WO2008063093A1 - Procédé de surproduction d'une protéine cible dans un végétal - Google Patents

Procédé de surproduction d'une protéine cible dans un végétal Download PDF

Info

Publication number
WO2008063093A1
WO2008063093A1 PCT/RU2006/000627 RU2006000627W WO2008063093A1 WO 2008063093 A1 WO2008063093 A1 WO 2008063093A1 RU 2006000627 W RU2006000627 W RU 2006000627W WO 2008063093 A1 WO2008063093 A1 WO 2008063093A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
protein
target protein
plant cell
coding rna
Prior art date
Application number
PCT/RU2006/000627
Other languages
English (en)
Russian (ru)
Inventor
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 (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut Fiziko-Khimicheskoi Biologii Im. A.N.Belozerskogo Mgu, Frolova, Olga Yurievna filed Critical Institut Fiziko-Khimicheskoi Biologii Im. A.N.Belozerskogo Mgu
Priority to PCT/RU2006/000627 priority Critical patent/WO2008063093A1/fr
Publication of WO2008063093A1 publication Critical patent/WO2008063093A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins

Definitions

  • 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.
  • 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 ⁇ lapt Sreteie ⁇ admir 122, 101-108).
  • the level of accumulation of the target protein is determined by the efficiency of: (a) transcription of the binary vector (Rombouts et al., 2003 ⁇ lapt ⁇ resumehusiol 132, 1162-1176), (b) replication ( ⁇ surmepti et al, 2004 Rgos Natl Acad Sretei USA 101, 141515 -1420, Marillopet et al, 2005 Nature ⁇ otheshp 23, 718-723), (c) protect the exogenous transcriptome from degradation (Voshitet al, 2003 ⁇ lapt J 33, 949-956), as well as the stability of the target protein
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • silepsipg silencing suppressors
  • the present invention relates to a method for producing a target protein in a plant cell, which comprises:
  • the plant cell is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.
  • the method of the invention is characterized in that the non-coding RNA is of natural origin.
  • the non-coding RNA is U6 nuclear RNA.
  • the method of the invention is characterized in that the non-coding RNA is of artificial origin.
  • the non-coding RNA is a module (GAAA) n , where n is an integer from 16 to 128.
  • the method is characterized in that the introduction of these vectors into the plant cell is carried out by agroinjection.
  • the method of the present invention is characterized in that the administration of expression vectors leads to a stable transformation of the plant.
  • the method of the present invention is characterized in that the administration of expression vectors results in transient transformation of the plant.
  • the method of the present invention is characterized in that vectors based on non-amplifying and viral vectors are used.
  • the method of the present invention is characterized in that the target protein is a drug protein.
  • the target protein is a human granulocyte colony stimulating factor.
  • the target protein is an Ag85B tuberculosis vaccine protein.
  • the method of the present invention is characterized in that the target protein is a fusion protein.
  • the fusion protein is an ESAT6 tuberculosis vaccine protein: Ag85B.
  • 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.
  • the use is characterized in that the non-coding RNA is of natural origin.
  • the non-coding RNA is U6 nuclear RNA.
  • the use is characterized in that the non-coding RNA is of artificial origin.
  • the non-coding RNA is a module (GAAA) n , where n is an integer from 16 to 128.
  • 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.
  • the genetically modified cell is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.
  • the genetically modified cell is characterized in that the non-coding RNA is of natural origin.
  • the non-coding RNA is U6 nuclear RNA.
  • 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.
  • the genetically modified cell is characterized in that said vectors are intended to be introduced into the plant cell by agroinjection.
  • the genetically modified cell is characterized in that it is stably transformed by expression vectors.
  • a genetically modified cell is characterized in that it is transiently transformed with expression vectors.
  • the genetically modified cell is characterized in that the vectors used are vectors based on non-amplifying and viral vectors.
  • the genetically modified cell is characterized in that the target protein is a drug protein.
  • the target protein is a human granulocyte colony stimulating factor.
  • the target protein is an Ag85B tuberculosis vaccine protein.
  • the genetically modified cell is characterized in that the target protein is a fusion protein.
  • the fusion protein is an ESAT6 tuberculosis vaccine protein: Ag85B.
  • 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.
  • 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.
  • 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.
  • the method is characterized in that said vectors are introduced into the plant cell by agroinjection.
  • the method is characterized in that expression vectors are introduced into the cell of the plant to ensure stable transformation.
  • the method is characterized in that expression vectors that provide transient transformation are introduced into the plant cell.
  • the method is characterized in that vectors based on non-amplifying and viral vectors are used.
  • the method is characterized in that the target protein is a drug protein.
  • the target protein is a human granulocyte colony stimulating factor.
  • the target protein is an Ag85B tuberculosis vaccine protein.
  • the method is characterized in that the target protein is a fusion protein.
  • the fusion protein is an ESAT6 tuberculosis vaccine protein: Ag85B.
  • 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
  • LoxP is the c-recombinase site
  • ⁇ 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
  • FIG. 4 Vectors expressing short non-coding RNAs stimulate the accumulation of GFP in leaves agroinjected with the “strong” vector c ⁇ V GFP (Intg)
  • FIG. 5 Vectors expressing short non-coding RNAs contribute to the early accumulation of GFP in leaves agroinjected with the “strong” vector c ⁇ V GFP (Intr)
  • 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)
  • 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
  • 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
  • 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
  • TM- massive multi-tenant 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 c ⁇ G-CSF expressing G-CSF and vectors expressing short non-coding RNA U6 (lane 1) and (GAAA) ⁇ 6 (lane 3)
  • Leaf samples agro-injected with r ⁇ V 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. sol ⁇ , producing T- CSF
  • 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
  • non-coding RNAs can be natural, for example, nuclear RNAs.
  • 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
  • Non-coding RNA can be of artificial origin.
  • 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.
  • 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.
  • all these DNA objects can be delivered using Agrobaster titefaseps.
  • the Agroinfiltration method and , 1997 ⁇ lapt Sreteie ⁇ admir 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)
  • 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). )
  • 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,
  • vaccine proteins are examples of drug proteins that can be obtained by the method of the present invention.
  • 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.
  • Gut Calmette-Gu ⁇ ripe (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.
  • BCG infection which was called BCG infection.
  • 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.
  • the invention provides a method for superproduction of vaccine proteins M. tuberculosis, ESAT6 ( ⁇ réellerlêt ⁇ pointedumble field Kunststofftechnik department ⁇ Fort ⁇ Mercedes 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.
  • 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.
  • G-CSF human granulocyte colony stimulating factor
  • Example 7 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.
  • T-CSF T-CSF
  • 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.
  • 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).
  • 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 U6 sequence with a length of 102 nt was obtained using primers (see Table 2 and Figure 1)
  • Agrobaster test tithaspeps with binary vectors expressing non-coding RNA (Fig. 1) and binary vector c- ⁇ V 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.
  • Non-coding RNAs stimulate faster GFP accumulation (Figure 5) by directly comparing with r ⁇ V GFP (Intr) in the presence (right) and in the absence (left) of a vector expressing short non-coding RNA
  • Non-coding RNAs stimulate the accumulation of GFP in leaves agroinjected with vectors created on the basis of XBK.
  • Non-coding RNAs stimulate the accumulation of GFP using a different vector than the previously described vectors (Example 3) created on the basis of the krVTM genome.
  • Example 3 created on the basis of the krVTM genome.
  • 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 ⁇ perfume ⁇ r в Example 5.
  • Non-coding RNAs stimulate the accumulation of tuberculosis vaccine proteins in leaves agroinjected with an XBK-based vector.
  • Non-coding RNAs stimulate the accumulation of tuberculosis vaccine proteins in leaves agroinjected with a vector created on the basis of krVTM.
  • Non-coding RNAs stimulate the accumulation of G-CSF in leaves agroinjected with a vector created on the basis of krVTM.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne la biotechnologie, le génie génétique et la médecine et met en oeuvre un procédé de production (de surproduction) dans la cellule d'un végétal de protéines cibles exogènes ou endogènes. La surproduction de protéines cibles est obtenue grâce à l'expression dans la cellule du végétal de vecteurs dirigeant la synthèse d'ARN d'un type spécial, désignés comme 'ARN non codants' (ou ARNnc), à savoir des ARN non capables de coder une protéine. Nous avons démontré que les ARNnc (autant d'origine naturelle qu'artificielle) stimulent radicalement la fabrication de protéines cibles. La procédé a un caractère universel et permet d'assurer la stimulation de l'accumulation dans les cellules de végétaux de n'importe quelles protéines cibles, y compris les protéines cibles de vaccin ou de traitement.
PCT/RU2006/000627 2006-11-24 2006-11-24 Procédé de surproduction d'une protéine cible dans un végétal WO2008063093A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/RU2006/000627 WO2008063093A1 (fr) 2006-11-24 2006-11-24 Procédé de surproduction d'une protéine cible dans un végétal

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/RU2006/000627 WO2008063093A1 (fr) 2006-11-24 2006-11-24 Procédé de surproduction d'une protéine cible dans un végétal

Publications (1)

Publication Number Publication Date
WO2008063093A1 true WO2008063093A1 (fr) 2008-05-29

Family

ID=39429942

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2006/000627 WO2008063093A1 (fr) 2006-11-24 2006-11-24 Procédé de surproduction d'une protéine cible dans un végétal

Country Status (1)

Country Link
WO (1) WO2008063093A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2128704C1 (ru) * 1992-01-09 1999-04-10 Новартис Аг Днк-последовательность для экспрессии генов в тканях и органах
NZ532689A (en) * 2000-06-20 2005-09-30 Arborgen Llc Polynucleotide regulatory sequences that modify expression of endogenous and/or heterologous polynucleotides in transgenic plants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2128704C1 (ru) * 1992-01-09 1999-04-10 Новартис Аг Днк-последовательность для экспрессии генов в тканях и органах
NZ532689A (en) * 2000-06-20 2005-09-30 Arborgen Llc Polynucleotide regulatory sequences that modify expression of endogenous and/or heterologous polynucleotides in transgenic plants

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
REINHART B.J. ET AL.: "MicroRNAs in plants", GENES & DEV., vol. 16, no. 13, 2002, pages 1616 - 1626 *
ROZHEN A.: "Za chto Alfred polyubil Violettu, ili Pochemu tuberkulez i sifilis ostavili zameny sled v mirovoi literature, a SPID-net", ZERKALO NEDELI / CHELOVEK/, NAUKA, no. 23(602), 17 June 2006 (2006-06-17) - 23 June 2006 (2006-06-23), pages 1 - 7, Retrieved from the Internet <URL:http://www.zn.ua/3000/3100/53647> *
VAZQUEZ F. ET AL.: "Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs", MOL. CELL, vol. 16, no. 1, 2004, pages 69 - 79 *

Similar Documents

Publication Publication Date Title
Laere et al. Plant-based vaccines: production and challenges.
Mardanova et al. Efficient transient expression of recombinant proteins in plants by the novel pEff vector based on the genome of potato virus X
Peyret et al. The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants
Bock et al. Solar-powered factories for new vaccines and antibiotics
Scotti et al. Production of foreign proteins using plastid transformation
US7081567B2 (en) Transgenic dunaliella salina as a bioreactor
JP5360727B2 (ja) 細菌毒素ワクチン
Lentz et al. High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants
Tyulkina et al. New viral vector for superproduction of epitopes of vaccine proteins in plants
JP2014508512A (ja) Ｄｎａ発現構築物
Bai et al. Construction of a fusion anti‐caries DNA vaccine in transgenic tomato plants for PAcA gene and cholera toxin B subunit
CN105939599A (zh) 使用柑橘衰退病毒载体进行外源基因表达
Naseri et al. Virus-based vectors: A new approach for the production of recombinant proteins
Elmer et al. Applying horizontal gene transfer phenomena to enhance non-viral gene therapy
WO2008063093A1 (fr) Procédé de surproduction d&#39;une protéine cible dans un végétal
CN106566842B (zh) 一种蛋白或多肽的植物表达载体及其应用
CN111849927B (zh) 高效产生重组不分节段负义rna病毒的方法及重组病毒
CN106939317A (zh) 一种提高植物抵御rna病毒的能力的方法
Ravin et al. Complete sequencing of potato virus X new strain genome and construction of viral vector for production of target proteins in plants
Šutković et al. The methods behind transgenic plant production: A review
RU2370280C2 (ru) СПОСОБ СУПЕРПРОДУКЦИИ В РАСТЕНИИ АНТИТЕЛ ПРОТИВ ОНКОГЕНА HER2/neu
SP et al. Molecular Pharming: A New Approach for a Healthy Future by a Vast Development in the Pharmaceutical Industry
WO2024075729A1 (fr) Procédé de fabrication de protéines, plante et procédé de production de vecteur viral végétal
CN116904489B (zh) 一种鸭坦布苏病毒核酸疫苗及应用
Ravin et al. Highly efficient expression of Escherichia coli heat-labile enterotoxin B subunit in plants using potato virus X-based vector

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06849629

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06849629

Country of ref document: EP

Kind code of ref document: A1