JP2005102652A - Transformed cell, method for producing protein by using such cell and kit for producing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a system for producing a protein having a large-scale production capability, a high production efficiency and also having a high safety, a method for producing the protein and a kit for performing the method for producing the protein. <P>SOLUTION: This expression vector is constructed by introducing a cDNA of a recombinant tomato mosaic virus (ToMV) having a suppressor of virus resistant reaction with a GFP (green fluorescent protein) gene and obtained by substituting an envelope protein gene of the ToMV into the downstream of a promotor capable of performing a transcriptive induction with a steroid hormone. By performing the transcriptive induction by the steroid hormone for a transformed tobacco BY-2 cells obtained by introducing the above expression vector into the tobacco BY-2 cells, it becomes possible to perform the amplification of mRNA of the GFP gene and the inductive expression of the GFP. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention introduces an expression vector comprising a gene encoding a protein to be expressed and linking a plant virus gene having a suppressor of virus resistance reaction (silencing reaction) and a promoter transcriptionally induced by a hormone. The present invention relates to a transformed cell, a protein production method using the transformed cell, and a kit for performing the protein production method.

  More specifically, transcription induction by a tomato mosaic virus (hereinafter referred to as ToMV as appropriate) gene having a suppressor for a virus resistance reaction (silencing reaction) including a gene encoding a protein to be expressed, and a steroid hormone. A transformed tobacco BY-2 cell introduced by the Agrobacterium method, a method for producing a protein using the transformed tobacco BY-2 cell, and the protein It is related with the kit for performing the production method.

  In recent years, attention has been focused on the development of efficient production methods for useful proteins including pharmaceuticals. In particular, development is underway with the aim of using plants not only as food, but also as a production plant for useful proteins such as pharmaceuticals. Such an attempt is called “molecular agriculture” and is expected as next-generation agriculture.

  Currently, the production of useful proteins in plants is carried out by either a method using a transformed plant (for example, see Non-patent Document 5) or a method for infecting a plant with a viral vector (for example, Non-Patent Documents 1 and 2). reference).

  The present inventor has previously been able to incorporate a plant virus (brom mosaic virus) replication enzyme gene and a useful protein gene amplified thereby into the plant chromosome, and to control the expression of the replication enzyme gene. Thus, a gene expression system capable of synthesizing useful proteins (hereinafter referred to as a high-efficiency mRNA induction amplification system) was constructed. The high-efficiency mRNA-inducing amplification system is applied to Nicotinia benthamiana plants, and the gamma interferon gene is expressed at the RNA level by inducing and expressing the 1a protein that is one of the subunits of the replication enzyme in the steroid hormone control system. It became possible to amplify (refer nonpatent literature 1 and 2).

  Furthermore, the present inventor used a tomato mosaic virus (ToMV), which is a genus Tobamo mosaic virus, which is a single-stranded RNA virus having a suppressor for virus resistance reaction (silencing reaction) and high replication ability, as a steroid. A system that amplifies foreign protein mRNA with high efficiency by hormonal induction was constructed. As a result of applying this system to a Nicotine benzamiana plant and trying to induce expression in a steroid hormone control system using a Green Fluorescent Protein gene (hereinafter referred to as GFP gene) as a reporter gene, amplification of the gene at the RNA level, And GFP expression was confirmed (see Non-Patent Document 2).

On the other hand, production of useful proteins using plant-derived cultured cells has also been attempted. For example, Non-Patent Document 3 reports the production of recombinant proteins in tobacco BY-2 cells using the cauliflower mosaic virus 35S promoter. Further, in Non-Patent Document 4, tobacco BY protoplasted using a mutant viral RNA vector in which the target peptide gene is linked to the 3 ′ end of the coat protein gene of tobacco mosaic virus (hereinafter referred to as TMV). -2 cells are inoculated to express a fusion protein with the coat protein.
Ishikawa Agricultural College Agricultural Resource Research Institute Annual Report 2000, No. 9, 2000, p. 16-18 (issue date: October 25, 2001) Ishikawa Agricultural College Agricultural Resource Research Institute Annual Report 2001, No. 10, 2001, p. 13-16 (issue date: September 25, 2004) Matsumoto S, Ikura K, Ueda M, Sasaki R. “Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells.” Plant Mol boil. 1995 Mar; 27 (6): 1163-72. Takamatsu N, Watanabe Y, Yanagi H, Meshi T, Shiba T, Okada Y. “Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector.” FEBS Lett. 1990 Aug 20; 269 (1): 73-6. Transgenic plants as factories for biopharmaceuticals Glynis Giddings, Gordon Allison, Douglas Brooks & Adrian Carter Nature Biotechnology (2000) 18: 1151-1155.

  Among the protein production methods in the plant, the method using a transformed plant can produce a protein only by cultivating the plant, but has a problem that the production efficiency per cell is extremely low. On the other hand, the method of infecting plants with viral vectors has high production efficiency, but it is difficult to achieve large-scale production due to poor work efficiency due to the necessity of inoculation work and safety problems such as virus scattering to the outside world. It has become.

  In the case of the high-efficiency mRNA-inducing amplification system using the brom mosaic virus constructed by the inventor, the recombinant virus cannot be formed into particles because the brom mosaic virus has no suppressor for the virus resistance reaction (silencing reaction). When is used, viral RNA is degraded by silencing reaction (virus resistance reaction). In other words, in a high-efficiency mRNA-inducing amplification system using brom mosaic virus, the gene encoding the target protein is amplified at the RNA level, but the RNA is degraded by virus resistance reaction (silencing reaction) over time. Therefore, there is a drawback that continuous high protein production cannot be performed. Further, in this system, a transcription factor activated by steroid hormone has a disadvantage that it causes plant yellowing and the like and adversely affects plant growth.

  On the other hand, when a protein is produced in a plant individual using a ToMV system that has a suppressor for virus resistance (silencing) reaction, RNA degradation due to silencing can be avoided and protein can be produced with high efficiency. However, it is difficult to scale up because it requires plant cultivation equipment, etc., it takes a relatively long time to produce the plant, and it is safe due to the scattering of seeds and pollen of the transformed plant. There are various drawbacks, such as a problem with respect to performance and a complicated operation.

  In addition, the protein production system using tobacco BY-2 cells listed in Non-Patent Documents 3 and 4 can produce proteins by cell culture in a medium, is easy to scale up, and has a high growth rate. The time merit is great. Furthermore, since it is a cultured cell, even if it leaks to the outside world, it is immediately killed, so it is excellent in terms of safety. However, these methods also have problems such as low production efficiency and complicated operation (protoplastization, inoculation operation).

  As explained above, the conventional protein production methods and protein production systems using plants and plant-derived cells have various drawbacks and have never been satisfactory.

  The present invention has been made in view of the above-described conventional problems, and its purpose is to produce a protein production system having both the advantages of protein production by a transformed plant and the advantages of protein production using a viral vector, that is, large-scale production. The object is to provide a protein production system, production method and the like with high capacity, high production efficiency, and high safety.

  As a result of intensive investigations to solve the above-mentioned problems, the present inventors have found that a recombinant ToMV cDNA in which a coat protein gene is replaced with a GFP gene downstream of a promoter capable of inducing transcription with a steroid hormone. As a result of the transcriptional induction with steroid hormones in transformed tobacco BY-2 cells introduced into tobacco BY-2 cells, the expression vector constructed by introducing the gene can be surely amplified viral RNA and induced expression of GFP As a result, the present invention has been completed.

  That is, the present invention includes the following inventions.

  (1) An expression vector in which a plant virus gene having a suppressor of virus resistance reaction including a gene encoding a protein to be expressed and a promoter transcriptionally induced by a hormone is introduced into a biological cell. A transformed cell characterized by comprising:

  (2) The transformed cell according to (1), wherein the plant virus is a virus belonging to the genus Tobamovirus.

  (3) The transformed cell according to (2), wherein the virus belonging to the genus Tobamovirus is tobacco mosaic virus or tomato mosaic virus.

  (4) The transformed cell according to any one of (1) to (3), wherein the hormone is a steroid hormone.

  (5) The transformed cell according to any one of (1) to (4), wherein the organism-derived cell is a plant-derived cell.

  (6) A transformed cell, wherein the plant-derived cell of (5) is a tobacco-derived cell.

  (7) A transformed cell, wherein the tobacco-derived cell of (6) is a tobacco BY-2 cell.

  (8) The transformed cell according to any one of (1) to (7), wherein the protein expression vector is introduced by the Agrobacterium method.

  (9) A method for producing a protein, comprising using the transformed cell of any one of (1) to (8) above.

  (10) The protein production method according to (9), wherein the protein production method includes a step of culturing transformed cells.

  (11) The method for producing a protein according to (10), wherein the method further comprises a hormone-induced transcription induction step.

  (12) The method for producing a protein according to (11), wherein the hormone-induced transcription step is a steroid hormone-induced transcription step.

  (13) A protein production kit for performing the protein production method of any one of (9) to (12) above.

  (14) The protein production kit according to (13), wherein the protein production kit includes the expression vector according to any one of (1) to (8).

  (15) The protein production kit according to (13) or (14), wherein the protein production kit further comprises a hormone.

  (16) The protein production kit according to (15), wherein the hormone is a steroid hormone.

  (17) The protein production kit according to any one of (13) to (16), wherein the protein production kit further comprises a biological cell serving as a host.

  (18) The protein production kit according to (17), wherein the organism-derived cells are plant-derived cells.

  (19) The protein production kit according to (18), wherein the plant-derived cells are tobacco-derived cells.

  (20) The protein production kit according to (19), wherein the tobacco-derived cells are tobacco BY-2 cells.

  As described above, the transformant according to the present invention is an expression obtained by linking a gene of a plant virus having a suppressor of a virus resistance reaction including a gene encoding a protein to be expressed and a promoter transcriptionally induced by a hormone. The vector is a transformed cell characterized by being introduced into a biological cell.

  Similarly, the method for producing a protein according to the present invention uses the transformed cell according to the present invention, and is preferably composed of a step of culturing the cell and a step of inducing transcription by a hormone.

  The protein production kit according to the present invention is a kit for carrying out the protein production method according to the present invention, and is preferably a host into which the expression vector, a hormone for performing transcription induction, and an expression vector are introduced. It is composed of cells.

  According to the said structure, mRNA of the gene which codes an expressed protein can be highly amplified by the high replication capability of a virus. In addition, since the mRNA is amplified due to a virus having a suppressor of virus resistance reaction, degradation is suppressed by the virus resistance reaction of the host cell. Therefore, the target protein can be continuously produced at a high yield. Furthermore, since the host into which the expression vector has been introduced is not an individual organism but an organism-derived cell, the culture can be performed by liquid culture, and the culture scale can be increased inexpensively and easily. In addition, the transformed cells cannot be self-generated, and even if the transformed cells leak to the outside, they can be said to be safe because they die.

  Therefore, the transformed cell according to the present invention, the protein production method according to the present invention, and the protein production kit according to the present invention have large-scale production capacity and high production efficiency, and can produce a protein with higher safety. It has the effect of becoming.

  An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.

  In the present invention, (A) an expression constructed by linking a plant virus gene having a suppressor of a virus resistance reaction (silencing reaction) containing a gene encoding a protein to be expressed and a hormone transcription-inducible promoter. A transformed cell obtained by introducing a vector into an organism-derived cell (hereinafter referred to as a transformed cell according to the present invention), (B) a protein production method using the transformed cell (hereinafter referred to as a protein production method according to the present invention) And (C) a kit for performing the protein production method (hereinafter referred to as a protein production kit according to the present invention).

  Each invention will be described below.

(A) Transformed cell according to the present invention The transformed cell according to the present invention is a plant virus gene having a suppressor of virus resistance reaction (silencing reaction) containing a gene encoding a protein to be expressed, and transcribed with a hormone. An expression vector constructed by linking an inducible promoter is introduced into a biological cell.

<Plant virus having suppressor of virus resistance reaction>
A virus resistance reaction (silencing reaction) is a function of various organisms such as nematodes, drosophila, mice, humans, and plants that efficiently eliminate RNA inappropriate for their life activity. In particular, it refers to a defense mechanism against viral infection in living organisms.

  More specifically, when a living organism is infected with a virus, a large amount of virus-derived RNA (mRNA or viral genomic RNA) that is undesirable for living cells is produced. For this reason, a biological cell infected with a virus functions a virus resistance reaction (silencing reaction), drives out such virus-derived RNA, and prevents the spread of virus infection.

  On the other hand, many viruses have the ability to suppress virus resistance reactions (silencing reactions) in living cells. That is, the virus expresses a suppressor (suppressor) against the growth suppression by the virus resistance reaction (silencing reaction).

  The transformed cell according to the present invention is obtained by introducing an expression vector containing a plant virus gene, and finally used for expression of a target protein contained in the vector. It is necessary to have a suppressor (hereinafter simply referred to as a suppressor).

  If the plant virus used in the present invention does not have such a suppressor, as mentioned in the above-mentioned conventional technology, the RNA of the target protein is resistant to virus over time even though the gene of the target protein is amplified at the mRNA level. It is decomposed by the reaction (silencing reaction), and continuous protein production cannot be performed.

  Therefore, it is preferable that the plant virus used in the present invention has a suppressor.

  Here, the plant virus having a suppressor used in the present invention is not particularly limited, and examples thereof include a genus Potyvirus and a genus Cucumovirus (for example, cucumber mosaic virus (CMV). Abbreviation)), Potexvirus virus (eg Potato virus X (abbreviated as PVX)), Tombusvirus virus (eg Tomato bushy stunt virus (abbreviated as TBSV)), Cymbidiumu ringspot virus (Abbreviated as CymRSV)), Carmovirus virus (eg, Turnip rinkle virus (abbreviated as TCV)), Tobamovirus virus (eg, tobacco mosaic virus (abbreviated as TMV), tomato mosaic virus) (Abbreviated as ToMV)).

  In the examples described below, an expression vector is constructed using ToMV, but the virus (a) has higher growth ability than other viruses and can be expected to produce a large amount of protein. Adopted because of its high growth ability in tobacco BY-2 cells used as a host in the examples, and (c) it has been widely used in the past and has excellent handleability, versatility, and applicability.

  By using the plant virus having the suppressor, the amplified mRNA of the target protein can be continuously produced at a high level without being decomposed by a virus resistance reaction (silencing reaction).

  Here, the plant virus is a general term for viruses having a plant as a host, and in a narrow sense, refers to a virus of a higher plant. Plants infected with viruses show various different symptoms depending on the combination of virus types and parasitic plants, such as mosaic, chlorosis, yellowing, malformation, curly leaf, hatching, necrosis, etc. Cause tremendous damage to the harvest. Virus propagation patterns include sap propagation, insect propagation, soil propagation, grafting, and seed propagation. Some viruses also propagate in the vector insect and can be transmitted through eggs.

<Construction of expression vector>
The target protein to be expressed is not particularly limited as long as it can be expressed in a biological cell. Examples include useful enzymes that can be used in pharmaceuticals, interferons, allergen proteins, pathogen antigens, erythropoietin, enkephalins, cell growth factors, antibodies (immunoglobulins), and albumins. By ingesting an appropriate amount of the organism-derived cell in which the protein is expressed, it can be used as a pharmaceutical product such that the same effect as ingesting the protein can be obtained.

  The gene encoding the target protein to be expressed may be introduced using a conventional gene recombination technique such as replacement with a part of the above-described suppressor-containing plant virus gene or ligation to the virus gene.

  The expression vector to be introduced into the transformed cell according to the present invention may be constructed by linking a recombinant plant virus gene into which the gene encoding the target protein is introduced downstream of a promoter transcriptionally induced by a hormone. By using such a hormone-inducible promoter, a protein derived from a plant virus linked downstream thereof is induced and expressed with high efficiency.

  Such promoters and base vectors are not particularly limited, and conventionally known promoters and vectors may be used. For example, Ti plasmid pTA7001 (McNellis TW, Mudgett MB, Li K, Aoyama T, Horvath D, Chua NH, Staskawicz BJ. Glucocorticoid-inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death. See Plant J. 1998 Apr; 14 (2): 247-57.) and pTA7001 (Stu) constructed by the present inventors, and Ti plasmid pER8 (Zuo, having a promoter estrogen-induced by estrogen). J., Niu, QW, and Chua, NH (2000) .An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants.See Plant J. 24, 265-273.) . As other examples of transformation vectors, other transformation vectors include plasmids having promoters that are transcriptionally induced by ecdysone, pES60 and pES46 (The Plant Journal (1999) 19: 97-106. Ecdysone agonist inducible transcription in transgenic tobacco plants Alberto Martinez, Caroline Sparks, Cliff A. Hart, John Thompson and Ian Jepson).

  The expression vector preferably contains a transcription factor for the promoter. This is because the transcription factor regulates the transcription of the gene of the plant virus linked downstream of the promoter, and it becomes possible to induce transcription more efficiently. The transcription factor may be selected according to the promoter used. For example, when the Ti plasmid pTA7001 (Stu) having a promoter induced by transcription with steroid hormone is used, the transcription factor is GVG, and when the Ti plasmid pER8 having a promoter induced by estrogen is used, XVE.

  The expression vector may contain various other DNA segments. An example of the DNA segment is a terminator. In addition, in order to express a chimeric protein, a gene encoding another protein and a restriction enzyme recognition site (such as a multicloning site) for introducing such a gene may be included as needed.

  The method (construction method) for constructing the expression vector is not particularly limited, and a known recombinant DNA technique may be used by combining a DNA segment such as a recombinant virus gene and a promoter and the above-described base vector. You can connect using. In addition, a method for propagating the constructed expression vector (production method) is not particularly limited, and a known method can be used. In general, E. coli may be used as a host and propagated in the E. coli cells. At this time, a preferred E. coli type may be selected according to the type of vector.

<Transformation method>
The specific technique for introducing the constructed expression vector into a plant-derived cell serving as a host is not particularly limited, and an appropriate transformation method according to the type of plant-derived cell serving as a host is used. Good. As a general transformation method for plant-derived cells, there can be mentioned a transformation method using Agrobacterium (Agrobacterium method). As shown in Examples described later, Agrobacterium is also used in the present invention. The method can be suitably used. In addition, conventionally known methods such as a particle gun method, a protoplast / spheroplast method, an electroporation method (electroporation method), a calcium phosphate method, a liposome method, and a DEAE dextran method can be suitably used.

  The method for confirming whether or not a recombinant plant virus gene (expression vector) has been introduced into a host cell, and whether or not it is reliably expressed in the host cell is not particularly limited, and various known methods The method can be used. Specifically, various markers may be used. For example, a gene deleted in the host cell is used as a marker, and a plasmid containing this marker and a recombinant plant virus gene is introduced into the host cell as an expression vector. Thus, the introduction of the gene of the present invention can be confirmed from the expression of the marker gene.

For example, in Examples described later, a drug resistance marker (hygromycin resistance gene, Hyg ^r) is used to, in a medium containing hygromycin, by culturing the transformed candidate strain, cell lines have been grown It becomes possible to select as a transformant. As other markers, bialaphos resistance markers, kanamycin resistance markers, etc. are effective for selection of plant cells, and puromycin resistance markers, bleomycin resistance markers, XGPRT gene, DHFR gene, thymidine kinase gene, etc. are used for selection of animal cells. It is valid. Furthermore, in yeast and the like, auxotrophic markers such as uracil auxotrophic markers can be used for selection. However, the method for selecting these transformants is not limited, and may be appropriately selected and used depending on the host into which the expression vector is introduced.

  In addition, there can be mentioned a so-called genomic PCR method in which genomic DNA prepared from a host cell is used as a template and the entire gene length of the introduced protein is specifically amplified. If it can be confirmed by electrophoresis or the like that the gene encoding the target protein is amplified by this method, the introduction of the gene can be confirmed.

  As another method, the target protein may be expressed as a fusion protein. For example, the green fluorescent protein (GFP) derived from Aequorea jellyfish is used as a marker, and the target protein is expressed as a GFP fusion protein. May be. Furthermore, a gene for visualizing and monitoring an expression site in a transformed plant cell can be introduced into the expression vector. An example of such a gene is the β-glucuronidase (GUS) gene.

<Host cell>
The host cell into which the expression vector is introduced is not particularly limited as long as it is a biological cell, and may be an animal-derived cell or a plant-derived cell. However, the plant-derived cells are more preferably plant-derived cells in that the growth rate is high and the risk of contamination is low as compared to animal-derived cells, and the medium preparation cost is very low. Here, animal-derived cells and plant-derived cells are meant to include animal individuals and cells, tissues, and organs excluding plant individuals, respectively. In particular, cells that can be cultured in a liquid medium or the like are preferable.

  In addition, the animal to be derived is not particularly limited, but fish such as humans, monkeys, dogs, sheep, goats, rabbits, mice, rats, guinea pigs, Chinese hamsters, cattle, horses, pigs, medaka fish and zebrafish , Silkworm, and night pod (Spodoptera frugiperda). Moreover, as a plant used as origin, although not specifically limited, a rice, a Syringa, a barley, a wheat, tobacco, a tomato, a cucumber, a soybean, a potato, a corn, a periwinkle, an Arabidopsis thaliana, and an alfalfa are mentioned.

  In addition, unicellular organisms such as fungi such as Bacillus subtilis and lactic acid bacteria and yeast can be used as host cells.

  Examples of animal-derived cells are not particularly limited, but include HeLa cells, CHO cells, melanoma cells, and mouse 3T3 cells. Examples of plant-derived cells include tobacco BY-2 cells, potato-derived, rice-derived, sweet potato-derived, soybean-derived, parsley-derived, Arabidopsis-derived, wheat-derived, corn-derived cells, and periwinkle-derived cells.

  In Examples described later, tobacco BY-2 cells are used as a host. Tobacco BY-2 Cell Line as the “Hela” Cell in the Cell Biology of Higher Plants ”International Review of cytology, vol.132, pp 1-30 (Toshiyuki nagata, Yasuyuki Nemoto, and Seiichiro Hasezawa 1992) and http://wwwriken.go.jp/r-wprld/info/release/press/2003/030620/) are the most widely used plant cell lines in the world. It was adopted because it has the fastest growth rate, easy genetic manipulation, and easy mass culture.

  In the present invention, not a plant individual but a plant-derived cell is used as a host because it has the following advantages.

  (a) Compared with a case where a plant individual is used as a host, the growth rate is high, and transformed cells can be grown in a short period of time. Large scale and mass production are also easy.

  (b) Compared to the case where a plant individual is used as a host, it does not require a vast space, facility (field, greenhouse) or the like for growth. In addition, the culture apparatus can be easily increased in size.

  (c) Compared to the case where a plant individual is used as a host, there is no step of differentiating a plant individual from callus, a step of blooming a seed, and a step of sowing and growing the seed. Production, screening and protein production periods can be significantly reduced.

  (d) Protein-induced expression by an inducer such as a chemical substance can be easily performed. When a plant individual is used as a host, it is necessary to uniformly administer a chemical substance or the like to the plant individual by a method such as direct application and spraying to the plant individual, which is very laborious and time consuming. On the other hand, when plant-derived cells are used as a host, it is sufficient to add the inducer or the like to the medium during cell culture, and it is possible to induce the pre-cells simultaneously and uniformly. .

  (e) A plant individual is difficult to strictly control the surface temperature due to transpiration or the like, but in the case of cultured cells grown in a liquid medium, temperature control and the like are easy. When a promoter that is highly sensitive to various stresses due to changes in environmental conditions or the like is used, protein production can be performed stably.

  (f) Unlike plant individuals composed of various tissues, the cultured cells are homogeneous, so there is no tissue-specific effect and protein expression control is easy.

  (g) Since the cell cycle can be highly synchronized, strict protein expression can be controlled.

  (h) Since it is a liquid culture, the protein can be easily recovered and purified by secreting the secreted protein into the medium. Even when the target protein is not a secretory protein, the same effect can be obtained by adding a secretory tag to the target protein.

  (i) Unlike a plant individual, a cultured cell does not require light for growth, so that it is possible to reduce lighting equipment and lighting costs. It is also advantageous when expressing light-sensitive proteins.

  (j) The protein to be produced can be chemically modified by adding various chemical substances or the like to the medium. For example, by adding a radioisotope, a radiolabeled protein can be easily prepared.

  (k) Even when a transformant leaks to the outside world, unlike a plant individual, it cannot grow by itself and is safe to die. This can reduce facilities, costs, and time required for prevention of scattering of plant individuals, seeds, pollen, etc., safety testing, and risk management for genetically modified organisms.

(B) Protein production method according to the present invention The protein production method according to the present invention is performed using the above-described transformed cell according to the present invention. In the protein production process according to the present invention, the protein in the cells may be obtained without culturing the transformed cells obtained at the time of transformation. However, the protein production method according to the present invention preferably includes a step of culturing the transformed cell according to the present invention (hereinafter referred to as a cell culture step). This is because the number of transformed cells into which the gene encoding the target protein has been introduced can be increased by such a culturing step, and the production amount of the protein can be increased.

  The cell culturing method in the cell culturing step is not particularly limited, and a medium and culturing conditions suitable for the cells to be cultured may be applied. The medium for culturing plant cells is not limited, but may contain inorganic salts, carbon sources, vitamins, and amino acids. In addition, coconut milk or yeast extract may be added to promote growth. In addition, plant hormones such as auxin and cytokinin, gibberellin, abscisic acid, and ethylene may be added. Moreover, although it is culture conditions, what is necessary is just to employ | adopt the optimal thing according to the cell to culture | cultivate light, temperature, the presence or absence of aeration, etc. For example, in Examples described later, when culturing tobacco BY-2 cells, 370 mg / l potassium dihydrogen phosphate, 1 mg / l thiamine hydrochloride, 3% sucrose, 0.2 mg / l 2,4-D 1/100 volume is subcultured every week after the shaking culture at 26 ° C. and 135 rpm in the dark using the MS medium containing.

On the other hand, a medium for culturing animal cells is not limited, but serum may be added to amino acids, vitamins, glucose, and salts. In addition, a bicarbonate / carbon dioxide buffer solution is used as a buffer solution, and a CO ₂ incubator is used as an incubator. In addition, phenol red may be added for pH monitoring. The culture condition is generally cultured at 37 ° C., but depending on the cell line, it may be 28 ° C. or 40 ° C.

  In the protein production method according to the present invention, in addition to the cell culture step, it is preferable to further include a step of performing transcription induction with hormones on the cultured cells (hereinafter referred to as transcription induction step). Since the expression vector introduced into the above-described transformed cell according to the present invention contains a promoter that is transcriptionally induced by a hormone, the transcription induction step initiates transcription of the gene encoding the target protein, transcription. The amount can be increased and controlled, and the amount of protein produced can be increased and the amount of protein produced can be controlled.

  The hormone used in this transcription induction step is not particularly limited, and may be appropriately selected according to the promoter contained in the expression vector introduced into the transformed cell according to the present invention. For example, in addition to steroid hormones, estrogens, ecdysone, etc., ethanol can also be used for transcription induction.

  In the examples described later, since a protein expression vector is constructed based on Ti plasmid pTA7001 (Stu) having a promoter that is transcriptionally induced by steroid hormones, induction is performed by steroid hormones.

  The amount of hormone used in this transcription induction step may be appropriately selected according to the type of promoter, the type of cell line, the culture phase of the cell line, the cell culture conditions, and the like. For example, in the examples described later, this transcription induction step is performed using a steroid hormone (dexamethasone) at a concentration of 30 μM.

  Further, the timing for performing this transcription induction step is not particularly limited, and may be appropriately selected according to the type of cell line, the culture phase of the cell line, the cell culture conditions, and the like. For example, in the examples described later, when the transcription induction was performed on the fifth day after the subculture, the ratio of the cells that expressed the reporter protein GFP was the highest. From this, it can be said that the physiological conditions of the cultured cells on the fifth day of the preculture were suitable for the induction of transcription and the expression of the protein under the conditions of this example.

(C) Protein production kit according to the present invention The protein production kit according to the present invention is not particularly limited as long as it includes reagents, instruments, devices and the like that can perform the protein production method according to the present invention. Although it is not a thing, it is preferable to contain the expression vector introduce | transduced into the transformed cell concerning this invention. In this case, the target protein can be produced by introducing a gene encoding the target protein to be expressed in the expression vector using a general gene recombination technique such as ligation or substitution.

  For example, an expression vector constructed by introducing a recombinant ToMV cDNA in which the coat protein gene is replaced with the GFP gene may be included downstream of a promoter capable of inducing transcription with steroid hormones. In such a case, the GFP gene and the target protein gene to be expressed may be replaced or linked to the GFP gene. When linked to the GFP gene, the target protein is obtained as a fusion protein with GFP, and protein production becomes possible while monitoring expression using GFP fluorescence as an index.

  Further, the protein production kit according to the present invention preferably further contains a hormone for performing the above-described transcription induction step. The hormone contained in the kit may be appropriately selected according to the promoter contained in the expression vector introduced into the transformed cell according to the present invention, as described above. For example, there are steroid hormones, estrogen, ecdysone and the like. In addition, ethanol or the like can be used for induction of transcription and may be included in the kit.

  For example, as shown in Examples below, when a protein expression vector is constructed based on a Ti plasmid pTA7001 (Stu) having a promoter that is transcriptionally induced by steroid hormone, it is preferable that steroid hormone is contained. I can say that.

  Moreover, the protein production kit according to the present invention may further include a biological cell serving as a host. The host cell contained in the kit is not particularly limited as long as it is a biological cell, and may be an animal-derived cell or a plant-derived cell. In addition, the animal to be derived is not particularly limited, but fish such as humans, monkeys, dogs, sheep, goats, rabbits, mice, rats, guinea pigs, Chinese hamsters, cattle, horses, pigs, medaka fish and zebrafish , Silkworm, and night pod (Spodoptera frugiperda). Moreover, as a plant used as origin, although not specifically limited, a rice, a Syringa, a barley, a wheat, tobacco, a tomato, a cucumber, a soybean, a potato, a corn, a periwinkle, an Arabidopsis thaliana, and an alfalfa are mentioned.

  Other examples of host cells include fungi such as Bacillus subtilis and lactic acid bacteria, and unicellular organisms such as yeast.

  Examples of animal-derived cells are not particularly limited, but include HeLa cells, CHO cells, melanoma cells, and mouse 3T3 cells. Examples of plant-derived cells include tobacco BY-2 cells, potato-derived, rice-derived, sweet potato-derived, soybean-derived, parsley-derived, Arabidopsis-derived, wheat-derived, corn-derived cells, and periwinkle-derived cells.

  In addition, the protein production kit according to the present invention may also include a medium for culturing biological cells, an incubator, and the like.

  According to the invention described above, it is apparent that it is possible to provide a protein production system, a production method, and the like with large-scale production capacity, high production efficiency, and high safety.

  The present invention is not limited to the embodiments described above, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments can be appropriately combined. Such embodiments are also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and FIGS. 1-4, this invention is not limited to this.

[Example 1: Induced expression of protein by steroid hormone in tobacco BY-2 cells]
<Construction of expression vector pTA7001-ToMV-erG3 (SF3)>
As a viral vector, a ToMV mutant (ToMV-erG3 (SF3)) in which the coat protein gene was replaced with the GFP gene was used. The ToMV-erG3 (SF3) was sold by Dr. Tetsuo Ii (Kyoto University Graduate School).

  On the other hand, a Ti plasmid pTA7001 (Stu) having a promoter and transcription factor (GVG) that is transcriptionally induced by steroid hormone was used as a transformation vector. The vector was prepared by introducing a StuI site into the transcription start point of pTA7001 using the PCR method. The pTA7001 was sold by Dr. Chua (Laboratory of Plant Molecular Biology, The Rockefeller University).

  Next, ToMV-erG3 (SF3) cDNA was introduced downstream of the promoter induced by transcription with the steroid hormone of pTA7001 (Stu) to construct an expression vector pTA7001-ToMV-erG3 (SF3). The construction of the vector will be explained in more detail as follows. PiL.erG3 (SF3) containing ToMV mutant cDNA (Atsushi Tamai and Tetsuo Meshi, “Tobamoviral movement protein transiently expressed in a single epidermal cell nctions beyond multiple plasmodesmata and spreads multicellularly in an infection-coupled manner” Molecular Plant-Microbe interaction (2001) 14: 6-134), a plasmid piL.erG3 (SF3) (Avr) was prepared by replacing the MluI site with the AvrII site using a linker. Furthermore, a DNA fragment of about 1600 base pairs of ToMV cDNA 5 'end into which SnaBI site was introduced at the 5' end using piL.erG3 (SF3) as a template was cleaved with SnaBI and SpeI. A DNA fragment of about 1220 base pairs obtained by this operation was inserted between the StuI site and SpeI site of pTA7001 (Stu) to prepare plasmid pTA7001-ToMV5'-Spe. After cleaving pTA7001-ToMV5′-Spe with SpeI and AvrII, an approximately 5200 base pair fragment containing the 3 ′ end portion of the ToMV mutant cDNA was inserted into the SpeI site of pTA7001-ToMV5′-Spe, and the expression vector pTA7001 -ToMV-erG3 (SF3) was created.

  FIG. 1 shows the constructed expression vector pTA7001-ToMV-erG3 (SF3).

<Transformation of tobacco BY-2 cells>
The expression vector pTA7001-ToMV-erG3 (SF3) was introduced into tobacco BY-2 cells by the Agrobacterium method. Specifically, it was as shown below.

  The expression vector was introduced into Agrobacterium Tumefacience EHA105 strain by electroporation. This was precultured in AB sucrose medium containing kanamycin (50 mg / l). Next, the mixture was mixed with tobacco BY-2 cells, transferred to a petri dish, and allowed to stand in a dark place at 26 ° C. for 42 to 48 hours to transform tobacco BY-2 cells. After washing with a medium for tobacco BY-2 cells, it is spread on a solid medium for tobacco BY-2 cells containing carbenicillin (100 mg / l) and hygromycin (20 mg / l) to grow transformed tobacco BY-2 cells. It was.

<Transcription induction by steroid hormone>
Transcription induction was performed on the obtained transformed tobacco BY-2 cells by steroid hormone treatment (hereinafter referred to as DEX treatment as appropriate). Specifically, steroid hormone (dexamethasone) was added to a culture solution of transformed tobacco BY-2 cells to a concentration of 30 μM.

  As a result of the induction of transcription, GFP fluorescence was observed 48 hours later using a solid fluorescence microscope apparatus (manufactured by Olympus). Further, total RNA 48 hours after transcription induction was extracted by the trisol method and used for Northern analysis. For Northern analysis, an RNA probe complementary to about 200 bases of the 3 'untranslated region of ToMV was used. Probe labeling was performed using a DIG RNA Labeling Kit from Roche Diagnostics. The detection was carried out using the company's DIG Luminescent Ditection Kit and CDP-Star according to the manual of each kit.

  FIG. 2 shows the result of Northern transcription analysis of GFP gene mRNA transcription with and without DEX treatment. As is clear from FIG. 2, the transcriptional induction of GFP gene mRNA by DEX treatment was confirmed.

  FIG. 3 shows the result of fluorescence microscope observation of transformed tobacco BY-2 cells after DEX treatment. As a result of detecting the fluorescence by GFP, the induction | guidance | derivation expression of GFP by DEX processing was confirmed as shown in FIG.

<Examination of pre-culture conditions for tobacco BY-2 cells>
The number of days before subculture to DEX treatment and the expression rate of GFP were examined. The method was performed as follows.

  Transformed cells were subcultured in 1/100 volume and precultured for 3 days, 5 days and 7 days. Steroid hormone (dexamethasone) was added to 30 μM, and the cells were further cultured for 48 hours, and then the cells were observed with a Nikon upright fluorescence microscope. The cells with and without GFP fluorescence were counted, and the expression rate was calculated.

FIG. 4 shows the results of examining the relationship between the number of days precultured and the expression rate of GFP. As a result, the expression rate of GFP when pre-cultured for 5 days showed the highest value of about 4%. This is thought to be because changes in the physiological state of the cells accompanying proliferation affect the replication of viral sequences and the expression of GFP. As explained above, downstream of the promoter that can induce transcription with steroid hormones. In addition, transcriptional induction by steroid hormone was performed on transformed tobacco BY-2 cells into which expression vectors constructed by introducing recombinant ToMV cDNA with the coat protein gene replaced with the GFP gene were introduced into tobacco BY-2 cells. As a result, reliable viral RNA amplification and GFP induced expression were achieved. Therefore, by using the transformed cell according to the present invention, the protein production method according to the present invention, and the protein production kit according to the present invention, a protein having a large-scale production capacity and a high production efficiency, and a higher safety. It can be said that it can be produced.

  As described above, according to the present invention, the present invention can produce a large amount of protein at a low cost, and the obtained protein is effective in a wide range of fields including the pharmaceutical, chemical and food industries. Available to: Furthermore, since expression vectors, host cells and the like introduced into the transformed cells according to the present invention can be marketed as protein expression kits, it can be applied to the reagent industry for experiments and research. Become.

1 is a schematic diagram showing the structure of pTA7001-ToMV-erG3 (SF3), which is an example of an expression vector introduced into a transformed cell according to the present invention. In the Examples, transcription of GFP gene mRNA in the presence or absence of steroid hormone treatment (DEX treatment) of transformed tobacco BY-2 cells into which the expression vector pTA7001-ToMV-erG3 (SF3) was introduced was analyzed by Northern analysis. It is a figure which shows the result of having investigated. In Examples, when steroid hormone treatment (DEX treatment) was performed on transformed tobacco BY-2 cells into which the expression vector pTA7001-ToMV-erG3 (SF3) was introduced, the GFP expression of the cells was measured using a fluorescence microscope. It is a figure which shows the result observed. In an Example, it is a line graph which shows the result of having investigated the relationship of the expression rate of GFP with the preculture days (the 3rd day of preculture, the 5th day, the 7th day) before subculture and DEX processing.

Claims (20)

A gene of a plant virus having a suppressor of a virus resistance reaction including a gene encoding a protein to be expressed;
An expression vector formed by linking a promoter that is transcriptionally induced by a hormone,
A transformed cell, which is introduced into a biological cell.   The transformed cell according to claim 1, wherein the plant virus is a virus belonging to the genus Tobamovirus.   The transformed cell according to claim 2, wherein the virus belonging to the genus Tobamovirus is tobacco mosaic virus or tomato mosaic virus.   The transformed cell according to any one of claims 1 to 3, wherein the hormone is a steroid hormone.   The transformed cell according to any one of claims 1 to 4, wherein the organism-derived cell is a plant-derived cell.   6. A transformed cell, wherein the plant-derived cell according to claim 5 is a tobacco-derived cell.   The transformed cell according to claim 6, wherein the tobacco-derived cell is a tobacco BY-2 cell.   The transformed cell according to any one of claims 1 to 7, wherein the protein expression vector is introduced by the Agrobacterium method.   A method for producing a protein, comprising using the transformed cell according to any one of claims 1 to 8.   The method for producing a protein according to claim 9, comprising a step of culturing transformed cells.   The method for producing a protein according to claim 10, further comprising a step of inducing transcription by a hormone.   12. The method for producing a protein according to claim 11, wherein the transcription induction step by the hormone is a transcription induction step by a steroid hormone.   A protein production kit for performing the protein production method according to any one of claims 9 to 12.   The protein production kit according to claim 13, wherein the protein production kit comprises the expression vector according to any one of claims 1 to 8.   The protein production kit according to claim 13 or 14, wherein the protein production kit further comprises a hormone.   The protein production kit according to claim 15, wherein the hormone is a steroid hormone.   The protein production kit according to any one of claims 13 to 16, wherein the protein production kit further comprises an organism-derived cell serving as a host.   The protein production kit according to claim 17, wherein the biological cell is a plant-derived cell.   The protein production kit according to claim 18, wherein the plant-derived cell is a tobacco-derived cell.   The protein production kit according to claim 19, wherein the tobacco-derived cells are tobacco BY-2 cells.

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