WO2008063093A1

WO2008063093A1 - Method for hyperproducing target protein in a plant

Info

Publication number: WO2008063093A1
Application number: PCT/RU2006/000627
Authority: WO
Inventors: Jury Leonidovich Dorokhov; Tatyana Valerievna Komarova; Iosif Grigorievich Atabekov
Original assignee: Institut Fiziko-Khimicheskoi Biologii Im. A.N.Belozerskogo Mgu; Frolova, Olga Yurievna
Priority date: 2006-11-24
Filing date: 2006-11-24
Publication date: 2008-05-29

Abstract

The invention relates to biotechnology, gene engineering and to medicine, in particular to a method for producing (hyperproducing) foreign or endogenous target proteins in a plant cell. The hyperproduction of the target proteins is obtained by the expression, in the plant cell, of vectors leading synthesis of ribonucleic acids of a specific type designated as noncoding RNA, i.e the RNA incapable to code protein. The applicants disclose that noncoding RNA substantially promote the production of target proteins, wherein the stimulating activity is exhibited by natural and synthetic noncoding RNA. The inventive method is embodied in the form of a multi-purpose method and promotes the accumulation of any type of target proteins, including vaccinal and therapeutic target proteins, in cells of plants.

Description

METHOD OF SUPERPRODUCTION IN TARGET PROTEIN PLANT

FIELD OF THE INVENTION

The invention relates to biotechnology, genetic engineering and medicine and can be used to create plants - superproducers of target proteins, in particular drug proteins, which can be used for therapeutic or prophylactic purposes.

State of the art

Plants have several advantages compared with bacteria, yeast and mammalian cells for the production of foreign proteins. Plants as “factories” (a) do not contain viruses and prions pathogenic for humans, and (b) do not require the use of expensive equipment (fermenters), culture media, and sterility systems; the cost of growing the original experimental plants is incomparably lower than the cost of cultivating bacterial, yeast, or animal cells. The technology of “temporary” production of protein in a plant allows you to produce a foreign protein in an amount reaching 10% - 30% of the total soluble protein of the plant in a short time (5 -10 days), when the target gene is included in the binary vector with its subsequent delivery to the cells of the infected plant by agroinoculation or agroinfiltration (Karila et al, 1997 Рlapt Sсieпсе 122, 101-108). Under these conditions, the level of accumulation of the target protein is determined by the efficiency of: (a) transcription of the binary vector (Rombouts et al., 2003 Рlapt Рсhusiol 132, 1162-1176), (b) replication (Аsurmepti et al, 2004 Rgos Natl Acad Sсi USA 101, 141515 -1420, Marillopet et al, 2005 Nature Віotheshp 23, 718-723), (c) protect the exogenous transcriptome from degradation (Voshitet al, 2003 Рlapt J 33, 949-956), as well as the stability of the target protein The term “target” (hepe of iptest) can mean both “foreign” (foreig), that is, a gene that is not part of the genome of a given plant, and also “endogenous” (ephepous), that is, a gene representing portion of the plant genome term "vipyc-vektop" may denote the structure at the level of DNA containing a complete copy of the modified viral genome or RNA transcript of the DNA capable of self-replicating in a plant cell expressing the target protein (CB)

Recently developed systems of transient expression of viral vectors are aimed, first of all, to increase the stability of viral RNA transcripts by suppressing the cellular mechanism of silencing, or silencing, genes using suppressor proteins (Voipet, 2005. Nat 6, 206-220; Qu apd Morris, 2005. FEBS Let. 579, 5958-5964; Roth et a!., 2004 Viru Res J 02, “7-108: Missiard apt Voipet, 2004. MoI. Riath Pat. 5, 71-82). The action of viral suppressors is expressed in the suppression of the destruction of viral RNAs and the accumulation of specific short (21-25 nt) interfering double-stranded RNAs or short / smarter ip RNA, siRNA and miRNA. Most of the known suppressors exhibit their properties when they bind to siRNA and miRNA. This applies, first of all, to the well-studied Tombusvirus protein P 19 (Voipet et al, 1999. Rros. Natl. Acad. Ssi. USA 96, 14147-14152), closterovirus P21 (Reed et al, 2003. Virolog 306, 203- 209), cucumovirus 2b (Vesliet et al, 1998. Virology 252, 313-317) and potiviral HcPro (Aplaxmi et al, 1998. Proc. Natl. Acad.Sci.USA 95, 13079-13084). The binding of suppressors to siRNA and miRNA leads to the suppression of (a) the binding of short RNAs to Dicers and Argonaut proteins; (b) stopping their local and systemic distribution; (c) the blockade of siRNA and miRNA methylation and, consequently, the stimulation of their degradation (Yu et al, 2006 FEBS Let. 580, 3117-3120). Another way to increase protein accumulation is to increase its intracellular stability by optimizing the codon composition of the gene or by removing, for example, the transmembrane domain.

However, the use of gene silencing suppressor proteins and known methods for increasing the stability of the target protein may not achieve the expected effect, especially when it is necessary to express the target proteins, in particular drug proteins. The present invention provides a new method for superproduction of a target protein. The use of vectors expressing non-coding RNAs allows one to sharply increase the accumulation of drug proteins in the plant, in particular, such as the ESAT6 tuberculosis vaccine proteins: Ag85B and Ag85B and human granulocyte colony-stimulating factor (G-CSF).

Disclosure of invention

The inventors of the present invention unexpectedly found that the introduction of nucleic acid into the nucleus of a plant cell, which ensures the appearance of non-coding RNA in the cytoplasm, leads to superproduction of the target protein directed by another expression vector. Thus, the authors created the present invention by developing a simple previously undescribed method of superproduction in a plant cell target proteins.

The present invention provides a new method and technology for overexpression and accumulation in a plant cell of a target protein of various origins. The proposed method differs from the known technologies associated with the use of silencing suppressors (silepsipg) of genes (US6395962, WO02057301, WO0138512, WO2006032713) in that the effect is achieved by using non-protein molecules, namely short non-coding RECs. Thus, in a first aspect, the present invention relates to a method for producing a target protein in a plant cell, which comprises:

(a) providing an expression vector directing in the cell the synthesis of non-coding RNA,

(b) providing an expression vector directing the synthesis of the target protein in the cell;

(c) introducing said vectors into a plant cell;

(g) co-expression in the cell of these vectors.

In one embodiment, the plant cell is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.

In one embodiment, the method of the invention is characterized in that the non-coding RNA is of natural origin. In one preferred embodiment, the non-coding RNA is U6 nuclear RNA.

In a further embodiment, the method of the invention is characterized in that the non-coding RNA is of artificial origin. In one preferred embodiment, the non-coding RNA is a module (GAAA) _n , where n is an integer from 16 to 128.

In yet another embodiment, the method is characterized in that the introduction of these vectors into the plant cell is carried out by agroinjection.

In one embodiment, the method of the present invention is characterized in that the administration of expression vectors leads to a stable transformation of the plant.

In a further embodiment, the method of the present invention is characterized in that the administration of expression vectors results in transient transformation of the plant.

In one embodiment, the method of the present invention is characterized in that vectors based on non-amplifying and viral vectors are used. In one embodiment, the method of the present invention is characterized in that the target protein is a drug protein. In one preferred embodiment, the target protein is a human granulocyte colony stimulating factor. In a further preferred embodiment, the target protein is an Ag85B tuberculosis vaccine protein.

In a further embodiment, the method of the present invention is characterized in that the target protein is a fusion protein. In one preferred embodiment, the fusion protein is an ESAT6 tuberculosis vaccine protein: Ag85B.

In a further aspect, the present invention relates to the use of non-coding RNA as a means for increasing the production of a target protein in a plant cell.

In one embodiment, the use is characterized in that the non-coding RNA is of natural origin. In one preferred embodiment, the non-coding RNA is U6 nuclear RNA.

In another embodiment, the use is characterized in that the non-coding RNA is of artificial origin. In one preferred embodiment, the non-coding RNA is a module (GAAA) _n , where n is an integer from 16 to 128.

In a further aspect, the present invention relates to a genetically modified plant cell producing a target protein and comprising an expression vector directing non-coding RNA synthesis in the plant cell and an expression vector directing the synthesis of the target protein in the plant cell.

In one embodiment, the genetically modified cell is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.

In a further embodiment, the genetically modified cell is characterized in that the non-coding RNA is of natural origin. In one of the most preferred embodiments, the non-coding RNA is U6 nuclear RNA.

In yet another embodiment, the genetically modified cell is characterized in that the non-coding RNA is of artificial origin. AT One of the most preferred embodiments of non-coding RNA is a module (GAAA) _n , where n is an integer from 16 to 128.

In a further embodiment, the genetically modified cell is characterized in that said vectors are intended to be introduced into the plant cell by agroinjection.

In one embodiment, the genetically modified cell is characterized in that it is stably transformed by expression vectors.

In yet another embodiment, a genetically modified cell is characterized in that it is transiently transformed with expression vectors.

In a further embodiment, the genetically modified cell is characterized in that the vectors used are vectors based on non-amplifying and viral vectors.

In one embodiment, the genetically modified cell is characterized in that the target protein is a drug protein. In one of the most preferred embodiments, the target protein is a human granulocyte colony stimulating factor. In another of the most preferred embodiments, the target protein is an Ag85B tuberculosis vaccine protein.

In yet another embodiment, the genetically modified cell is characterized in that the target protein is a fusion protein. In one of the most preferred embodiments, the fusion protein is an ESAT6 tuberculosis vaccine protein: Ag85B.

In a further aspect, the present invention relates to a method for producing a genetically modified plant cell producing a target protein. The method includes introducing into the cell an expression vector directing the synthesis of non-coding RNA in the plant cell and an expression vector directing the synthesis of the target protein in the plant cell.

In one embodiment, the method is characterized in that the plant cell is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.

In one embodiment, the method is characterized in that the non-coding RNA is of natural origin. In one of the most preferred embodiments, the non-coding RNA is U6 nuclear RNA In yet another embodiment, the method is characterized in that the non-coding RNA is of artificial origin. In one of the most preferred embodiments, the non-coding RNA is a module (GAAA) _n , where n is an integer from 16 to 128.

In a further embodiment, the method is characterized in that said vectors are introduced into the plant cell by agroinjection.

In one embodiment, the method is characterized in that expression vectors are introduced into the cell of the plant to ensure stable transformation.

In yet another embodiment, the method is characterized in that expression vectors that provide transient transformation are introduced into the plant cell.

In yet another embodiment, the method is characterized in that vectors based on non-amplifying and viral vectors are used.

In a further embodiment, the method is characterized in that the target protein is a drug protein. In one of the most preferred embodiments, the target protein is a human granulocyte colony stimulating factor. In another of the most preferred embodiments, the target protein is an Ag85B tuberculosis vaccine protein.

In one embodiment, the method is characterized in that the target protein is a fusion protein. In one of the most preferred embodiments, the fusion protein is an ESAT6 tuberculosis vaccine protein: Ag85B.

Brief description of the figures:

FIG. 1. Scheme of oligonucleotide primers for cloning cDNA expressing U6.

FIG. 2. The structure diagram of binary vectors expressing non-coding RNAs, where RB and LB are the right and left parts of the site of embedding the binary vector into the plant genome, respectively; 35S — transcriptional promoter of cauliflower mosaic virus (VMCC); PoIyA is the polyadenylation signal of the MCC. In parentheses, the length (in nucleotides, pt) of the cDNA expressing non-coding RNA is shown.

FIG. 3. Vectors expressing short non-coding RNAs stimulate the accumulation of GFP in leaves agroinjected with a “weak” vector created on the basis of krBTM. (A) Schematic diagram of the structure of the crTMV: GFP vector, a binary vector based on the infectious cruciferous tobamovirus cDNA, krVTM, where (from left to right) LB is the left part of the site of insertion of the binary vector into the plant genome, Nos-t is the nopaline synthase terminator, and NTPTP is the neomycin phosphotransferase, Nos-p - nopaline synthase transcriptional promoter, Arab. Act2 is the actin transcriptional promoter 2 from Arabidopsis, RePcase is the crBTM replicase gene, MP (tgps) is the transport crBTM protein with a C-terminal deletion that does not affect the protein’s transport properties, LoxP is the c-recombinase site, and Ω is the omega-translational enhancer BTM, GFP - green fluorescent protein from Aqiorea, NTR - 3 'untranslated region of the genome of krVTM RNA, RB - the right side of the site of integration of the binary vector into the plant genome

(B and C) Photograph of Nepthatapa leaf 4 (B) and 5 (C) days after agroinjection of the viral vector cТТМV GFP (V, in the middle of the sheet) at a dilution of 100 times and with the joint agroinjection of the viral vector сТТМV GFP and vectors expressing short non-coding RNA U6, (GAAA) _n (64 and 128 pt long) and хМir (chimeric miR171 / 159) Vectors encoding suppressor proteins P19 and НсРrо were used as a control

FIG. 4. Vectors expressing short non-coding RNAs stimulate the accumulation of GFP in leaves agroinjected with the “strong” vector cТТМV GFP (Intg)

(A) Diagram of the structure of the cТТМV GFP vector (Intr) Introns not shown in the figure are introduced into the replicase and transport protein (MP) genes. Other designations are explained in the caption to Fig. 2

(B-D) Photograph of leaf N atptspa 5 (B) and 6 (C, D) days after agroinjection of the viral vector c-ТМV GFP (Intr) (V, in the middle of the sheet) at a dilution of 100,000 times and with joint agroinjection of the viral vector c-ТТМV GFP (Intr) and vectors expressing short non-coding RNA U6, (GAAA) _n (64 and 128 pt long) and хМir (chimeric miR171 / 159) Vectors encoding the suppressor proteins P19 and HcPrо were used as a control

FIG. 5. Vectors expressing short non-coding RNAs contribute to the early accumulation of GFP in leaves agroinjected with the “strong” vector cТТМV GFP (Intr)

(AB) Photograph of leaf of Nepthate 3 (A), 4 (B) and 6 (C) days after agroinjection of the viral vector cТТМV GFP (Intr) (left) and сТТМV GFP (Intr) + vector expressing (GAAA) ib ( 64 pt long) Dilution (from 10 ^"1 to 10 ^{" 6} ) of agrobacteria cТТМV GFP (Intr) is indicated in the middle of the sheet

FIG. 6. Vectors expressing short non-coding RNAs stimulate the accumulation of GFP in leaves agroinjected with a vector created on the basis of PVX (PVX GFP)

(A) Diagram of the structure of the vector PVX GFP, a binary vector based on the infectious potato X virus cDNA (X-BK), where PVX PoI is replicase X-BK, 25K, 12K, 8K are transport genes X-BK, sgp is a subgenomic promoter X-BK envelope protein, Pvx ptr - non-translated region of X-BK

(B) A photograph of leaf Nptpa 5 days after agroinjection of the viral vector PVX GFP (V, in the middle of the leaf) at a 100-fold dilution and when agroinjection of the viral vector PVX GFP and vectors expressing short non-coding U6 RNA, (GAAA) _n ( 64 and 128 pt long) and xMir (chimeric miR171 / 159) Vectors encoding the suppressor proteins P19 and HcPro were used as a control

FIG. 7. Vectors expressing short non-coding RNAs contribute to increased accumulation of tuberculosis vaccine protein ESAT6 Ag85B in leaves agroinjected with the vector PVX-ESAT6 Ag85B

(A) PVX-ESAT6 Ag85B Vector Structure Diagram.

(B) Western analysis of leaf proteins of N. bepatiapa 5 days after joint agroinjection of the viral vector PVX-ESAT6 Ag85B and vectors expressing short non-coding RNA U6, (GAAA) _n (64, 256 and 512 pt lengths) Vectors encoding P suppressor proteins 19 and НсРrо, used as a control M - positive control, ESAT6 Ag85B (60 ng) synthesized in E coli On the autograph, the arrow (on the right) shows the position of the plant ESAT6 Ag85B detected by specific antibodies to the bacterial ESAT6 Ag85 In the preparation of a recombinant protein synthesized in E colι, usually ESAT6 Ag85B is present in the form of monomer (lower band) and dimer (upper band) The different mobility of the bacterial and plant protein ESAT6 Ag85B is due to the deletion introduced into the Ag85B protein when the transmembrane domain is removed

(B) Test for the amount of protein introduced into the gel (loving control) A photograph of the zones of the RUBISCO protein, usually present in the preparation of total soluble protein of plant leaves, is shown

FIG. 8. Photograph of Coomassie stained gel after electrophoresis of proteins of the cell walls of leaves of N. bepthciash 5 days after joint agroinjection сrТМV Ag85B-His (TM-) viral vector and vectors expressing short non-coding U6 RNA, (GAAA) ib (64 pt long) Vectors encoding P 19 suppressor proteins were used as a control. Track designated as ESAT6 Ag85B (0.5 mcg) is a positive control, i.e. a protein synthesized in E. colg The arrow shows the position of Ag85B

FIG. 9 Western analysis of leaf proteins of N. bepatiapa 5 days after the joint agroinjection of the viral vector cТТМ G-CSF expressing G-CSF and vectors expressing short non-coding RNA U6 (lane 1) and (GAAA) ι ₆ (lane 3) Leaf samples agro-injected with rТМV G-CSF in the presence of a vector encoding P19 (lane 2) or a vector without an insert (empty vector, lane 4) were used as a positive and negative control, respectively. The arrow shows the position of G-CSF detected by specific antibodies to G - CSF isolated and purification nnogo from recombinant bacteria E. solι, producing T- CSF

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the fact that the authors unexpectedly discovered that superproduction of the target protein in a plant cell encoded by an expression vector can be achieved by introducing a nucleic acid into the nucleus of a plant cell that provides short non-coding RNAs in the cytoplasm

In the context of the present invention, non-coding RNAs can be natural, for example, nuclear RNAs. In particular, nuclear RNA U6 (Example 2) can stimulate protein production directed by the viral vector (see Examples 3-6) of U6 RNA, which is normally transcribed by RNA polymerase III and does not leave the nucleus of a plant cell (Norrer, 2006 Critical Revues Vuschemistr apd Molesulur Vulogu, 41, 3-19), provides increased synthesis of the target protein when RNA transport from the nucleus into the cytoplasm is included in the signal vector

Another class of non-coding RNA can be of artificial origin. In particular, an artificial sequence such as (GAAA) _n , where n is an integer from 16 to 128 (Example 1), can be used to effectively increase the production of a protein directed by a viral vector (see Examples 3-7)

The proposed method involves (a) the formation in plant cells of non-coding RNAs and (b) the synthesis of mRNAs that direct superproduction of target proteins in the mRNA cell can be formed both from replicating vectors created on the basis of viral genomes (see Examples 3-7), and from non-amplifying mono- or polycistronic RNAs

A plant cell producing the target protein can be in the form of an isolated cell devoid of a cell wall (protoplast), or as part of a cultured explant (tissue, organ), or a whole plant Cell cultivation under artificial conditions occurs in a sterile medium of known composition and under controlled conditions at a constant temperature (26-28 ⁰ C) and illumination The present invention suggests the possibility of using non-coding RNA to stimulate the production of the target protein in cultured cells as in in suspension culture, and in plant explants cultivated on a solid medium, or in a whole plant grown in a suitable medium, such as a natural substrate (e.g. soil, soil substitutes), or in a hydroponic culture, The target protein can accumulate inside a plant cell or by introducing a signal sequence into its composition, it can be exported to the culture medium. The selection of the target protein can occur during the destruction of culture cells or directly from the culture medium

Foreign DNA can be introduced into the plant cell using electroporation, micro-bombardment bombardment, microinjection and the virus. In addition, all these DNA objects can be delivered using Agrobaster titefaseps. For transient expression of a foreign gene in leaf tissue, the Agroinfiltration method and , 1997 Рlapt Sсieпсе 122, 101-108) The transformation of plant cells can be stable, accompanied by the integration of the introduced DNA into the plant genome. The desired effect can also be achieved. bend when introduced into the nucleus of DNA and transient (temporary) synthesis of RNA without stable integration of DNA into the host genome (see Examples 3-7)

One of the advantages provided by the present invention is that non-coding RNAs are able to stimulate the production of the target protein to a greater extent than the most effective gene silencing suppressor, P19 Tomato Dwarf Virus Protein P19 (BKKT) (see Examples 3-7). )

Another advantage provided by the present invention is that non-coding RNAs can stimulate the production of any target Proteins: both the model GFP protein, which, due to its structure, is characterized by extremely high stability in plant cells, as well as drug proteins.

Target proteins useful for medical (therapeutic or prophylactic) use that can be obtained by the method of the present invention include, without limitation: growth factors, for example, vascular endothelial growth factor (VEGF), platelet growth factor (PDGF), hepatic growth factor (HGP ), placental growth factor (PLGF), tumor growth factor (TGF), in particular tumor growth factor beta (TGF-beta), insulin-like growth factor (IGF), thrombopoietin (TPO), erythropoietin (EPO), stem cell factor (SCF) ), fibroblast growth factor (FGF), l ycotic inhibitory factor (LIF), interleukins 1 to 8 (EL-I - GL-8) and 12 (BL-12), antigens of pathogens of various bacterial, viral, protozoal infections, such as dysentery, meningitis, typhoid and rash, plague, cholera, smallpox, measles, brucellosis, malaria, AIDS, flu, rubella, mumps, poliomyelitis, yellow fever, diphtheria, whooping cough, gonorrhea, syphilis, tetanus.

One example of drug proteins that can be obtained by the method of the present invention are vaccine proteins. As vaccine proteins, the production of which can be carried out by the method of the present invention, of particular interest are vaccine proteins that protect humans from tuberculosis. Human tuberculosis (PM), caused by the intracellular bacterium Musobasterite tuberculosis, occupies a leading position in human mortality (3 million people per year) among infectious diseases. Vasile Calmette-Guέripe (BCG vaccine), a live attenuated vaccine based on Musobasterite bovis, obtained by French bacteriologists AIb ert Calmette and Camille Guéripe in the 1920s, is now widely used in healthcare. This vaccine has been used for decades, although its effectiveness is not entirely satisfactory. BCG vaccine is effective against miliary PM and tuberculous meningitis in children, but it does not protect well from adult pulmonary tuberculosis. In addition, in recent years there have been widespread cases of illness in children as a result of BCG vaccination, which was called BCG infection, or “BCG”. The situation does not improve with BCG revaccination, but rather increases the risk of pulmonary PM in adults. The general opinion is that we need a new effective vaccine and an immunization strategy. Difficulties in developing a new vaccine are explained to a large extent by the peculiarities of biology and genetics of M. tubercylosis. It was shown that 129 genes of M. tuberculosis are absent in the genome of the BCG vaccine strain. Among them, a number of genes encoding secreted proteins were identified, which can be considered as candidate vaccine proteins.

That is why, in one of its most preferred embodiments, the invention provides a method for superproduction of vaccine proteins M. tuberculosis, ESAT6 (Еаrlу Сесретдтигепіс Target-6, with a molar mass of 6 kDa), Ag85B, (mycolyltransferase with a molar mass of 35 kDa) in a plant cell and chimeric artificial fusion protein ESAT6: Ag85B, which can be used for vaccination with the aim of creating specific immunity against the human tuberculosis pathogen caused by M. tuberculosis.

The inventors showed (Examples 5 and 6) that, in accordance with the method of the present invention, non-coding RNAs can stimulate the accumulation of ESAT6: Ag85B and Ag85B in a plant cell, at least not less efficiently than the known protein silencing suppressors, P19 and Hcrro.

Another non-limiting example of a drug protein that can be superproduced in a plant cell by the method of the present invention is a human granulocyte colony stimulating factor (G-CSF) (Example 7). G-CSF is a glycoprotein with a molecular weight of about 19 kDa, produced by macrophages, fibroblasts and endothelial cells. It is an important factor in hematopoiesis and is characterized by a wide spectrum of biological activity, including a rapid increase in the number of neutrophilic granulocytes, stimulation of terminal stages of differentiation, and an increase in the activity of mature neutrophils. These properties of T-CSF open up broad prospects for its use in the treatment of certain forms of cancer to reduce the duration of neutropenia after bone marrow transplantation and intensive chemotherapy, as well as to increase resistance to bacterial and viral infections, including AIDS. Plants producing G-CSF should be widely used as a source of cheap, functionally active protein for use in medicine.

Genes encoding target proteins for production in a plant cell by the method of the present invention may, in addition to the coding sequence, further comprise coding and / or non-coding sequences. Such coding sequences are well known to those skilled in the art and include, for example, leader peptides that can be used to synthesis of the target protein in the form of a space with the aim of its subsequent secretion into the intercellular space. Production of target proteins in the form of fusion proteins is also possible. Fusion partners are well known in the art and include, for example, sequences that facilitate subsequent highly efficient purification of the target protein, or provide better solubility thereof, etc. As the first, we can give examples of a polyhistidine cluster (His) b or (His) ω, which can be used for purification by affinity chromatography on an immobilized metal, or a fragment of glutathione-S-transferase, which can be used for purification by affinity chromatography on a resin containing bound glutathione, or maltose binding protein MBP, cellulose binding protein CBP, etc. (Matsudaira, PT 1993. A rgastisal guide to rotei apd rertide rurifiсiopi fomisroseksepsipg. Asademis Rgess, Sap Diego; Shhaferet al., 2002. J. 13. Timo.

Non-coding sequences are also well known to those skilled in the art and may include sequences that increase transcript output (strong promoters, enhancers, aptamers), or provide a choice between constitutive gene expression or inducible expression that is regulated by an external factor (for example, physical or chemical effects) , or specific micro-RNAs (Gooddrive ap Kugel, 2006. NATURE Revises, MoI. CeIl Biol. 7, 612-615). Examples of such sequences include more than 75 enhancers of transcription of the human genome (Repassio et al., 2006. Natury Nov 5; Epub achad of frag).

Examples

The following examples reveal the most preferred embodiments of the present invention and are provided solely for the purpose of better clarifying its essence. One skilled in the art will recognize that many modifications can be made both to the means and materials used, and to the methods used, without departing from the scope of the invention.

Example 1. Cloning of vectors expressing non-coding RNA based on the GAAA module. cDNA expressing the module (GAAA) ib was obtained using the polymerase chain reaction (TTTIP) using oligonucleotide primers (see Table 1). The (GAAA) ₁₆ module was used to create the Bin 35S- (GAAA) _n construct, where n = 32 (128 nt), 64 (256 nt), 128 (512 nt). Each version was made using the previous

Table 1 The sequence of oligonucleotide primers used to obtain the module (GAAA) ₁₆

Restriction sites are underlined

Example 2. Cloning of vectors expressing non-coding RNAs based on nuclear RNA U6.

The U6 sequence with a length of 102 nt was obtained using primers (see Table 2 and Figure 1)

Table 2 The sequence of oligonucleotide primers used to obtain cDNA encoding U6

Kprl and SaII restriction enzyme sites are underlined

The resulting PCR product was cloned into the pGEMZ subclone at the Acc65I (Kpn) + SalI sites. Then, the U6 sequence was cloned into the pFF19 subclone containing the 35S sample at the Acc65I (Kpn) + SalI sites. After that, a fragment of this construct containing the 35S sample and located following it, the U6 sequence was cloned into the Bm 19 vector at the Nhеh-EcoRI sites, resulting in the construction of 10840 Example 3. Non-coding RNAs stimulate the accumulation of GFP in leaves agroinjected with vectors created on the basis of krVTM.

Agrobaster test tithaspeps with binary vectors expressing non-coding RNA (Fig. 1) and binary vector c-ТМV GFP (Fig. 3 A) were seeded into 2-YT liquid medium and grown overnight at 28 ^° C. Night culture was precipitated by centrifugation at 5 thousand rpm 3 min. Then, the precipitate was resuspended in agroinfiltration buffer containing 10 mM MgCl ₂ and HUMM MES pH 5.0, and introduced into the sheet at the appropriate dilution. Fig 3 (B and C) shows the accumulation of GFP in N. leaves of the liver through 4 (B) and 5 (B) days after agroinjection of the viral vector cТТМV GFP (V, in the middle of the leaf) in a dilution 100 times and with the joint agroinjection of the viral vector c-ТМV GFP and the vector expressing short non-coding RNA U6, (GAAA) _n (64 and 128 pt long) and хМir (chimeric miR171 / 159) с-ТМV GFP is considered “weak”, because for quick visualization of the expression result of a gene encoding GFP requires the introduction of sufficiently “gytx” initial suspensions of agrobacteria into the leaf (only 100-fold dilution) It can be seen that non-coding RNAs [U6 and (GAAA) _n , where n = 64 or 128 pt] stimulate the accumulation in leaves of GFP in an amount exceeding the accumulation of GFP during co-injection with protein upressorami silencing P 19 and NsRro

A similar effect is observed when the vectors expressing U6 or (GAAA) _n (where n = 64 or 128 pt) are injected into the leaves, together with the “strong” cТТМV GFP (Iptr) vector (Fig. 4 A), which is characterized by early GFP synthesis at the introduction into the leaves of diluted (dilution 10 ⁵ ) suspensions of agrobacteria

Non-coding RNAs stimulate faster GFP accumulation (Figure 5) by directly comparing with rТМV GFP (Intr) in the presence (right) and in the absence (left) of a vector expressing short non-coding RNA

Example 4. Non-coding RNAs stimulate the accumulation of GFP in leaves agroinjected with vectors created on the basis of XBK.

The ability of non-coding RNAs to stimulate the accumulation of GFP was shown using a different vector than the previously described vectors (Example 3) created on the basis of the krVTM genome. Here we used the PVX GFP vector created on the basis of the XBK genome (Fig. 6A). It can be seen (Fig. 6B). that vectors created on the basis of the (GAAA) module of ib and U6, as well as the precursor of the chimeric miRNA (XmiR, miR171 / 159) sharply stimulate the accumulation of GFP, comparable to its accumulation in the presence of protein suppressors of silencing P19 and НсРrо Example 5. Non-coding RNAs stimulate the accumulation of tuberculosis vaccine proteins in leaves agroinjected with an XBK-based vector.

Our method can be used for the production of tuberculosis vaccine proteins in plants using the PVX-ESAT6 Ag85B vector (Fig. 7A). It can be seen (Fig. 7B) that vectors created on the basis of the (GAAA) ₁ O and U6 module sharply stimulate the accumulation of chimeric tuberculosis vaccine protein ESAT6 Ag85B, comparable to its accumulation in the presence of protein suppressors of silencing P 19 and Hcrro

Example 6. Non-coding RNAs stimulate the accumulation of tuberculosis vaccine proteins in leaves agroinjected with a vector created on the basis of krVTM.

This example shows that our method can be used to produce tuberculosis vaccine proteins in plants using the cGTMV GFP vector expressing Ag85B instead of GFP. It can be seen (Fig. 8) that the vectors created on the basis of the module (GAAA) i _b and U6, sharply stimulate the accumulation of chimeric tuberculosis vaccine protein Ag85B, comparable to its accumulation in the presence of protein suppressors of silencing P19 The level of accumulation of vaccine protein is 1 g per 1 kg of sheet material

Example 7. Non-coding RNAs stimulate the accumulation of G-CSF in leaves agroinjected with a vector created on the basis of krVTM.

This example shows that our method can be used to produce human granulocyte colony stimulating factor (G-CSF) in a plant using the cGTMV GFP vector expressing G-CSF instead of GFP. It can be seen (Fig. 9) that the vectors created on the basis of the module (GAAA) ₁₆ (lane 3) and U6 (lane 1), sharply stimulate the accumulation of G-CSF, comparable to its accumulation in the presence of the protein suppressor silencing P 19 (lane 2)

All patents, publications, scientific articles, and other documents and materials cited or referenced herein are hereby incorporated by reference to the same extent as if each of these documents were individually incorporated by reference or are provided in their entirety here

Specific genes, vectors, plant species, uses used The materials and methods described herein relate to preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. When studying this description, other objects, aspects, and embodiments will come to mind to those skilled in the art, and they are encompassed by the scope of this invention. Specialists in this field will be clear that various replacements and modifications can be made in relation to the invention described here without deviating from its scope and scope, which are determined by the attached claims.

Claims

Claim

1. The method of production of the target protein, which includes:

(a) providing an expression vector directing the synthesis of non-coding RNA in a plant cell,

(b) providing an expression vector directing the synthesis of the target protein in the plant cell;

(c) introducing said vectors into a plant cell;

(g) cultivating the plant cell under conditions providing co-expression of the indicated vectors in the cell.

2. The method of claim 1, wherein the plant cell is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.

3. The method according to p. 1, characterized in that the non-coding RNA is of natural origin.

4. The method according to p. 3, characterized in that the non-coding RNA is a nuclear RNA U6.

5. The method according to p. 1, characterized in that the non-coding RNA is of artificial origin.

6. The method according to p. 5, characterized in that the non-coding RNA is a module (GAAA) _n , where n is an integer from 16 to 128.

7. The method according to any one of the preceding paragraphs, characterized in that the introduction of these vectors into the plant cell is carried out by agroinjection.

8. The method according to any one of the preceding paragraphs, characterized in that the introduction of expression vectors leads to stable transformation of plant cells.

9. The method according to any one of paragraphs. 1-7, characterized in that the introduction of expression vectors leads to transient transformation of plant cells.

10. The method according to any one of the preceding paragraphs, characterized in that the use of vectors based on non-amplifying and viral vectors.

11. The method according to any one of the preceding paragraphs, characterized in that the target protein is a drug protein.

12. The method according to p. 11, characterized in that the target protein is a granulocyte colony-stimulating factor in humans.

13. The method according to p. 11, characterized in that the target protein is a tuberculosis vaccine protein Ag85B.

14. The method according to any one of paragraphs. 1-11, characterized in that the target protein is a fusion protein The method according to claim 14, characterized in that the fusion protein is a tuberculosis vaccine protein ESAT6 Ag85B The use of non-coding RNA as a means to increase the production of the target protein in a plant cell The use according to claim 16, characterized in that the non-coding RNA is of natural origin The use according to claim 17, characterized in that the non-coding RNA is a nuclear RNA U6 The application according to claim 16, characterized in that the non-coding RNA is of artificial origin the claim according to claim 19, characterized in that the non-coding RNA is a module (GAAA) _n , where n is an integer from 16 to 128 A genetically modified plant cell is a producer of the target protein containing an expression vector directing the synthesis of non-coding RNA in the plant cell, and expression vector directing the synthesis of the target protein in a plant cell The genetically modified cell according to claim 21, which is located in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part of it A cell modified according to claim 21, characterized in that the non-coding RNA is of natural origin. A genetically modified cell according to claim 23, characterized in that the non-coding RNA is nuclear RNA. U6 A genetically modified cell according to claim 21, characterized in that the non-coding RNA is of artificial origin. the genetically modified cell according to claim 25, characterized in that the non-coding RNA is a module (GAAA) _n, where n is an integer from 16 to 128 genetically modified to a net according to any one of paragraphs 21-26, characterized in that said vectors are intended to be introduced into a plant cell by agroinjection A genetically modified cell according to any one of paragraphs 21-27, characterized in that the plant cell is stably transformed by expression vectors Genetically modified cell according to any from paragraphs 21-27, characterized in that the plant cell is transiently transformed with expression vectors

30. The genetically modified cell according to any one of paragraphs 21-29, characterized in that the vectors used are vectors based on non-amplifying and viral vectors.

31. The genetically modified cell according to any one of paragraphs 21-30, characterized in that the target protein is a drug protein.

32. The genetically modified cell according to p. 31, characterized in that the target protein is a granulocyte colony-stimulating factor in humans.

33. The genetically modified cell according to p. 31, characterized in that the target protein is a tuberculosis vaccine protein Ag85B.

34. The genetically modified cell according to any one of paragraphs 21-31, characterized in that the target protein is a fusion protein.

35. The genetically modified cell according to p. 34, characterized in that the fusion protein is a tuberculosis vaccine protein ESAT6: Ag85B.

36. A method of producing a genetically modified plant cell producing the target protein, comprising introducing into the cell an expression vector directing the synthesis of non-coding RNA in the plant cell and an expression vector directing the synthesis of the target protein in the plant cell.

37. The method of claim 36, wherein the plant cell is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.

38. The method according to p. 36, characterized in that the non-coding RNA is of natural origin.

39. The method according to p. 38, characterized in that the non-coding RNA is a nuclear RNA U6.

40. The method according to p. 36, characterized in that the non-coding RNA is of artificial origin.

41. The method according to p. 40, characterized in that the non-coding RNA is a module (GAAA) _n , where n is an integer from 16 to 128.

42. The method according to any one of paragraphs 36-41, characterized in that said vectors are introduced into the plant cell by agroinjection.

43. The method according to any one of paragraphs 36-42, characterized in that expression vectors are introduced into the cell of the plant to ensure stable transformation.

44. The method according to any one of paragraphs. 36-42, characterized in that the plant cell introducing expression vectors providing transient transformation The method according to any one of paragraphs 36-44, characterized in that vectors based on non-amplifying and viral vectors are used. The method according to any one of paragraphs 36-45, characterized in that the target protein is a drug protein. The method according to claim 46, characterized in that the target protein is a granulocyte colony-stimulating human factor. The method according to claim 46, characterized in that the target protein is a tuberculosis vaccine lon gest protein Ag85B method according to any one of claims 36-46, characterized in that the target protein is a fusion protein The method according to claim 49, characterized in that the fusion protein is a tuberculosis vaccine protein ESAT6 Ag85B