RU2390563C1 - Recombinant viral vector for producing red rot viral protein e1 in plants (versions) and red rot viral protein e1 expression system in plant cells (versions) - Google Patents

Abstract

FIELD: chemistry, biology.

SUBSTANCE: synthetic genes which code modified proteins E1 and E12 of the red rot virus provided. In order to produce these proteins in plants, synthetic genes are incorporated into the viral vector based on the potato virus X genome. Plants are infected with agrobacteria containing the obtained recombinant viral vector. Reproduction of the viral vector and synthesis of the product on a high level take place in the plants.

EFFECT: production of E1 and E12 using designed viral expression systems in Nicotiana benthamiana plants can be used to make a potential recombinant vaccine for red rot.

14 cl, 4 dwg, 4 ex

Description

Application area

The present invention relates to immunology and biotechnology, in particular to methods for producing immunogenic preparations and vaccines that can be used to prevent rubella.

Relevance

Rubella virus is a small, single-stranded (+) RNA-containing virus and is a pathogen dangerous to humans. The Russian rubella vaccine market has a capacity of about $ 8 million. Today in the Russian Federation there is no domestic rubella vaccine. The imported rubella vaccine RUDIVAX ($ 4.40 per dose) is produced by Aventis.

The vaccine strain of rubella virus RA27 / 3 has been used in vaccination programs for more than 30 years, but along with its undoubted effectiveness, quite serious contraindications and side effects have also been identified. Of these, post-vaccination arthritis and arthralgia are more common than others. An attenuated vaccine virus causes a subclinical infection and can persist for a long time in the immunized organism, which, in turn, does not exclude unpredictable consequences. In general, a live rubella vaccine, like many other live virus vaccines, can cause side effects, in particular, the introduction of foreign RNA into the human body. Serious concerns are about the use of the live rubella vaccine for HIV-infected, pregnant women and newborns. Therefore, the task of creating a recombinant rubella vaccine that does not show negative effects due to the use of live virus continues to be relevant.

State of the art

Recombinant Protein Production Methods

One of the most striking and convincing achievements of modern biotechnology was the creation and rapid development in recent decades of a new area of the world economy - the biopharmaceutical and biomaterial industries, aimed at the production of a fundamentally new class of drugs - recombinant medical proteins. Various vaccine proteins, interferons, erythropoietins, growth factors, antibodies, previously available only in analytical quantities, are now included in the list of vital medicines, are produced in volumes of up to several tons per year, and are firmly included in the daily practice of modern medicine, food and pharmaceutical industries . In the vast majority of cases, the source of these products are cells of bacteria, animals or yeast.

The use of plants as “biofactories” for the production of foreign proteins has several advantages compared with cells of bacteria, yeast, and mammals:

(1) The production of target proteins in plants does not require the use of expensive equipment (fermenters), culture media and a sterility system. Plant growth requires only soil, water and the sun, so the cost of growing them is incomparably lower than the cost of cultivating cells of bacteria, yeast or animals. As a result, the cost of proteins obtained in plants is already 10-30 times lower than the cost of similar proteins obtained from bacteria (Giddings et al., 2000).

(2) Plant cells are biologically safe because plants and humans do not share common pathogens. Therefore, the products obtained in plants do not contain viruses and prions that are dangerous to humans.

(3) Unlike bacteria and yeast, plants and animals have a similar system of post-translational protein modifications. Therefore, in plant cells, those human and animal proteins can be obtained that cannot be correctly expressed in microorganisms.

Currently used technologies for the expression of recombinant proteins in plants, as a rule, involve the production of a transgenic producer plant. Already today there are a number of examples of commercially successful production of proteins in plants in this way. For example, trypsin, avidin, and glucuronidase obtained in transgenic maize plants are now produced and sold by Sigma Aldrich. Aprotinin (ProdiGene), collagen (ProdiGene, Medicago, Meristem Therapeautics), lipase (Meristem Therapeautics), lactoferrin (Ventria, Meristem Therapeutics), lysozyme (Ventria), vaccine against the gastroenteritis virus TGEV (ProdiGene antibodies), mono monok are close to the market against caries and herpes virus (Planet Biotechnology, Epicyte Pharmaceutical). The first plant-derived vaccine for Newcastle disease developed by Dow Agroscience was successfully registered in the United States in 2006, more than 20 biopharmaceuticals obtained in plants are currently in various stages of clinical trials (http://www.molecularfarming.com/news2. html).

It should be noted that obtaining transgenic plants is not the best way to create plant “biofactories”, since the level of production of target proteins by transgenic plants is usually very low (about 0.1% of the total protein). The low level of expression determines the high cost of cleaning the product and, ultimately, the competitiveness of the plant expression system. In addition, the cultivation of transgenic plants is associated with a number of formal limitations.

Improving the economic efficiency of using plants as “biofactories” - protein producers requires the development of new production technologies in non-transgenic plants at a high level of target proteins. Such technologies will make it possible to obtain inexpensive and safe proteins in plants and can become a competitive alternative to traditional methods based on the use of bacteria, yeast or animal cells.

An alternative to transgenic plants as producers of recombinant proteins may be expression systems based on recombinant plant viruses (vector viruses). Many plant viruses, such as the tobacco mosaic virus and the potato X virus, multiply in large quantities when infected, and the level of production of the virus's own proteins reaches 70% of the plant cell proteins. The meaning of this method is to integrate the gene encoding the necessary foreign protein into the genome of the virus and to infect the modified plant cell virus. An infection synthesizes not only its own proteins of the virus, but also the target protein, the gene of which was specially inserted into the genome of the virus. The level of recombinant protein production in this case can be up to 20% of the total protein (Marillonnet et al., 2005), which corresponds to 1 gram of protein per 1 kilogram of plant biomass, which is more than 100 times higher than the levels of production usually achieved in transgenic plants.

Rubella virus proteins that can be used to create a candidate vaccine

The genome of the rubella virus is a single-stranded + RNA strand 9762 nucleotides long, containing two open reading frames (OPC). The OPC RNA closest to the 5 ′ end encodes two proteins involved in viral RNA replication, P150 and P90. The second OPC encodes three protein components of the viral particle — capsid protein C, as well as surface glycoproteins E1 and E2.

During rubella infection, antibodies to proteins C, E1 and E2 are formed. While anti-E2 antibodies disappear within a few months after infection, anti-E1 antibodies persist in the human body for decades and remain active (Nates et al., 1989). Various studies have shown that infection protection is provided mainly by neutralizing antibodies against E1, while cellular immunity systems do not significantly contribute to infection protection. It has been shown that E1 glycoprotein can be the basis of a recombinant rubella vaccine (Perrenoud et al., 2004). Individual peptide fragments of this protein can also be used as the basis of a candidate vaccine.

To date, expression systems in insect and mammalian cells have been used for the production of E1 protein [Seppänen et al., 1991; Hobman et al., 1994; Johansson et al., 1996]. These methods require the use of special media and equipment, and the risk of product contamination by human pathogenic viruses remains, etc. We do not know published data on the production of rubella virus proteins in plants (both in transgenic plants and using recombinant vector viruses). Therefore, it remains relevant to create alternative methods for the production of rubella virus vaccine proteins, one of the most promising of which is plant expression systems.

Disclosure of invention

The present invention was tasked with creating a viral vector that can provide the rubella virus protein (SeqID01) and the E12 polypeptide (hereinafter E12 protein), comprising amino acids 153 to 239 of the E1 protein, in plants E1.

In fact, the problem was solved by:

a) design and synthesis of genes encoding proteins E1 and E12, modified by the addition of amino acid sequences to them, increasing their stability in the plant cell and facilitating purification,

b) cloning the synthesized genes in a vector virus based on the potato virus genome X,

c) development of infection protocols for the recombinant plant vector virus Nicotiana benthamiana and subsequent production of recombinant rubella virus E1 and E12 proteins in plants.

The first aspect of the present invention relates to the construction of synthetic genes encoding modified E1 and E12 proteins, to which at the N-terminus is attached a sequence of 6 amino acid residues of histidine, and at the C-terminus is a sequence of a localization signal in the SEKDEL endoplasmic reticulum.

A second aspect of the invention is the production of expression vector viruses that produce modified E1 and E12 proteins in plants. For this, the synthetic genes mentioned above were cloned into the pA7248amvT viral vector.

Accordingly, a third aspect of the invention relates to optimizing the conditions for the infection of Nicotiana benthamiana plants with recombinant vector viruses and subsequent production of rubella virus recombinant proteins E1 and E12 in plants.

A fourth aspect of the invention is the confirmation of the fact of the production of E1 and E12 proteins in plants as a result of Western blot analysis of protein preparations isolated from producer plants. It was shown that both recombinant proteins, E1 and E12, are actually synthesized in the leaves of producer plants.

Brief Description of the Drawings

Figure 1 - structure of artificial genes encoding the modified proteins E1 and E12 of the rubella virus.

The nucleotide and corresponding amino acid sequences of the genes are shown. The nucleotide sequences encoding the original E1 and E12 are highlighted in gray, the amino acid numbers in the E1 protein are indicated. The start and termination codon sequences are in italics, the sequence encoding 6 histidines is underlined, the sequence encoding the localization signal in the SEKDEL endoplasmic reticulum is highlighted in a rectangle.

Figure 2 - structure of the viral vectors pA7248amvE1 and pA7248amvE12, providing the production of vaccine proteins of the rubella virus in plants. The T-DNA structure of the region of the pA7248amvT viral vector is shown, regions of the vector outside the T-DNA are shown by a dashed line. The boundaries of T-DNA (border, "B") are marked with bold vertical lines. 35S - promoter, 35S-T - terminator 35S RNA of cauliflower mosaic virus. RDRP polymerase gene, Sgp1 - the first promoter of potato virus subgenomic RNA X. Tet is a tetracycline resistance gene. AMV is a DNA copy of the alfalfa mosaic virus RNA leader sequence.

Figure 3 - Western blot analysis of protein preparations isolated from plants producing E1.

Protein preparations are applied to the gel:

1. Molecular weight marker, band sizes are in kilodaltons.

2. Non-inoculated leaves of N. benthamiana (control) - membrane fraction.

3. Leaves of N. benthamiana inoculated with the pA7248amvE1 vector virus — membrane fraction.

No E1 protein was found in the soluble fraction.

Figure 4 - Western blot analysis of protein preparations isolated from plants producing E12.

Protein preparations are applied to the gel:

1. Molecular weight marker, band sizes are in kilodaltons.

2. Non-inoculated leaves of N. benthamiana (control) - membrane fraction.

3. Leaves of N. benthamiana inoculated with the pA7248amvE12 vector virus — membrane fraction.

E12 protein was not found in the soluble fraction.

The implementation of the invention.

Example 1. Design and synthesis of genes encoding modified for expression in plants proteins E1 and E12.

The construction of the artificial E1 gene was carried out in two stages. At the first stage, the E1 gene sequence was obtained by PCR using E1FBg primers (CGT AGA TCT GAG GAG GCT TTC ACC TAC CT) and E1RSm (ATA GAG CTC STA AAG TTC ATC TTT CTC AGA GCG AGG CGC TAT AGC G) and DNA copies of the rubella virus genome as a matrix. PCR was performed under the following conditions: (1) 96 ° C for 40 sec, (2) 72 ° C for 180 sec, steps 1-2 were repeated 40 times. Note that a nucleotide sequence encoding the SEKDEL peptide was introduced (in italics) into the reverse primer E1RSm sequence. After electrophoretic separation of PCR products, a 1.5 kbp fragment was isolated that was digested with restriction enzymes BglII and Ecl136II and ligated with DNA vector pQE30 (QIAGEN) pretreated with restriction enzymes BglII and Ecl136II. The resulting plasmid, which was selected according to the results of restriction analysis, was designated pQE-E1. At the second stage, the modified E1 gene sequence was obtained by PCR using pQtagF primers (AC TGG CGC GCC AAA ATG GCG CAT CAC CAT CAC CAT CAC GG) and pQtagR (ATA GAG CTC CTA AAG TTC ATC TTT CTC AGA) and plasmid DNA pQE-El as a matrix. PCR was performed under the following conditions: (1) 96 ° C for 40 sec, (2) 72 ° C for 180 sec, steps 1-2 were repeated 40 times. After electrophoretic separation of the PCR products, a 1.5 kb fragment was isolated containing an artificial gene encoding an E1 protein with the attached sequences MAHHHHHHGS (at the N-terminus) and SEKDEL (at the C-terminus). The structure of the artificial E1 gene is shown in Figure 1, and the amino acid sequence of its product is presented in the sequence listing in SeqID02.

The construction of the artificial E12 gene was carried out in a similar way, except that at the first stage of construction, instead of the E1FBg and E1RSm primers, the primers E12FBg (CGT AGA TCT ACC GTC CGG GTC AAG TTC CA) and E12RSm (A GAG CTC CTA AAG TTC ATC TTT CTC AGA GGA ATG GCG TTG GCA AAC CG) respectively. The structure of the artificial E12 gene is shown in Figure 1, and the amino acid sequence of its product is presented in the sequence listing in SeqID03.

Example 2. Construction of expression vector viruses that ensure the production of modified E1 and E12 proteins in plants.

The viral vector pA7248 (PVXdt_GFP) described in [Komarova et al., 2006], based on the genome of the potato X virus of the UK3 strain [the nucleotide sequence of the genome of this virus is given in GenBank number M95516], was used as the basis for creating the vector. This vector includes the 5′-untranslated portion of the PVX genome, the polymerase gene, the first promoter of the subgenomic RNA, the target GFP gene, the last 60 nucleotides of the coat protein gene, and the 3′-untranslated portion of the potato X virus genome. We modified this vector by replacing the GFP gene with a tetracycline resistance (Tet) gene by introducing unique AscI and SmaI restriction sites flanking the Tet gene, as well as introducing between the first promoter of the subgenomic RNA and the AscI site a nucleotide sequence corresponding to the alfalfa mosaic virus RNA leader sequence ( vector pA7248amvT. This whole construct is placed between the 35S promoter and the 35S terminator and cloned into the binary vector pBIN19. When delivered to cells by leaf agroinfiltration, infection occurs for more than an hour ty of leaf cells in the infected area, replication of the viral vector in individual cells and high level product synthesis.

To create a viral producer vector of the modified E1 protein, the pA7248amvT vector DNA was treated with AscI and SmaI restriction enzymes and ligated with a DNA fragment containing the artificial E1 gene treated with these restriction enzymes. The resulting recombinant vector containing the E1 gene in place of the Tet gene, which was selected by restriction analysis, was designated pA7248amvE1.

To create a viral producer vector of the modified E12 protein, the DNA of the pA7248amvT vector was treated with AscI and SmaI restriction enzymes and ligated with the DNA fragment treated with these restriction enzymes containing the artificial E12 gene. The resulting recombinant vector containing E12 in place of the Tet gene, which was selected by restriction analysis, was designated pA7248amvE12.

The structure of the viral vectors pA7248amvE1 and pA7248amvE12, providing for the production of modified E1 and E12 proteins in plants, is shown in FIG. 2.

Example 3. Infection of Nicotiana benthamiana plants with recombinant vector viruses and the production of rubella virus proteins E1 and E12 in plants.

For experiments on the expression of E1 protein in N. benthamiana plants, the viral vector pA7248amvE1 was introduced into the Agrobaterium tumefaciens GV3101 strain, recombinant agrobacteria were used to infiltrate N. benthamiana leaves. Agrobacteria containing recombinant binary vectors were grown for 12 hours on a shaker at 30 ° C. Cells (1.5 ml) were pelleted by centrifugation (4000g, 5 min), the pellet was resuspended in 1.5 ml of buffer containing 10 mM MES (pH 5.5) and 10 mM MgCl ₂ , the optical density was adjusted to OD ₆₀₀ = 0.2. Leaves of N. benthamiana plants were injected with a suspension of agrobacteria using a syringe without a needle. Infiltrated leaves were left on growing plants.

It is known that one of the main factors limiting viral infection and, in particular, the expression of target proteins in viral expression systems is the development of post-transcriptional gene silencing (PTGS) activated by double-stranded replicative forms of viral RNA [MacDiarmid, 2005]. To prevent this phenomenon, along with agrobacteria containing the pA7248amvE1l virus vector, agrobacteria containing the P19 protein vector of the bushy dwarf tomato virus, a known suppressor of gene silencing suppressor, were infiltrated into plant leaves.

After infiltration of N. benthamiana leaves with agrobacteria with the vector virus, most of the leaf cells are infected in the infected area, T-DNA is transferred to the nucleus, and a copy of the viral RNA is transcribed from the 35S promoter. At the next stage, the viral vector is replicated and the product is synthesized at a high level [Komarova et al., 2006]. The maximum synthesis of the product is achieved on 6-10 days after infiltration.

The production of E12 protein in plants was carried out in the same way using the pA7248amvE12 virus vector.

Example 4. Detection of the E1 and E12 proteins produced in plants by Western blot analysis.

The presence of the target protein in the leaves of producer plants was recorded using Western blot analysis. On the 6-10th day after infection, a leaf fragment was triturated in 0.4 M sucrose buffer, 50 mM Tris pH 8.0, 10 mM KCl, 5 mM MgCl ₂ , 10% glycerol, 10 mM β-mercaptanol. The extract obtained was centrifuged for 10 minutes at 14000g, the supernatant (“soluble fraction”) and the dissolved precipitate (“membrane fraction”) were analyzed by Western blot using antibodies obtained with recombinant E1 protein or by polyacrylamide (PAGE) gradient electrophoresis (8- 20%) Lammley gel and stained with Coomassie R-250. The results of the analysis are presented in Figure 3 (for protein E1) and Figure 4 (for protein E12).

As can be seen from the results presented in Figure 3, in plants infiltrated with the pA7248amvE1 vector virus in the membrane fraction of the cells, a product corresponding to a molecular weight of 53 kDa and reacting with antibodies to the E1 protein was found. In the control uninfiltrated plant, this protein was absent. The product yield (E1) was about 5% of the fraction of insoluble proteins.

In E12 producing plants, a target product with a molecular weight of approximately 11.5 kDa was also detected in the membrane fraction by Western blotting (Figure 4). The size of this protein coincided in electrophoretic mobility with the control recombinant E12 polypeptide. The product yield (E12) was about 1% of the fraction of insoluble proteins.

Thus, the recombinant proteins E1 and E12 are synthesized in the corresponding producer plants.

LITERATURE

1. Hobman TC, Lundstrom ML, Mauracher CA, Woodward L, Gillam S, Farquhar MG. (1994) Assembly of rubella virus structural proteins into virus-like particles in transfected cells. Virology, 202 (2): 574-85.

2. Johansson T, Enestam A, Kronqvist R, Schmidt M, Tuominen N, Weiss SA, Oker-Blom C. (1996) Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein. J Biotechnol. 50 (2-3): 171-178.

3. Giddings G, Allison G, Brooks D, Carter A. (2000) Transgenic plants as factories for biopharmaceuticals. Nat. Biotechnol. 18 (11), 1151-1155.

4. MacDiarmid R. (2005) RNA silencing in productive virus infections. Annu. Rev. Phytopathol., 43, 523-544.

5. Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y. (2005) Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat. Biotechnol., 23, 718-723.

6. Nates SV, Mersich SE, Damonte EB, Zapata MT. (1989) Comparison of immune response to rubella virus proteins in early and late natural infections. Microbiologica, 12, 335-338.

7. Perrenoud G, Messerli F, Thierry AC, Beltraminelli N, Cousin P, Fasel N, Vallet V, Demotz S, Duchosal MA, Moulon C. (2004) A recombinant rubella virus E1 glycoprotein as a rubella vaccine candidate. Vaccine 23, 480-488.

8. Seppänen H, Huhtala ML, Vaheri A, Summers MD, Oker-Blom C. (1991) Diagnostic potential of baculovirus-expressed rubella virus envelope proteins. J. Clin. Microbiol 29 (9): 1877-1882.

9. Komarova T.V., Skulachev M.V., Zvereva A.S., Schwartz A.M., Dorokhov Yu.L., Atabekov I.G. (2006) New virus vector for efficient production of target proteins in plants. Biochemistry, 71 (8), 1043-1049.

Claims (14)

1. Recombinant viral vector for the production of rubella virus protein E1 in plants, containing a promoter that is functionally active in plant cells, a 5'-untranslated portion of the potato X virus genome, a potato X virus polymerase gene, the first promoter of the potato X virus subgenomic RNA, a nucleotide a sequence encoding rubella virus E1 protein, a 3'-untranslated portion of the potato X virus genome, and a transcription terminator. 2. The recombinant viral vector according to claim 1, in which between the first promoter of the subgenomic potato X virus RNA and the nucleotide sequence encoding the rubella virus E1 protein, a nucleotide sequence corresponding to the sequence of the 5'-untranslated alfalfa mosaic virus RNA region is introduced. 3. The recombinant viral vector according to claim 2, wherein the promoter is a promoter of the 35S cauliflower mosaic virus 35S gene. 4. The recombinant viral vector according to claim 3, in which the transcription terminator is represented by the terminator of the 35S cauliflower mosaic virus 35S gene. 5. The recombinant viral vector according to claim 4, which is a plasmid pA7248amvEl, in which the elements are combined, as shown in figure 2. 6. The expression system of the rubella virus E1 protein in plant cells, which is a Nicotiana benthamiana plant, into the cells of which a fragment of the recombinant viral vector according to claim 1, comprising a promoter, 5'-untranslated portion of the potato X virus genome, is functionally active in plant cells, potato X virus polymerase gene, the first promoter of the potato X virus subgenomic RNA, the nucleotide sequence encoding the rubella virus E1 protein, the 3'-untranslated portion of the potato X virus genome, and the transcription terminator. 7. The expression system according to claim 6, which is a Nicotiana benthamiana plant, into the cells of which, using the bacterium Agrobacterium tumefaciens, a fragment of the recombinant viral vector pA7248amvEl shown as t-DNA in FIG. 2 was introduced. 8. Recombinant viral vector for the production of E12 polypeptide in plants, including amino acids 153 to 239 of the rubella virus E1 protein, containing a promoter that is functionally active in plant cells, a 5'-untranslated portion of the potato X virus genome, the potato X virus polymerase gene, first a potato X virus subgenomic RNA promoter, a nucleotide sequence encoding a rubella virus E12 polypeptide, a 3'-untranslated portion of the potato X virus genome, and a transcription terminator. 9. The recombinant viral vector of claim 8, in which between the first promoter of the potato X virus RNA subgenomic RNA and the nucleotide sequence encoding the rubella virus E12 polypeptide, a nucleotide sequence corresponding to the sequence of the 5'-untranslated alfalfa mosaic virus RNA region is introduced. 10. The recombinant viral vector according to claim 9, in which the promoter is a promoter of the gene 35S RNA of the cauliflower mosaic virus. 11. The recombinant viral vector of claim 10, in which the transcription terminator is represented by the terminator of the cauliflower mosaic virus 35S gene. 12. The recombinant viral vector according to claim 11, which is a plasmid pA7248amvE12, in which the elements are combined, as shown in figure 2. 13. The system of expression of the rubella virus E12 polypeptide in plant cells, which is a Nicotiana benthamiana plant, into the cells of which a fragment of the recombinant viral vector of claim 8 is introduced, including a promoter functionally active in plant cells, a 5'-untranslated portion of the potato X-virus genome, potato X virus polymerase gene, the first promoter of the potato X virus subgenomic RNA, the nucleotide sequence encoding the rubella virus E12 polypeptide, the 3'-untranslated portion of the potato X virus genome and the trans terminator ripts. 14. The expression system of claim 13, which is a Nicotiana benthamiana plant, into the cells of which, using the bacterium Agrobacterium tumefaciens, a fragment of the recombinant viral vector pA7248amvE12 is introduced, shown as t-DNA in FIG.

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