequence is functional in eukaryotic cells and organisms.

Advantageously, said expression cassette It also includes a marker or gene that codes for a reason or for a phenotype that allows the selection of the host cell transformed with said expression cassette. Illustrative examples of said markers that could be present in the cassette of expression of the invention include resistance genes to antibiotics, resistance genes to toxic compounds, and in general all those that allow plants to be selected genetically transformed

The DNA construct of the invention, or the expression cassette provided by this invention, can be inserted into an appropriate vector. Therefore, in another aspect, the invention relates to a vector, such as an expression vector, comprising said DNA construct or said expression cassette. The choice of the vector will depend on the host cell into which it will be subsequently introduced. By way of example, the vector where said DNA sequence is introduced can be a plasmid or a vector that, when introduced into a host cell, is integrated or not into the genome of said cell. The obtaining of said vector can be carried out by conventional methods known to those skilled in the art [Sambrok et al ., 1989, cited supra ]. In a particular embodiment,
said recombinant vector is a viral vector, preferably, a viral vector capable of infecting plants or algae.

Said viral vector can be used to infecting cells susceptible to being infected by said virus. Therefore, in another aspect, the invention relates to a cell infected with said viral vector. In one embodiment in particular, said infected cell is an infected plant cell with an appropriate viral vector, said plant cell being infected capable of expressing the product of interest. Cells Vegetables infected with recombinant viral vectors can obtained after infection of a plant or a protoplast with said recombinant viral vector. An interesting application of the Infectious viral vectors of plants is the expression in plants of proteins or epitopes of animal pathogens in order to produce edible vaccines against these pathogens.

The recombinant vectors provided by this invention can be used to transform eukaryotic or prokaryotic cells. Therefore, in another aspect, the invention relates to a transformed cell comprising said recombinant vector, or said DNA construct provided by this invention, or said expression cassette provided by the invention. Transformed cells can be obtained by conventional methods known to those skilled in the art [Sambrok et al ., 1989, cited supra ]. In a particular embodiment, eukaryotic or prokaryotic cells are co-infected with two or more recombinant vectors provided by this invention. Said recombinant vectors may contain sequences encoding the same products of interest whose expression results in homotetramers in the cells co-transformed with said vectors. Alternatively, said recombinant vectors may contain coding sequences of different products of interest whose expression results in tetramers of 2 different peptides or proteins in cells co-transformed with said vectors, which may be interesting, for example, in multiple immunization developments. . Therefore, in a particular embodiment, one of said vectors (vector I) contains a DNA construct of the invention comprising a nucleotide sequence encoding one or more products of interest while another of the vectors (vector II) contains a DNA construct of the invention comprising a nucleotide sequence that codes for one or more products of different interest (s) to the product or products of interest encoded (s) by the nucleotide sequence contained in the DNA construct present in the vector I.

Accordingly, in another aspect, the invention relates to a transformed cell comprising at least one DNA construct of the invention, or a recombinant vector provided by this invention or an expression cassette provided by this invention. In a particular embodiment, said transformed cell comprises two or more DNA constructs of the invention, or recombinant vectors or cassettes, the same or different, preferably different, provided by this invention. Transformed cells can be obtained by conventional methods known to those skilled in the art [Sambrok et al ., 1989, cited supra ].

In another aspect, the invention relates to a transgenic cell comprising, inserted in its genome, at less, a DNA construct of the invention, or a cassette of expression provided by this invention. In one embodiment in particular, said transgenic cell comes from a plant cell and comprises, inserted in its genome or in the genome of a chloroplast, at least one DNA construct of the invention or a expression cassette provided by the invention. From said transgenic plant cells or from material transgenic plant, transgenic plants can be obtained. By both, in another aspect, the invention relates to a plant transgenic comprising at least one transgenic plant cell provided by this invention. An interesting application of the transgenic plants is the expression in protein plants or animal pathogen epitopes in order to produce vaccines edible against such pathogens. Advantageously, said plants will express different products of interest, for example, different proteins or epitopes of animal pathogens, in order to simultaneously immunize animals against different pathogens

The DNA construct of the invention can be Used to produce products of interest. Therefore, in another aspect, the invention relates to a method of producing a product of interest that includes growing a cell or organism provided by this invention under conditions that allow production of said product of interest. The conditions for optimize the culture of said cell or organism will depend on the cell or organism used. If desired, the method to produce A product of interest provided by this invention includes, in addition, the isolation and purification of said product of interest. In this case, the DNA construct of the invention should advantageously including said nucleic acid sequence (E) [and (E ') where appropriate] previously defined comprising a sequence nucleotide encoding an amino acid sequence (E1) [and (E2) where applicable] that can be specifically split by  enzymatic or chemical means in order to release the product of interest once the fusion protein is isolated.

In general, the product of interest obtained by the method of the present invention is in form of a multimeric fusion protein, namely, dimeric or, mostly, tetrameric. In a particular embodiment, said product of interest is in the form of a homotetramer containing, for example, 4 residues of the same peptide or protein of interest (when using a DNA construct of the invention which contains only one nucleic acid sequence (A) that code for a product of interest) or 8 remains thereof peptide or protein of interest (when two are used DNA constructs of the invention containing each of them two nucleic acid sequences (A) and (A ') that code for the same product of interest) or in the form of a tetramer containing, for example, 2 remains of a first product of interest and another 2 remains of a second product of interest, where said first and second products of interest are products of interest different from each other (when two DNA constructs are used of the invention, each containing a nucleic acid sequence (A) encoding a product of different interest) or 4 remains of a first product of interest and 4 other remains of a second product of different interest, in where said first and second products of interest are products of interest different from each other (when two constructions are used of different DNAs of the invention each containing a nucleic acid sequence (A) encoding a product of different interest) or 4 different interest products (when two different DNA constructs of the invention are used each containing two nucleic acid sequences (A) and (A ') that code for different products of interest), etc., which may allow the development, for example, multivalent vaccines. Therefore, a characteristic of the product expression system of interest provided by this invention is its versatility, which allows to simultaneously express different products of interest.

The invention also provides, in another aspect, a method to express a gene that codes for a product of interest in a plant, which comprises transforming said plant with at least one DNA construct provided by this invention, or an expression cassette or a vector provided by this invention. The transformation of tissue cells Vegetables can be done by conventional methods. For one review of gene transfer to plants, including vectors, DNA transfer methods, etc., see, for example, the book entitled "Genetic engineering and gene transfer", of Marta Izquierdo, Ed. Pyramid (1999), in particular, Chapter 9, entitled "Gene transfer to plants", pages 283-316.

In another aspect, the invention relates to a fusion protein obtainable by expression of the sequence of nucleic acid contained in the DNA construct provided by this invention. More specifically, the invention provides a fusion protein comprising:

(i)

the amino acid sequence corresponding to a domain of tetramerization; Y

(ii)

the amino acid sequence of at least one product of interest.

In a particular embodiment, said domain of tetramerization comprises (or is constituted by) the domain of tetramerization of the p53 protein. Fusion protein can have the amino acid sequence of a single product of interest or Well the amino acid sequence of two products of interest.

Said fusion protein is a protein of multimeric fusion, namely, dimeric or, mostly, tetrameric Therefore, in a particular embodiment, said fusion protein comprises 4 residues of the same product of interest, or 8 remains of the same product of interest. In other particular embodiment, said fusion protein comprises 2 moieties of a first product of interest and another 2 remains of a second product of interest other than said first product of interest, for example, 4 remains of a first product of interest and another 4 remains of a second product of interest other than said first product of interest In another particular embodiment, said protein of fusion includes 4 different products of interest, or 8 products of different interest. Preferably, the protein of fusion provided by this invention comprises 4 residues of a same product of interest, or 8 remains of the same product of interest, or 4 remains of a first product of interest and another 4 remains of a second product of interest other than said first product of interest

Therefore, by way of illustration, in a particular embodiment, the invention provides a protein of fusion comprising:

(i)

the amino acid sequence corresponding to a domain of tetramerization;

(ii)

a amino acid sequence corresponding to a product of interest (A1); Y

(iii)

an amino acid sequence corresponding to a product of interest (A2).

Such products of interest (A1) and (A2) may Be the same or different. In a particular embodiment, said Products of interest (A1) and (A2) are different from each other. In a concrete embodiment, the tetramerization domain comprises (or it is constituted by) the tetramerization domain of the protein p53.

The fusion protein provided by this invention may also contain, if desired, (a) a peptide spacer between the product of interest and the domain of tetramerization; and / or (b) a peptide to facilitate isolation or purification of the peptide or fusion protein; and / or (c) a amino acid sequence capable of being cleaved specifically by enzymatic or chemical means.

In another aspect, the invention relates to a recombinant vaccine comprising a fusion protein provided by this invention, wherein said product of interest comprises an immunogenic peptide or protein, and, optionally, a pharmaceutically acceptable excipient. The vaccine recombinant provided by this invention can be univalent or multivalent, that is, can immunize against one or more pathogens simultaneously. By way of illustration, the fusion protein multimeric present in the recombinant vaccine provided by This invention may contain 4 residues of the same peptide or immunogenic protein, or 8 residues of the same peptide or immunogenic protein In another particular embodiment, said fusion protein comprises 2 residues of a first peptide or immunogenic protein and 2 other residues of a second peptide or different immunogenic protein, or 4 residues of a first immunogenic peptide or protein and other 4 residues of a second different immunogenic peptide or protein.

Therefore, in a particular embodiment, the The invention provides a vaccine comprising:

(i)

the amino acid sequence corresponding to a domain of tetramerization; Y

(ii)

the amino acid sequence of at least one peptide or protein immunogenic

In a particular embodiment, said domain of tetramerization comprises (or is constituted by) the domain of tetramerization of the p53 protein. Virtually any peptide or immunogenic protein (vaccine) can be used in the preparation of the recombinant vaccine provided by this invention; however, in a particular embodiment, they include, said immunogenic peptide or protein is selected from the peptide 2L21 of CPV or VP60 of RHDV.

In another particular embodiment, the vaccine provided by this invention comprises:

(i)

the amino acid sequence corresponding to a domain of tetramerization;

(ii)

the amino acid sequence of a first peptide or protein immunogenic; Y

(iii)

the amino acid sequence of a second peptide or protein immunogenic

In a specific embodiment, the domain of tetramerization comprises (or is constituted by) the domain of tetramerization of the p53 protein. Said first peptide or protein immunogenic may be the same or different from said second peptide or immunogenic protein, in order to obtain univalent vaccines or multivalent In a particular embodiment, said first peptide or immunogenic protein is different from said second peptide or immunogenic protein

As is known, the need to formulate more than one vaccine in the same vaccine preparation is frequent, both in human and animal health. The use of a tetramerization domain, such as the tetramerization domain of the p53 protein, offers very interesting possibilities in this regard since it is possible to postulate the inclusion of more than one vaccine antigen in the construction without interfering a priori in the formation of stable structures. Figure 7 illustrates the possibility of obtaining multivalent vaccines according to the present invention. The figure shows schematically the formation of tetramers containing 2 vaccine proteins or peptides from a single construct provided by this invention comprising the tetramerization domain of the p53 protein. The predicted structure that would be formed from a single expression cassette formed by two genes fused at the ends of the tetramerization domain is included.

The following Example serves to illustrate the invention and should not be considered as limiting the scope of the same.

Example

Multimerization of the 2L21 antigen and its use in immunization of animals I. Materials and methods 1. Growth of Arabidopsis thaliana

The model plant used has been Arabidopsis thaliana , ecotype Columbia. The genus Arabidopsis belongs to the family of the Crucifers ( Brassicaceae or Cruciferae ).

1.1 On land

For the growth of Arabidopsis plants on land, the seeds were sown on the surface, in plastic pots or alveoli, which contained a mixture of universal substrate and vermiculite (3: 1). The mixture was previously soaked in distilled water and sterilized in an autoclave at 101 kPa (1 atm) pressure for 20 minutes at 120 ° C. The pots or alveoli were placed in trays that were then covered with plastic to maintain adequate humidity and prevent contamination during germination. The trays were kept for 48 hours at 4 ° C and in darkness, to favor a homogeneous germination of the seeds. After this, the trays were taken to culture chambers at 22 ° C with a photoperiod of 16 hours of fluorescent light and 8 hours in darkness. After a week after planting, the plastic is removed, always keeping the tray with water. The plants were watered once a week with a minimum universal medium (Haughn and Somerville, (1986), Sulfonylurea resistant mutants of Arabidopsis thaliana. Molec. General Genetics 204: 430-434). The plants were kept in these conditions until flowering begins (6-7 weeks), ideal time for infiltration. Sometimes, to try to improve the yields of the infiltrations, some flowers are cut and the more numerous secondary inflorescences are expected to develop.

1.2 In petri dishes

For the germination of the seeds in plate, it used MS medium (Murashige T. and F. Skoog. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497, 1962), supplemented with sucrose 1% and solidified with 0.8% agar, and the antibiotic correspondent. The seeds were sterilized for 10 minutes in a solution of 30% sodium hypochlorite and Triton 0.01% X-100, and subsequently washed 5 times in sterile water. The seeds were sown on the petri dishes, which They were brought to 4 ° C and dark for 48 hours. Later they were taken to culture chambers under conditions of 22 ° C and 16 hours of light followed by 8 hours of darkness. After two weeks, the seedlings were transplanted to land and grown in the conditions discussed above.

2. Bacterial strains used, culture and obtaining competent

For the transformation and growth of the plasmids the strains of Escherichia coli , DH5-? And TOP-10 (Clontech) were used. The strains of Agrobacterium tumefaciens C58C1 and AGLO 5-? (Hellens R. and Mullineaux P. A guide to Agrobacterium binary Ti vectors. Trends in Plant Science 5: 446-451, 2000), were used for the infiltration of the flowers of A. thaliana .

one

E. coli cultures were performed in LB medium (Sigma) in the presence of the corresponding selective agent (Sambrook et al. 1989) for 14-16 hours at 37 ° C. The preparation of competent cells was carried out by the rubidium chloride method, described by Hanahan (Studies on transformation of E. coli with plasmids. J. Mol. Biol. 166: 557-580. 1983).

Cultures of the different strains of Agrobacterium were performed in liquid LB or plaque, supplemented with 50 µg / ml kanamycin and 50 µg / ml rifampicin (Sambrook et al. 1989), and were maintained 36-48 hours at 28 ° C .

Bacterial cultures were preserved long term in dimethyl sulfoxide (DMSO) at a final concentration of 6% at -80 ° C (Sambrook et al., 1989).

3. Bacterial transformation methods 3.1 E. coli transformation

Strains DH5-? And TOP-10, were used for the maintenance of plasmids and new generation. Both competent strains were transformed following the protocol of Birnboim and Dolly (Birnboin H.C. and Dolly J. A rapid alcaline extraction procedure for recombinant plasmid DNA screening. Nucl. Acid Res. 7: 1513-1516. 1979). Briefly, the plasmid DNA or ligation product was added to an aliquot of 100 µl of competent cells thawed and kept at 4 ° C for 30 minutes Then, a thermal shock was caused at 42 ° C, which which increases the permeability of the pores, 600 µl were added of LB and incubated with shaking at 37 ° C for 1-1.5 hours Bacterial cells sedimented by centrifugation for 3 minutes at 3,500 r.p.m. and they were plated in solid medium to which they had previously been added Selection antibiotics needed. The plates were incubated in stove at 37 ° C overnight.

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3.2 Transformation of A. tumefaciens

The C58C1 and AGLO strains were transformed with the binary plasmids. Briefly, 1 µg of DNA (binary plasmids) was added to an aliquot of 200 µl of said competent cells, without thawing and incubated for 5 minutes at 37 ° C, and then immediately incubated at 4 ° C for 30 minutes. Then, 1 ml of LB was added and cultured at 28 ° C for 3-4 hours. After centrifugation, they were plated in LB medium with kanamycin-rifampin. The plates were incubated at 28 ° C and the colonies took approximately 48 hours to appear. They were subsequently cultivated and used for the infiltration of A. thaliana plants.

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4. Plasmids used 4.1 Commercial plasmids

To obtain the different constructs expressing the different antigens, various commercial plasmids were used. For cloning and sequencing of PCR products, pGEM-Teasy was used. The binary plasmid PBI-121 and derivatives were used in the transformation of Agrobacterium and in the subsequent infiltration of A. thaliana plants. Plasmid pCMV-p53 was used as a template to obtain the sequence corresponding to the tetramerization domain of p53.

pBI-121 (Clontech Cat. 6018-1) : Derived from plasmid pBI-101. It contains the 35S promoter of the cauliflower mosaic virus (35S CaMV) directing the expression of the GUS gene and a 260 bp fragment (base pairs) containing the polyadenylation sequence of the nopalin synthetase (NOS-ter) gene of the Ti plasmid of Agrobacterium . It also contains an RK2 origin of replication (low copy number) and a kanamycin resistance gene.

pGEM-Teasy (Promega) : This plasmid is specially designed for cloning and sequencing of PCR products. It contains a region with multiple restriction sites (polylinker). The polylinker has been previously digested with EcoRV and subsequently 3 'thymidines have been added at both ends to facilitate cloning of the PCR products. In addition, it allows the selection of recombinants by means of the LacZ gene.

pCMV-p53 (CLONTECH Cat. K6004-1) : Plasmid pCMV-p53 is an expression plasmid that contains the protooncogene sequence that encodes the p53 tumor suppressor protein. This plasmid was used as a template to obtain the sequence corresponding to the tetramerization domain of p53.

In addition to these commercial plasmids, also plasmid p35S-TEV (4,051 bp) was used, generated in the laboratory of Dr. Escribano (INIA) and derived from plasmid pBI121 [collection of Dr. Escribano's laboratory (INIA)], which contains the 35S promoter of the mosaic virus cauliflower (35SCaMV), the enhancer sequence of the transcription (TEV) of tobacco mottling virus, after which a region of multiple cloning appears and the signal of Vsp polyadenylation. This plasmid was used in the subcloning of some constructions as a previous step to cloning in the binary plasmid.

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4.2 Transformation plasmids of developed plants during the implementation of this invention

For the realization of this example, the plasmid identified as pBI-TEV 2L21-DT, which was used in the transformation plant genetics The antigen expressed in said plasmid pBI-TEV 2L21-DT is the fusion of 2L21 peptide of canine parvovirus (CPV) to the domain of tetramerization of the p53 protein. The size of the transgene is approximately 0.2 kb.

The sequences encoding said antigen and their respective mergers were obtained by amplification by PCR. 2 types of commercial polymerases were used, the ECOTAQ (Ecogen), which was mainly used in the analysis of colonies, and the Pow DNA polymerase (Roche), which it presents corrective activity and was used to amplify the sequences of transgenes. The primers used were identified as:

p53 one way (5'-3 '): CCCGGG CAAACCACTGGATGGAG [SEQ ID NO: 1]; Y

p53 lap (5'-3 '): CCCGGG CCCTGGCTCCTT [SEQ ID NO: 2]

[The underlined corresponds to the targets of restriction]

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4.3 Obtaining the transformation plasmids of plants (cloning)

To obtain the transformation vector binary, enzymatic modifications of DNA and digestions with restriction endonucleases were performed according to the methods conventional (Sambrook et al. 1989) and following the recommendations of the manufacturers of the corresponding enzymes. The separation of the different DNA fragments was carried out in agarose gels (Pronadisa) of varying concentration between 0.7 and 2% (w / v), depending on the expected size of the fragments at solve. The buffer used was TBE (90 mM Tris, 90 boric acid mM, 1 mM EDTA). The purification of DNA fragments from the agarose gels was carried out with the "Gel Extraction Kit" (Quiagen).

The 5 'protuberant ends became blunt, when necessary, by filling the 3' end with the klenow fragment of E. coli DNA polymerase I (Roche). The incorporation of phosphate groups at the 5 'ends of the DNA was carried out with the T4 polynucleotide kinase (Roche diagnostic). In the ligation reactions, T4 DNA ligase (Roche diagnostic), or the ligation kit of pGEM-Teasy (Promega) was used.

Obtaining plasmid pBI-2L21-GUS is described in the publication of Gil et al. (Gil F., Brun A., Wigdorovitz A., Martínez-Torrecuadra JL, Catalá R., Casal I., Salinas J., Borca MV and Escribano JM Development of a high-yielding system for expression of peptide vaccines in transgenic plants FEBS Letters , 488: 13-17, 2001).

4.3.1 Obtaining the transformation vector pBI-TEV 2L21-DT

The tetramerization domain sequence of p53 protein was obtained by PCR amplification with specific primers, using the plasmid as a template pCMV-p53 (Clontech). The primers used were designed to obtain the tetramerization domain sequence of the p53 protein flanked by two restriction targets. Sayings primers were the oligonucleotides identified as p53 ida [SEQ ID NO: 1] and p53 turn [SEQ ID NO: 2]. After amplification by PCR, the sequence corresponding to the tetramerization domain was cloned into the pGEM-Teasy cloning plasmid (Promise), obtaining plasmid pGEM-DT. After sequencing and sequence verification, this was digested plasmid with the restriction enzyme KpnI and the 3 'protuberant ends by incubation with the klenow fragment of DNA polymerase (Roche).

Then, the vector was cleaned and cut with the restriction enzyme SmaI, releasing the fragment corresponding to the sequence of the tetramerization domain. This insert was ligated and cloned into the transformation plasmid of plants pBI-TEV-2L21-GUS previously digested with SmaI, constituting the plasmid pBI-TEV 2L21-DT [Figure 2]. The structure of the expression region of said binary plasmid pBI-TEV 2L21-DT is shown in the Figure 3. Said plasmid was introduced by transformation into A. tumefaciens for subsequent infiltration of plants.

5. Genetic transformation of plants mediated by A. tumefaciens

Agrobacterium cells previously transformed with the different plant transformation plasmids were grown in 500 ml of LB medium (Sigma), supplemented with kanamycin and rifampin (Sambrook et al. 1989), for 48 hours at 28 ° C. After centrifugation at 3,000 rpm for 20 minutes, the sediment (containing the bacteria) was resuspended in 200 ml of infiltration medium: 2.35 g / l DM (Murashige and Skoog, 1962), 5% sucrose, 10 g / l of 6-benzylaminopurine and 0.02% Silweet L-77 (Lehle Seeds, catalog number VIS-01).

5.1 Infiltration of flowers

In order to perform the infiltration, plants of 6-7 weeks of age were used, with the greatest possible number of inflorescences. Inflorescences were immersed in the infiltration medium containing Agrobacterium in a vacuum chamber, subjecting them to a pressure of 5 kPa (50 mbar) for 10 minutes (Bechtold N. and Pelletier G. In Agrobacterium -mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration Methods Mol. Biol. 82: 259-266, 1998). Once the time had elapsed, the vacuum was released as quickly as possible, the plants were washed with water and covered with plastic to prevent their drying out and possible contamination between the different infiltrated plants. Sometimes plants infiltrated in the absence of a vacuum (Clough SJ and Bent AF Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . Plant. J. 16: 735-743, 1998). They were kept in culture chambers, under controlled conditions of light, humidity and temperature until the development and maturation of silicones.

5.2 Means of growth and plant selection transgenic

Seeds from previously infiltrated A. thaliana plants were sterilized in a solution containing 30% bleach and 10% Triton X-100, and seeded in a petri dish in GM medium (MS 4.7 g / l, 1% sucrose, morpholin-ketanesulfonic acid (MES) 0.5 g / l, agar 8 g / l and pH 5.7) supplemented with ampicillin and kanamycin (Sambrook et al. 1989).

The T1 seeds were kept at 4 ° C temperature and darkness for 48 hours and subsequently in an in vitro culture chamber, under controlled conditions (16 hours of light and 8 hours of darkness, 22 ° C temperature). After 12-15 days, the transgenic seedlings (capable of growing normally in the presence of kanamycin) were transplanted to land and taken to culture chambers for growth and development.

6. Analysis of transgenic plants 6.1 Genomic DNA isolation, quantification, enzymatic modifications, electrophoresis and transfer to membranes

Arabidopsis genomic DNA was isolated according to the method described by Dellaporta et al. (Dellaporta SL, Wood JA, and Hicks JB A plant DNA minipreparation: version II. Plant Mol. Biol., Report. 1: 19-21, 1983). The DNA concentration was estimated by spectrophotometry (Sambrook et al. 1989) or on agarose gels with ethidium bromide, by comparing fluorescence of the test sample with a marker of known molecular weight (Gibco).

For its transfer to membranes, the DNA genomic, previously digested with the chosen enzymes, following manufacturer's instructions, agarose gels were used (Pronadisa) at 0.75% (w / v). The buffer used was TBE (90 mM Tris, 90 mM boric acid, 1 mM EDTA. For fragment detection specific DNA by Southern hybridization, the DNA is transferred to Hybond N + (Amersham) nylon membranes, with hydroxide 0.4 M sodium using conventional methods (Sambrook et al. 1989) for 14-16 hours.

6.2 Total RNA isolation, quantification, electrophoresis, membrane transfer, radioactive labeling of probes and nucleic acid hybridization

Total RNA was purified following the method of Guanidine Hydrochloride described by Logeman et al. (Logemann J., Schell J., and Willmitzer L. Improved method for the isolation of RNA from plant tissues. Anal. Biochem 163: 16-20, 1987). RNA quantification was performed by spectrophotometry

For its transfer to membranes, the RNA is resolved in 1.5% horizontal agarose gels in the presence of formaldehyde / formamide (Sambrook et al. 1989). RNA samples they were loaded with ethidium bromide, in order to visualize them by fluorescence and thus check its integrity. Subsequently, the RNA was transferred to Hybond N + membranes (Amersham) using hydroxide 0.05 M sodium by conventional methods (Sambrook et al. 1989).

All DNA probes were marked with 50 µCi of α32 P-dCTP (Amersham or ICN) by the extension method from random primers, using the Oligolabelling kit (Pharmacia Biotech). The Unincorporated nucleotides were removed by chromatography of molecular exclusion in S-200 columns (Pharmacia Biotech), following the manufacturer's instructions. The different DNA fragments used as probes (complete sequences of the fusion genes), as well as the method used to obtain them, Are detailed below.

2

The nucleic acid hybridizations are performed in glass tubes, containing 10-15 ml of prehybridization solution. The membranes were prehybridized for at least two hours at the hybridization temperature. After this time, the labeled probe was added radioactively denatured and the membranes remained in this solution from 16 to 20 hours.

Southern hybridizations at low astringency were carried out in a prehybridization solution with 5X SSPE (0.15 M NaCl, 10 mM NaH2PO4, 1 mM EDTA Na2), 5X Denhardt Solution (0.02% Ficoll, 0.02% Polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.5% sodium dodecyl sulfate (SDS) and denatured herring sperm DNA 0.5 mg / ml). Hybridization was carried out at 65 ° C. After hybridization, the membranes were subjected to successive washes with 5X SSPE and 0.5% SDS, decreasing the concentration of salts in each wash.

Northern hybridizations were performed at 42 ° C in 0.25 M phosphate buffer pH 7.2, 0.25 M NaCl, 1 mM EDTA, 7% SDS, 10% PEG 6000, 40% formamide and denatured herring sperm DNA 0 , 2 mg / ml. After hybridization, the membranes were subjected to several washes with 5X SSPE and 0.5% SDS, at 65 ° C. Each wash was approximately 20 minutes long. Sometimes it was necessary to wash with 0.5% SDS.

The membranes were exposed with film autoradiographic (Hyperfilm MP, Amersham) and screens intensifiers, at -80 ° C, for the time necessary to visualize the hybridization bands. In order to reuse the membranes, these were inhibited with a 5% SDS solution boiling, and stirring until the solution cools. For check that there was no radioactivity in the membrane, the filters they were exposed with autoradiographic film for several days.

6.3 Analysis of recombinant proteins produced in transgenic plants 6.3.1 Obtaining total soluble protein

Fresh plant material, usually from of the leaflets of the basal rosette of the plants, it was homogenized in protein extraction buffer (10 mM Tris pH 7.5, NaCl 500 mM, 0.1% Triton x-100, 1% β-mercaptoethanol and 1 mM PMFS as inhibitor of  proteases), after being cut and introduced into eppendorf tubes of 1.5 ml and kept on ice (4 ° C). After homogenization, centrifuged 10-15 minutes at 13,000 r.p.m. collecting the supernatant. In some cases material was used frozen. To solubilize some of the precipitated proteins and improve extraction yields a buffer was used containing urea. For Western blots under native conditions it used an extraction buffer without reducing agents or Denaturants: 10 mM Tris pH 7.5 and 500 mM NaCl.

Protein analysis was carried out in acrylamide-bisacrylamide electrophoresis gels (30: 1) in the presence of SDS, in gels under native conditions (structure formation analysis) or in tricine gels for low molecular weight antigens.

6.3.2 Electrophoresis in gels polyacrylamide-glycine in the presence of SDS

Protein electrophoresis in conditions denaturants were performed on discontinuous gels of acrylamide-bisacrylamide in the presence of SDS, following the conventional method (Sambrook et al. 1989). Be they prepared separating gels from acrylamide-bisacrylamide (Biorad or Serva), to Variable concentrations of 7 to 15% depending on weight expected molecular protein to be analyzed and caking gels with an arcrylamide-bisacrylamide concentration 3.5% (Sambrook et al. 1989). The electrophoresis developed at a constant voltage of 100 to 140 V in the case of minigeles (7x9 cm).

All samples were quantified, according to the Bradford-Lowry method (Biorad protein assay). They were solubilized in protein dissociation buffer (0.5 M Tris pH 8.0, 10% SDS, glycerol, β-mercaptoethanol, bromophenol blue 0.02%), heated 5 minutes at 100 ° C and subsequently they were applied to the gel.

6.3.3 Electrophoresis in polyacrylamide gels in native conditions

These gels were used to verify the possible multimerization and assembly of different antigens of fusion. For native gels a method based on a 15% SDS-PAGE but with ten times less SDS in the run buffer and avoiding the SDS and β-mercaptoethanol in the loading buffer. Low these conditions, in principle, do not dissociate proteins in subunits and the molecular weights of the oligoproteins In both cases the gels were run at 150 V of constant voltage

6.3.4 Electrophoresis in Tricine gels

Tricine allows the resolution of proteins from small molecular weight (5-35 kDa) in gels of acrylamide at higher concentrations than with the gels of polyacrylamide-glycine-SDS (in the that low molecular weight proteins are not resolved), without the use of urea is necessary, according to the method described by Schagger et al. (Schagger H. and von Jagow G. Tricine-sodium SDS-polyacrilamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem 166: 368-379, 1987).

10% caking gels were prepared and 20% separators. Samples were incubated for 30 minutes at 40 ° C in protein dissociation buffer (4% SDS, 12% glycerol (w / v), 50 mM Tris, 2% β-mercaptoethanol (v / v), 0.01% bromophenol blue, final pH 6.8). Electrophoresis is performed at a constant voltage of 100 V.

Two different buffers were prepared to be able to develop electrophoresis: anode buffer (Tris 0.2% pH 8.9), and cathode buffer (0.1% Tris, 0.1% Tricine, 0.1% SDS with a pH 8.25).

6.3.5 Transfer of proteins to filters nitrocellulose

The transfer of proteins to filters Nitrocellulose (Bio-Rad 2) was performed in cuvette wet at a constant voltage of 100 V for 1 hour, or by semi-dry transfer (Bio-Rad) for 25 minutes at 20 volts In both cases the same buffer was used. transfer (25 mM Tris, 151.8 mM glycine and 20% methanol (v / v)).

6.3.6 Red Ponceau staining of proteins transferred to nitrocellulose

Once the proteins are transferred to the filters of nitrocellulose, these were stained to check their integrity with a 0.2% solution of Ponceau (Sigma) in trichloroacetic acid 30% (w / v) and 30% sulfosalicylic acid (ply) for 1 minute per immersion. They were subsequently washed with distilled water to Remove excess dye.

6.3.7 Protein Analysis by Immunoblotting or Western Blotting

Once transferred and immobilized the proteins in nitrocellulose filters, said filters were blocked of nitrocellulose well for 1 hour at 37 ° C with skim milk in 2% powder (w / v) in PBS, or overnight at 4 ° C in stirring, with the same blocking solution. Following, the filters were incubated for 1 hour with the specific antibody diluted in 0.05% PBS-Tween 20 (v / v) and at adequate concentration for each test. The filters were washed 3 times for 10 minutes in wash buffer (PBS 1X-Tween) each time and incubated in it dilution solution with the corresponding secondary antibody, for 1 hour at room temperature. After repeating the washings proceeded to development using the appropriate substrate in accordance with the secondary antibody conjugation (NBT-BCIP (Roche), ECL ...).

7. Immunogenic analysis of proteins expressed in plants 7.1 Intraperitoneal immunization of mice with extracts of plants

Female females were immunized intraperitoneally 11-week-old mouse and Swisse lineage or Balb-c, on days 0, 7 and 14 with plant extracts, expressing the recombinant protein under study (0.5-3 mg of total soluble protein / dose).

Adjuvant was used for the first inoculation complete Freund (Sigma) and incomplete Freund's adjuvant (Sigma) for the rest.

Control mice were immunized. using the same protocol with plant extracts without transform or transformed with the vector without the transgene. Ten days after the last immunization the mice were bled and proceeded to obtain the sera for study.

7.2 Oral immunizations with fresh leaves of A. thaliana

Leaves of the basal rosette of Arabidopsis expressing the different recombinant antigens were used to feed mice. 5 doses of 1-3 g of fresh transgenic Arabidopsis leaves per mouse plus 10 µg of the B-subunit of the cholera toxin (Sigma) sprayed above the leaves were administered over 3 weeks. Fifteen days later, the mice were bled.

7.3 Obtaining sera

The sera were obtained by incubation of blood for 30 minutes at 37 ° C, followed by incubation 14 hours at 4 ° C. Blood clots and sera were clarified by centrifugation at 1,500 r.p.m during 10 minutes.

7.4 Detection of antibodies by ELISA

Wells of ELISA plates were upholstered with 0.2 µl of peptide 2L21 per well, diluted in buffer glued (carbonate / bicarbonate), for 12 hours at 4 ° C. It's blocked with a solution of PBS-Tween-20 0.05% (v / v) and fetal bovine serum or 5% BSA for 1 hour at 37 ° C in agitation After washing 3 times with a solution of PBS-Tween 20 0.05%, the sera were incubated at determine at different dilutions in PBS-Tween 20 0.05%, for 1 hour at 37 ° C under stirring. After 5 washes with PBS-Tween, wells were incubated with solution of PBS-Tween-20 0.05% containing a peroxidase-conjugated anti-mouse (Amersham) to a dilution of 1/500, for 1 hour at 37 ° C with stirring. The plates were washed and revealed using as substrate o-phenylenediamine (OPD) (Sigma-Aldrich), prepared in water containing water 0.1% oxygenated. The reactions were stopped with sulfuric acid 3 N then reading the absorbance of each well of the plate in an ELISA reader at a wavelength of 492 nm.

For the detection of antibodies against Canine parvovirus capsid the INGEZIM PARVO Kit was used CANINO 1.5.CPV.K.1 (Ingenasa), designed for detection and quantification of specific antibodies against parvovirus Canine in dog sera.

To determine the antibody titer of the sera analyzed by ELISA, serial dilutions of the sera to holder, expressing the title of each serum, as the inverse of the maximum dilution of said serum in which the absorbance at 492 nm was significantly greater than that of the negative controls (2 times greater).

7.5 Antibody characterization by Immunoblotting

Antigen specificity of antibodies from immunized animals was determined by immunoblotting The VP2 capsid protein produced in baculovirus (Ingenasa), was resuspended in dissociation buffer of  protein, boiled for 5 minutes and loaded in gel polyacrylamide-glycine in the presence of SDS and transferred to nitrocellulose membrane (Biorad). The sera of mouse were tested at dilution limit from 1/10, and the antibody secondary anti-mouse was conjugated with alkaline phosphatase (Roche). The substrate used for the reaction it was nitroblue-tetrazolum-4-chloro-3 indolfosphate (NBT-BCIP, Roche).

II. Results Obtaining of transgenic plants of A. thaliana producing the fusion protein 2L21-DT

The nucleotide sequence of the domain of tetramerization of p53 (DT), obtained by PCR from the coding gene for this protein, was fused to the 3 'region of the  CPV 2L21 peptide, previously adapted to the use of codons of plants and donated in the binary vector pBI 121. At the 5 'end of the fusion gene a 5 'UTR TEV sequence was incorporated to favor the beginning of the translation of messenger RNA. The resulting plasmid  It was named pBI-TEV 2L21-DT. Saying plasmid pBI-TEV 2L21-DT allows the selection of transformants in MS medium with kanamycin and the stable integration in nuclear chromosomal DNA of plants in the expression region between the left border (LB) and right (RB) of the plasmid.

Next, A. thaliana plants were transformed with plasmid pBI-TEV 2L21-DT by infiltration of the flowers mediated by A. tumefaciens (Clough and Bent, 1998). Around 30 plants, belonging to different antibiotic resistant clones and presenting a phenotype similar to that of wild plants, were analyzed in T1.

Expression of the 2L21-DT fusion antigen in transgenic plants

The transgenic plants were analyzed by Western blot with the monoclonal antibody 3C9 (Ingenasa), specific for peptide 2L21. This study revealed the accumulation in the soluble leaf protein fraction of a protein immunoreactive somewhat higher than 8 kDa, which did not appear in the Unprocessed or transformed plant controls with the plasmid pBIl21. The electrophoretic mobility of said protein corresponded to the expected monomer of the fusion protein 2L21-DT. Like he found the merger to GUS (Gil et al. 2001), a great variation in levels was observed of expression between the different transgenic lines. Approximately 20% of these lines had high levels of expression and accumulation of the fusion antigen and were selected for subsequent analyzes (Figure 4A).

In order to try to verify the integrity and functionality of the expressed chimeric protein and if the domain of tetramerization maintained the ability of multimerization in dimers and tetramers inside the plant cell, were performed acrylamide / bisacrylamide gels under native conditions. The Western blot on these native gels (Figure 4B) determined the presence of two high intensity immunoreactive bands, the which corresponded to the dimers and tetramers formed by the fusion peptide (2L21-DT). This results suggest that transgenic plants expressed the antigen of fusion in monomeric form and this oligomerized spontaneously in inside the cytoplasm of plant cells, in dimers and, mostly, in tetramers, which migrated as proteins of about 8 kDa for the monomeric form; 16.5 kDa for the dimeric form; and approximately 32 kDa for the form tetrameric [Figure 4].

An analysis of high expression plants by Northern blot to verify all the fundamental steps in gene expression, it revealed a good correlation between mRNA levels and accumulated protein [Figure 5], suggesting that plants that express the 2L21-DT antigen do not they present problems at the translational level.

Study of the stability to the extraction and storage of 2L21-DT peptide

The ELISA results showed results similar to those obtained by the Western technique blot, showing that the plants transformed with the plasmid pBI-TEV 2L21-DT expressed high 2L21-DT antigen levels. ELISAs with plant extracts were compared with others carried out with the 2L21 synthetic peptide. As expected, the antibody monoclonal 3C9 presented a great avidity for the pure peptide, decreasing greatly when diluted with extracts of plants, presenting some variation and lack of linearity, which prevented a more accurate quantification could be performed. Without However, it can be stated that in approximately 300 ng of protein Total soluble lines of highest antigen expression 2L21-DT, an absorbance similar to the obtained with approximately 1.5 ng of pure peptide, and that presented approximately the same absorbance as 7.5-8 ng of the peptide diluted in an extract of unprocessed plant proteins.

An important aspect for the possible application of vaccines produced in plants is the stability of recombinant proteins during extraction and storage. To verify the stability and accumulation of the 2L21-DT antigen, different ELISA tests were performed, using total soluble protein extracts from these plants over time once extracted and stored at 4 ° C. Therefore, studies were carried out to determine the stability of the 2L21-DT recombinant antigen once extracted, under different storage conditions. On the one hand, the antigen once extracted had a high degree of freeze / thaw tolerance, maintaining its antigenic characteristics in ELISA assays. In addition, the stability of the antigen once extracted and stored at 4 ° C was studied in the same extraction buffer. The results show
that the 2L21-DT antigen is stable and is maintained at the same levels, at least, for three weeks at 4 ° C.

Finally, leaves of these plants that express the 2L21-DT tetrameric antigen, were stored at -20ºC for 1 year and subsequently analyzed both in ELISA as in Western blot without differences obtained significant compared to freshly extracted protein extracts. These results suggest the stability of the structure tetrameric both in the cell cytoplasm and once extracted.

In conclusion, these results show manifested that peptide 2L21 fused to DT (2L21-DT) is very resistant to extraction and storage. These data highlight the possibility not only of increase expression levels of vaccine epitopes in plants by multimerization, but its high stability.

Antibody response obtained with the 2L21-DT peptide produced in A. thaliana plants

In order to test the immunogenicity of the recombinant peptide 2L21-DT produced in transgenic plants of A. thaliana , different groups of mice (Balb-c) were immunized intraperitoneally. One group of mice was immunized with crude extracts of soluble total protein (500 µg) from the maximum expression line of the 2L21-DT peptide (line 22) and another group of mice was inoculated in the same way with 500 µg of protein Total soluble of plants expressing the 2L21 antigen fused to the β-GUS protein (2L21-GUS). Both transgenic plants used contained equimolar amounts of the 2L21 peptide. The results of the analysis by ELISA (Ingenase) of the sera obtained show that all mice immunized intraperitoneally, both with extracts containing the 2L21-DT peptide or the 2L21-GUS peptide, presented specific antibodies against the synthetic 2L21 peptide. Antibody titer in mice immunized with extracts containing the 2L21-DT peptide varied between 1/640 and 1/7120 while in the case of GUS fusion (2L21-GUS) titers were obtained that varied between 1 / 320 and 1/1280. The group of negative control mice, immunized with unprocessed plant extracts, did not show specific antibodies against the synthetic peptide 2L21 [Figure 6A].

Then, the immunization of mice with the same transgenic plants as in the case anterior, but by oral administration, letting the mice consumed leaves of both types of plants. The result of said immunization can be seen in Figure 6B. Both groups of mice presented serum antibodies after immunization, being the titles of these by ELISA superior in the case of plants expressing 2L21-DT.

The data obtained seem to suggest that the tetrameric structure of the antigen allows an improvement in the immune context by possibly favoring presentation of the antigen to the immune system. Another possibility could be related to the half-life of the tetrameric antigen in the bloodstream once injected. Such a possibility could favor the uptake of the immunogen by the presenting cells of antigen.

With this strategy, the possibility of obtaining high levels of expression of the CPV 2L21 vaccine peptide fused to the tetramerization domain of p53 (2L21-DT) has been tested, based on the multimerization capacity of the fusion protein. The 2L21-DT monomer tends to oligomerize in dimers and tetramers, which accumulate in the cell cytoplasm. In fact, as observed in native gels, most of the detected protein is in tetrameric form, indicating both the functionality of the tetramerization domain expressed in plants, and the stability of this complex structure in A. thaliana plant cells . . Although it is not desired to be linked to any theory, it is believed that the tetramers formed could offer greater resistance to degradation by cellular proteases. In addition, these results indicate that the tetramerization domain spontaneously multimerizes in the cytoplasm of plant cells, without having to go to the endoplasmic reticulum to facilitate folding and shaping and without any modification of its sequence to adapt it to the use of plant codons .

From the point of view of the immune response obtained, the antigen fused to the DT of p53 produced in plants maintain their antigenic and immunogenic characteristics, both intraperitoneally and orally and, in both cases, improves response obtained with the peptide fused to a protein carrier, immunologically irrelevant.

These results open the possibility of use this domain in future overexpression strategies of vaccine epitopes and could even be used in the development of multicomponent vaccines in plants, by enabling fusion to TD domain of more than one protein / peptide that would be assembled in the same molecule (Figure 7).

Deposit of biological material

PBI-TEV plasmid 2L21-DT has been deposited in the Spanish Collection of Type Crops (CECT), Burjassot, Valencia, on November 11 2003, corresponding to the access number CECT 5853.

Claims (32)

1. A DNA construct comprising, Operationally linked, at least:

to)

a nucleic acid sequence (A) containing the sequence of nucleotides that it codes for a product of interest, said Product of interest is CPV 2L21 peptide.

b)

a nucleic acid sequence (B) containing the sequence of nucleotides encoding a tetramerization domain, said tetramerization domain is the tetramerization domain of the p53 protein.

where the 3 'end of said nucleic acid sequence (A) is attached to the 5 'end of said nucleic acid sequence (B). 2. Construction according to claim 1, which it comprises, in addition to said nucleic acid sequences (A) and (B), a nucleic acid sequence (A ') containing the sequence of nucleotides that it codes for a product of interest, where said product of interest is CPV peptide 2L21, where the end 5 'of said nucleic acid sequence (A') is' attached to the end 3 'of said nucleic acid sequence (B). 3. DNA construction according to claim 1, further comprising a nucleic acid sequence (C) that contains the nucleotide sequence that codes for a peptide spacer (C1) located between said nucleic acid sequences (A) and (B), wherein the 5 'end of said acid sequence nucleic (C) is attached to the 3 'end of said acid sequence nucleic acid (A) and the 3 'end of said nucleic acid sequence (C) is attached to the 5 'end of said nucleic acid sequence (B). 4. DNA construction according to claim, 2 further comprising a nucleic acid sequence (C ') that contains the nucleotide sequence that codes for a peptide spacer (C2) located between said nucleic acid sequences (A ') and (B), wherein the 5' end of said acid sequence nucleic (C ') is attached to the 3' end of said acid sequence nucleic (B) and the 3 'end of said nucleic acid sequence (C ') is attached to the 5' end of said nucleic acid sequence (TO'). 5. DNA construction according to claim 1, further comprising a nucleic acid sequence (D) that contains the nucleotide sequence that codes for a peptide liable to be used for isolation purposes or purification, wherein said nucleic acid sequence (D) is located in a position that does not alter the functionality of the tetramerization domain of p53 protein or peptide 2L21 of CPV 6. DNA construction according to claim 5, wherein said nucleic acid sequence (D) is located between said nucleic acid sequences (A) and (B), wherein the 5 'end of said nucleic acid sequence (D) is attached to the 3 'end of said nucleic acid sequence (A) and the 3' end of said nucleic acid sequence (D) is attached to the 5 'end of said nucleic acid sequence (B), or said sequence of nucleic acid (D) is located upstream of the 5 'end of said nucleic acid sequence (A), or said sequence of nucleic acid (D) is located downstream of the 3 'end of said nucleic acid sequence (B). 7. DNA construction according to claim 1, further comprising a nucleic acid sequence (E) that contains the nucleotide sequence that codes for a amino acid sequence (E1) capable of being cleaved specifically by enzymatic or chemical means, wherein said nucleic acid sequence (E) is found after the 3 'end of said nucleic acid sequence (A). 8. DNA construction according to claim 2, which further comprises a nucleic acid sequence (E ') that contains the nucleotide sequence that codes for a amino acid sequence (E2) capable of being cleaved specifically by enzymatic or chemical means, wherein said nucleic acid sequence (E ') is found after the 3 'end of said nucleic acid sequence (A'). 9. An expression cassette comprising a DNA construct according to any one of claims 1 to 8 operably linked to an expression control sequence that it comprises a promoter sequence, a sequence coding for transcriptional regulators, a ribosome binding sequence (RBS) and / or a transcription terminator sequence. 10. Expression cassette according to claim 9, further comprising a marker. 11. Expression cassette according to claim 9, wherein said expression control sequence is selected from a sequence of functional expression control in prokaryotic cells and organisms and a control sequence of functional expression in eukaryotic cells and organisms. 12. A recombinant vector comprising a DNA construct according to any one of claims 1 to 8, or an expression cassette according to any of the claims 9 to 11. 13. Vector according to claim 12, wherein said recombinant vector is a viral vector. 14. Vector according to claim 13, wherein said viral vector is a viral vector capable of infecting plants or algae. 15. A cell infected with a viral vector according to claim 13 or 14. 16. A transformed cell comprising a recombinant vector according to any of claims 12 to 14, or a DNA construct according to any of the claims 1 to 8, or an expression cassette according to any of claims 9 to 11. 17. Transformed cell according to claim 16, comprising two or more recombinant vectors containing coding sequences of the same products of interest where said products of interest correspond to peptide 2L21 of CPV 18 A transgenic cell comprising a DNA construct according to any one of claims 1 to 8, or an expression cassette according to any of the claims 9 to 11. 19. Transgenic cell according to claim 18, wherein said cell is a plant cell and comprises, inserted in its genome or in the genome of a chloroplast, said DNA construct according to any one of claims 1 to 8, or said expression cassette according to any of the claims 9 to 11. 20. A transgenic plant comprising, at less, a transgenic cell according to claim 18 or 19. 21. A method of producing a product of interest in growing a cell according to any of the claims 15 to 20 under conditions that allow for production of said product of interest, where said product of interest is peptide 2L21 of CPV. 22. Method according to claim 21, which It also includes the isolation and purification of said product of interest. 23. Method according to any of the claims 21 or 22, wherein said product of interest is found in the form of a dimeric fusion protein or tetrameric 24. A method to express a gene that encodes for CPV peptide 2L21 in a plant, which comprises transform said plant with a DNA construct according to any one of claims 1 to 8, or with a cassette of expression according to any of claims 9 to 11, or with a vector according to any of claims 12 to 14. 25. A fusion protein obtainable by expression of the nucleic acid sequence contained in a DNA construct according to any one of claims 1 to 8. 26. Fusion protein according to claim 25, comprising:

(i)

the amino acid sequence corresponding to a domain of tetramerization of the p53 protein; Y

(ii)

the amino acid sequence of at least one product of interest, where said product of interest is CPV peptide 2L21.

27. Fusion protein according to claim 26, which comprises the amino acid sequence of a single product of interest or the amino acid sequence of two products of interest. 28. Fusion protein according to claim 25, comprising:

(i)

the amino acid sequence corresponding to a domain of tetramerization of the p53 protein;

(ii)

a amino acid sequence corresponding to a product of interest (A1), where said product of interest is CPV 2L21 peptide; Y

(iii)

an amino acid sequence corresponding to a product of interest (A2) where said product Of interest is CPV peptide 2L21.

29. Fusion protein according to any of the claims 25 to 28, further comprising (a) a peptide spacer between the product of interest and the domain of tetramerization; and / or (b) a peptide to facilitate isolation or purification of the peptide or fusion protein; and / or (c) a amino acid sequence capable of being cleaved specifically by enzymatic or chemical means. 30. A recombinant vaccine comprising a fusion protein according to any of claims 25 to 29, wherein said product of interest comprises a peptide or immunogenic protein, wherein said peptide is peptide 2L21 of CPV, and, optionally, a pharmaceutically excipient acceptable. 31. Vaccine according to claim 30, selected between a univalent vaccine and a vaccine multivalent 32. Vaccine according to claim 30, which understands:

(i)

the amino acid sequence corresponding to a domain of tetramerization of the p53 protein; Y

(ii)

the amino acid sequence of at least one peptide or protein immunogenic where said peptide is peptide 2L21 of CPV.

33. Vaccine according to claim 30, which understands:

(i)

the amino acid sequence corresponding to a domain of tetramerization of the p53 protein; Y

(ii)

the amino acid sequence of a first peptide or protein immunogenic where said peptide is CPV 2L21 peptide; Y

(iii)

the amino acid sequence of a second peptide or protein immunogenic where said peptide is peptide 2L21 of CPV.