KR20050027839A - Human granulocyte macrophage colony stimulating factor expressed in plants - Google Patents

Abstract

A granulocyte macrophage colony stimulating factor expressed in plants is provided, which granulocyte macrophage colony stimulating factor(GM-CSF) is cheaply mass produced using plants, and has the identical biological properties and function to natural GM-CSF, so that the GM-CSF is useful for treatment of aplastic anemia, intrinsic and idiopathic neutropenia and Crohn's disease. A polynucleotide encoding the granulocyte macrophage colony stimulating factor(GM-CSF) expressed in plants has the nucleotide sequence set forth in SEQ ID NO:1. A recombinant plant expression vector of GM-CSF comprises (i) the polynucleotide of SEQ ID NO:1; (ii) a promoter operately linked to the polynucleotide and forming a RNA molecule in plant cells; and (iii) 3'-untranslated region which results in polyadenylation of 3'-terminal of the RNA molecule in plant cells. The method for producing the GM-CSF in plants comprises the steps of: (a) introducing the recombinant plant expression vector of human growth hormone into plant cells; (b) selecting the transformed plant cells; (c) regenerating the transformed plant cells to produce transformed plants; and (d) producing the GM-CSF in the transformed plants.

Description

Human Granulocyte Macrophage Colony Stimulating Factor Expressed in Plants

The present invention relates to a human granulocyte macrophage colony stimulating factor (GM-CSF) expressed in plants, and more particularly, a novel polynucleotide encoding human GM-CSF, a vector comprising the same, It relates to a plant to which the vector is introduced and a method for producing GM-CSF using the plant.

Plant genetic engineering has made remarkable developments in spite of its short history, enabling the development of transgenic plants with new traits by introducing useful genes from plants and microorganisms into plants. The attempted plant development by genetic manipulation has been focused on agricultural applications such as increased crop productivity by introduction of disease resistance, insect resistance, and herbicide tolerance genes, which formed the first generation of plant genetic engineering.

Recently, however, interest in development as a production system for the production of high value-added useful recombinant proteins, using the functions of various industrial materials and the advantages of higher organisms, rather than the agricultural application of plants as simply a food resource. This is concentrated. In other words, it is not intended for the plant itself, but for the use of the plant as a tool, rather than the concept of an input in which the introduced gene simply ends up being expressed. It is intended to be used as a concept, which is the basic concept of second generation plant genetic engineering.

Plants are particularly attractive as a mass production system for medical and industrial proteins, because they can be harvested and processed easily using traditional farming infrastructures that are already established, allowing for the mass storage of desired biomass. . In addition, unlike bacteria, which are mainly used as protein expression systems, plants have a eukaryotic protein synthesis pathway through which post-detox modification processes are essential for the activity of mammalian proteins (Cabanes-Macheteau et al ., Glycobiolgy 9: 365-372). (1999)).

On the other hand, the hematopoietic action of the human body is mainly made in the bone marrow and various types of blood cells are produced through various and complicated pathways. These hematopoiesis is regulated and influenced by hematopoietic microenvironment consisting of stromal cells in bone marrow, hemopoietic growth factor and various extracellular matrix. Hematopoietic growth factors involved in hematopoietic action are called granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony factor (macrophage) involved in the formation of macrophages depending on the stage and route of hematopoiesis. colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF) and interleukin-3 (Multi-CSF) involved in the formation of granulocytes It is mostly secreted from interstitial cells such as macrophage, endothelial cells and fibroblasts.

Granulocyte macrophage colony promoting factor (hereinafter referred to as "GM-CSF") is involved in the production of blood cells and platelets of the bone marrow, and also the separation of macrophages promotes the growth of white blood cells. As such, GM-CSF is not only a regulator of hematopoietic activity but also used as a drug to be administered after bone marrow transplantation in leukemia patients due to its effect of increasing the production of immune cells. . It has also recently been used to treat Crohn's disease, a chronic disease of the gastrointestinal system (Dieckgraefe et al ., Lancet 360 (9344): 1478-80 (2002)).

In general, GM-CSF is produced using animal cell lines, but maintenance costs are high, and there are difficulties in mass expression and separation and purification. In order to compensate for these disadvantages, a technique for separating and purifying glycoproteins having large-scale expression and biological activity using E. coli has been introduced. Less produced or produced polypeptides do not form a stable tertiary structure, are easily degraded, and also have a problem in that they are aggregated in an inactive form and exist as an inclusion body in cells.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made diligent research efforts to solve the above-mentioned problems of the prior art, and confirmed that the novel polynucleotides encoding human GM-CSF can be successfully introduced into plants, and functional GM-CSF can be obtained therefrom. Thus, the present invention has been completed.

Accordingly, it is an object of the present invention to provide novel polynucleotides encoding GM-CSF.

Another object of the present invention to provide a plant expression vector of recombinant human GM-CSF.

It is another object of the present invention to provide a method for producing a transient transfected plant that expresses recombinant human GM-CSF temporarily.

Another object of the present invention is to provide a transient transfected plant that expresses recombinant human GM-CSF temporarily.

Another object of the present invention is to provide a method for producing recombinant human GM-CSF.

Another object of the present invention is to provide a method for producing a transgenic plant stably expressing recombinant human GM-CSF.

Another object of the present invention is to provide a transgenic plant stably expressing recombinant human GM-CSF.

Another object of the present invention is to provide a recombinant human GM-CSF produced by the method of the present invention.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the invention provides a polynucleotide encoding GM-CSF comprising the nucleotide sequence set forth in SEQ ID NO: 1.

The novel polynucleotides of the present invention encode GM-CSF and have a modified nucleotide sequence to optimize expression in plants without amino acid substitutions.

The design of the polynucleotides of the invention is carried out by the following criteria: (i) to make the GC content at least 50%, (ii) to have the plant's preferred codons, and (i) to remove intron-like sequences. will be. Such plant-compatible sequences can increase the translation rate of genes (Kusnadi et al ., Biotechnol. Prog. 14: 149-155 (1998)). In addition, the intron-like sequence in the foreign gene to be introduced is removed in order to rule out the possibility that the arrangement of the transgene is recognized as an intron in the host plant, resulting in cleavage in the plant nucleus and a failure to produce the desired protein.

The novel polynucleotides of the present invention are to be interpreted to include not only the nucleotide sequence set forth in SEQ ID NO: 1, but also a nucleotide sequence exhibiting substantial identity to the nucleotide sequence described above. The substantial identity is that at least 90% of the phases when the nucleotide sequence of the present invention and any other sequence are arranged to correspond as closely as possible and the sequence is analyzed using algorithms commonly used in the art. By nucleotide sequence, which shows homology, more preferably 95% homology, most preferably 98% homology.

According to another aspect of the present invention, the present invention provides a kit comprising: (i) the nucleotide sequence set forth in SEQ ID NO: 1; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be any one commonly used in the art for gene introduction of a plant, for example, the Coliflora Mosaic Virus (CaMV) 35S promoter , Nopaline synthase (nos) promoter, pigwarm mosaic virus 35S promoter, sugacran basilisform virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carboxylase small sub Photoinducible promoters of ssRUBISCO, rice cytosol triosphosphate isomerase (TPI) promoters, adenine phosphoribosyltransferase (APRT) promoters of Arabidopsis and octopine synthase promoters.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site causing the polyadenylation suitable for the present invention is derived from the nopalin synthase gene of Agrobacterium turmeration (nos 3 'end) ( Bevan et al., Nucleic Acids Research , 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tuberculosis, of the protease inhibitor I or II gene of tomato or potato 3 'terminal portion and CaMV 35S terminator.

Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention may be selected as an indicator for antibiotics (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, and the like) and resistance genes (e.g. neomycin phosphotransferase ( nptII ) Mycin phosphotransferase ( hpt ), and the like).

According to the present invention, the production of plants in which the gene encoding GM-CSF has been introduced is largely achieved in two ways: transient transfected plants and transgenic plants.

As used herein, the term "transient transfected plant" means that the introduced gene is not transferred to the next generation as the plant into which the foreign gene has been introduced. In transiently transfected plants, foreign genes are generally present separately from the chromosome of the host cell. In contrast, the term “transgenic plant” refers to a plant having a foreign gene that is transmitted to the next generation. In transgenic plants, foreign genes are generally integrated into the chromosome of the host cell, becoming the genetic repertoire of the host cell, and reliably delivered to the next generation.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention into the plant cell of the plant; And (b) provides a method for producing a transient transfected plant to temporarily express recombinant human GM-CSF comprising the step of confirming whether the gene has been introduced into the plant cells.

According to another aspect of the present invention, the present invention provides a transient transfected plant that transiently expresses recombinant human GM-CSF prepared by the above method.

According to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention into plant cells; (b) confirming whether the gene has been introduced into the plant cell; And (c) provides a method for producing a recombinant human GM-CSF comprising the step of obtaining a recombinant human GM-CSF from a plant comprising a plant cell introduced gene.

In the method of the present invention, transient transfection of plant cells can be carried out according to conventional methods known in the art (Rainer Fisher et al., Biotechnol. Appl. Biochem. , 30: 113-116 (1999) ). Transient gene expression in plants is faster than in transformants with stable expression, and the result of protein expression can be confirmed as soon as possible. Therefore, whether the target protein obtained in plants prior to large-scale production using stable transgenic plants is functioning normally. Suitable for checking

Therefore, the present inventors expressed, isolated and purified GM-CSF using a transient gene expression method in plants for efficient mass production of GM-CSF having biological activity.

Transient gene expression methods are suitable approaches to test the functionality and stable expression of target genes before manufacturing plant transformants for mass production, which has the advantage of saving time and money. In particular, plant transformants produced by stable transformation show a chromosomal positional effect that is affected by the position at which the foreign gene is inserted. Transient transfection can rule out this effect. It is well suited for the efficient expression and isolation of foreign genes.

In transient transfection, there are three types of methods for delivering foreign genes to plant cells: particle bombardment, which coats and introduces naked DNA onto particles (Christou, P., Trends Plant Sci). 1: 423-431 (1996)), vacuum infiltration (vacuum infiltration) Agrobacterium-fill trafficking Orientation method (agroinfiltration containing the expression vector is introduced into plant tissue by Agrobacterium in the like) (Kapila et al, plant Sci. ., 122: 101-108 (1996)), and a modified plant virus virus vector method using a vector (viral vectors) (Scholthof, H. et al, Annu Rev. Phytopathol 34:... 299-323 (1996) ).

The transformation efficiencies of these three methods vary, so that in the case of DNA introduction by particle guns, genes are generally introduced into only a small number of cells and function only when DNA reaches the cell nucleus for transcription. This method can be used to test the stability of recombinant proteins prior to stable transformation, but is not suitable for mass expression of recombinant proteins.

Agroinfiltration allows genes to be introduced into more cells than the particle gun, whereby T-DNA containing the target gene is actively introduced into the nucleus with the help of several bacterial proteins. In this method, the introduced T-DNA is not integrated into the host cell's chromosome and continuously expressed, but is present in the nucleus independently and expresses the transient expression of the gene of interest. It is suitable for obtaining protein quickly.

In addition, the method using a viral vector introduces a foreign gene into the genome of a viral plant pathogene to be controlled by a strong promoter, and when the recombinant viral vector including the foreign gene is infected with a plant, the foreign gene introduced is During plant virus replication, they are amplified with high efficiency and then expressed to produce foreign proteins. However, when using a viral vector, the size of the protein to be produced is limited to less than 30 kDa, so the size of the gene to be introduced is difficult to use.

Therefore, in the present invention, the agroin fill method is most preferable. The agroin filtration method is generally carried out with leaves.

When the present invention is practiced in agrofiltration, more preferably, the Agrobacterium tumefaciens -binary vector system is used.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') Agrobacterium tumefaciens containing a vector having a sequence of Infiltrating tissue: a nucleotide sequence set forth in SEQ ID NO: 1; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts in plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') confirming the expression of the protein in the infiltrated leaves.

In one specific embodiment described above, suitable leaves include any tissue derived from the germinated seed, preferably the leaves produced before the peduncle rises, most preferably the leaves are too young or not mature. The younger the leaves, the higher the expression level, but the tissues are more susceptible to yellowing or dying. Seed germination is carried out under suitable dark / light conditions using a suitable medium.

Introduction of genes into plant cells is carried out with Agrobacterium trimers containing Ti plasmids (Depicker, A. et al., In Genetic Engineering of Plants, Plenum Press, New York (1983)). More preferably, binary vector systems such as pBin19, pRD400, pRD320 and pHS737 are used (An, G. et al., “Binary vectors” In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986); RK Jain, et al., Biochem. Soc. Trans. 28: 958-961 (2000).

Gene introduction into the plant cells in the leaves by Agrobacterium tufasion includes methods known in the art. Most preferably, the gene introduction process includes infiltration of a culture of Agrobacterium tumeration into the tissues of the leaves.

The infiltration process may be performed by a vacuum method or a syringe method. One specific embodiment when using a vacuum is as follows. The plant leaves are placed in a culture of Agrobacterium trimmers and vacuum is applied for a short time. Quickly release the vacuum, disinfect the leaves with sterile water, and place them on a damp piece of paper with the top of the leaves facing down. The leaves are then stored at about 22 ° C. under light conditions of about 16 hours. Then, after about 2-3 hours, the target protein expressed is identified.

On the other hand, a specific embodiment when using a syringe is as follows. The back of the plant leaves is infused with a syringe of Agrobacterium turmeration using a syringe, the plant is incubated for about 3-5 days, and then the expressed target protein is identified.

Agrobacterium tuberation cultures preferably include acetosyringone, to aid in the penetration of Agrobacterium into plants.

Plants transgenic according to the present invention are confirmed the expression of the protein of interest by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transgenic plants to identify foreign genes present in plant cells. Alternatively, Northern or Southern blotting can be performed to confirm gene introduction (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). On the other hand, if the vector used to introduce the gene has a β-glucuronidase gene, a part of the leaf tissue into which the gene is introduced is selected from X-gluc (5-Bromo-4-Chloro-3-Indole-β-D- By visually observing the color development by immersion in a substrate solution such as Glucuronic acid) it can be easily confirmed whether the gene introduced (Jefferson, RA, Plant Mol. Biol. Rep. , 5: 387 (1987)).

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

GM-CSF can be obtained from the tissues of transgenic plants of the present invention, ie, transiently transfected plants, such as leaves, and the extract obtained from the tissues can be obtained through conventional purification. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

In a specific embodiment of the present invention, when there is 6 x His at the N-terminus of the recombinant GM-CSF protein of the present invention, a Ni-NTA His-binding resin column is used to quickly and rapidly produce the desired recombinant GM-CSF protein. It can be easily separated and purified.

According to the method of the present invention, not only can the recombinant GM-CSF protein be produced in a short time and at low cost by using a transient transfected plant, but the produced GM-CSF has the same biological properties as the natural-occurring GM-CSF. And functions.

Another approach for producing plants in which genes encoding GM-CSF have been introduced is to use transgenic plants that stably express GM-CSF.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant, thereby providing a method for producing a transformed plant stably expressing the recombinant human GM-CSF.

According to another aspect of the present invention, the present invention provides a transgenic plant stably expressing the recombinant human GM-CSF produced by the method of the present invention described above.

According to another aspect of the invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) provides a method for producing a recombinant human GM-CSF comprising the step of obtaining a recombinant human GM-CSF from the transgenic plant.

According to another aspect of the present invention, the present invention provides a recombinant human GM-CSF prepared by the above method.

In the method of the present invention, the transformation of plant cells can be carried out according to a conventional method known in the art, which is electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982) And Saul, M. et al., Plant Molecular Biology Manual , Vol. A1, Kluwer Academic Publishers, Dordrecht (1988)), particle gun method (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568- 9572 (1990) and Christou, P., Trends Plant Sci. 1: 423-431 (1996)) and Agrobacterium-mediated transformation (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011). Of these, Agrobacterium-mediated transformation is most preferred. Agrobacterium-mediated transformation is generally carried out with leaf discs and other tissues (cotyledon and hypocotyls). Agrobacterium-mediated transformation is most efficient in dicotyledonous plants.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that are transformed and stably contain marker genes that confer selective resistance are grown and divided in the cultures described above. Exemplary labels include, but are not limited to, glycophosphate resistance genes and neomycin phosphotransferase (nptII) systems.

Methods for the development or regeneration of plants from plant protoplasts or various exploits are well known in the art (Bhojwani, SS et al., Plant Tissue Culture: Theory and Practice , Elsevier Science, New York, (1983); and Lindsey , K., Ed., Plant Tissue Culture Manual , Kluwer Academic Publishers, Dordrecht, The Netherlands, (1991)). The transformed root shoots formed are imbedded in a suitable plant growth medium. Development or regeneration of plants comprising foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

On the other hand, the present inventors have made extensive research efforts to develop novel transgenic plants (eg, tobacco, melons, cucumbers, watermelons and rapeseeds), and as a result, have established the most efficient method for the transformation of specific plants. Such a method is disclosed in PCT / KR02 / 01461, PCT / KR02 / 01462 and PCT / KR02 / 01463, which patent documents are incorporated herein by reference.

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens -binary vector system.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') an Agrobacterium tumer embedded in a genome DNA of a plant cell and having a vector having the sequence Infecting the plant's plant with Agrobacterium tumefaciens : (i) the nucleotide sequence encoding the GM-CSF described in SEQ ID NO: 1; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; (Iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') re-differentiating the infected implant in a regeneration medium to obtain a redifferentiated shoot; And (c ') culturing the re-differentiated shoots in a rooting medium to obtain a transformed plant.

In one specific embodiment described above, suitable implants for transformation include any tissue derived from the germinated seed, preferably cotyledon and hypocotyl, most preferably cotyledon. Seed germination is carried out under suitable dark / light conditions using a suitable medium. Transformation of plant cells is carried out with Agrobacterium trimers comprising Ti plasmids (Depicker, A. et al., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983) ).

More preferably, binary vector systems such as pBin19, pRD400 and pRD320 are used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986)). Binary vectors suitable for include (i) a promoter operating in a plant, (ii) a structural gene operably linked to said promoter, and (iii) a polyadenylation signal sequence Optionally, said vector comprises a reporter molecule (eg : Additionally carries genes encoding luciferase and glucuronidase) Examples of promoters used in binary vectors include the CaMV 35S promoter, 1 promoter, 2 promoter and nopaline synthase (nos) promoter. It is not limited to this.

Infection of implants with Agrobacterium trimmers includes methods known in the art. Most preferably, the infection process comprises coculture by impregnating cotyledon in a culture of Agrobacterium tumerification. As a result, Agrobacterium trimmers are infected into plants.

Explants transformed with Agrobacterium trimerization are re-differentiated in regeneration medium, which successfully leads to regeneration of shoots. Finally, transgenic plants are produced by the rooting of shoots that have been regenerated in the rooting medium.

Plants transformed according to the present invention is confirmed whether or not transformed by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transformed plants to identify foreign genes inserted into the genome of the transformed plants. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

GM-CSF expressed in the transformant can be obtained from tissues derived from various organs (e.g., stems, leaves, roots, fruits, seeds, etc.) of the transformed plant, and the extract obtained from the tissues is subjected to conventional purification. Can get through. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

According to the method of the present invention, not only can GM-CSF be produced in large quantities at low cost, but the produced GM-CSF exhibits the same biological properties and functions as natural-occurring GM-CSF. Therefore, the GM-CSF of the present invention is useful as a therapeutic agent for various diseases such as aplastic anemia, congenital and idiopathic neutropenia, and Crohn's disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1 Synthesis of Novel Genes of GM-CSF Protein

While encoding 128 amino acids (see SEQ ID NO: 2 sequence) identically to the native GM-CSF gene, it designed the optimal nucleotide sequence preferred by plants, unlike the nucleotide sequence encoding the existing GM-CSF. . The design criteria for the new gene is first, to make the GC content above 50%, to have the plant's preferred codons, and to remove intron-like sequences. The nucleotide sequences thus designed were chemically synthesized at the Plant Biotechnology Institute (hereinafter referred to as "PBI") and the National Research Center (SK, Canada). DNA encoding the synthesized GM-CSF protein is described in SEQ ID NO: 1.

Example 2: Preparation of plant expression vector of GM-CSF protein gene

The synthetic gene encoding the recombinant GM-CSF is composed of 384 bp, and synthesized to have a recognition site of Xba I at the 5'-end and Bam HI at the 3'-end.

PHS737 (PBI, National Research Center, Canada), a plant expression binary vector, was digested with restriction enzymes Xba I and Bam HI enzymes, digested, and isolated and purified. The 384 bp synthetic gene and the cleaved pHS737 vector were mixed and added to the ligation buffer (KOSCO, Korea) and T4 ligase (KOSCO, Korea) and reacted at 16 ° C. for at least 1 hour. The reactants were then transformed into competent cells treated with CaCl ₂ ( E. coli strain DH5α (Promega, USA) and then antibiotic resistant strains were selected in LB medium containing the antibiotic kanamycin (100 mg / ml).

Plasmid DNA was isolated from the selected clones using an alkaline method and polymerized using forward primer 5'-AAT CTA GAA ATG GCT ACT AT-3 'and reverse primer 5'-AAG GAT CCC CGA GCT TGA GA-3'. Enzyme chain reaction (PCR) was performed. The enzyme used in the PCR amplification was Taq polymerase (Solgent, Korea), and the temperature conditions were set as follows: predenature for 2 minutes at 96 ° C, denature for 1 minute at 94 ° C, and connect for 1 minute at 55 ° C. The reaction was repeated 35 times at 72 ° C. for 2 minutes, and further extended at 72 ° C. for 10 minutes.

The amplification product was then analyzed on a 1.0% agarose gel to confirm that the desired insert sequence was in the plasmid.

Example 3: GM-CSF gene introduction into plants

Example 3-1 Preparation of Agrobacterium tumefaciens GV3101 Culture

The recombinant pHS737 / hGM-CSF vector prepared in Example 2 was conjugated to Agrobacterium tuberization GV3101 (Mp90) ( Plant-cell-rep. , 15 (11): 799-803 (1996)). Introduced. Escherichia coli S17-1 with recombinant pHS737 / hGM-CSF vector and Agrobacterium Tumperfections GV3101 (Mp90) were each cultured in liquid LB medium to log phase. Each of these cells were mixed together 1: 1 in an Eppendorf tube and then centrifuged for 30 seconds to concentrate the cells, followed by stationary culture at 1-2C for 1-2 days. The Eppendorf tube containing the cells over time was gently shaken to float the concentrated cells, and then plated in LB solid medium to which 50 mg / L kanamycin and 30 mg / L gentamicin were added. Colonies were selected after incubation for one day (Alt-Morbe et al ., J. Bacteriol. 178: 4248-4257 (1996)). The strain into which the vector was introduced was named Agrobacterium turmation GV3101-pHS737 / hGM-CSF.

Agrobacterium trimmers GV3101-pHS737 / hGM-CSF with pHS737 / hGM-CSF were inoculated in liquid LB medium and incubated at 28 ° C. for 2 days to be used as seed culture. The fully cultured spawn was added to 1/50 of the final culture volume and antibiotics (50 mg / L Kanamycin) in liquid LB medium and incubated for about 18 hours until the absorbance reached 1.5 at 600 nm. The culture solution was centrifuged at 3500 rpm for 15 minutes at room temperature to remove the supernatant, and then the cells were suspended in a 10 mM MgCl ₂ solution (containing 0.2 ppm of BA and 150 μM of acetosyringone) and allowed to acclimate at room temperature for at least 3 hours. It was used for infection of plants.

Example 3-2 Preparation of Plant Cells and Agroinfiltration

Seeds of the first sterilized tobacco ( Nicotina bentamiana ) were sown and incubated for at least 3 weeks and then purified in a greenhouse. Agrobacterium tuberation GV3101 (Mp90) having a pHS737 vector (pHS737 / hGM-CSF) having GM-CSF gene on the leaves of purified plants was prepared by the method described in Example 3-1. The needle was inserted into a 1 ml syringe (Note Green Cross) with the needle removed and injected until it spreads evenly on the back of the leaf. Injecting the fungus into the entire leaf may cause the plant to fall down due to heavy leaves or to break the leaf disease.The plant injected with Agrobacterium restores its original condition after 10 minutes to 1 hour. Done. Plants injected with Agrobacterium were harvested within 5 days after 3 days of culture and analyzed using the method of Example 4.

Example 4 Label Factor Analysis for Confirming Transgene Expression in Transgenic Plants

Confirmation of the gene expression of the plant obtained in Example 3 was carried out as follows: beta-glucuronidase (β- glucuronidase, GUS) After extracting the leaves to confirm the expression of the reporter gene, GUS analysis solution 200 μl of (X-gluc (5-Bromo-4-Chloro-3 3-Indole-β-D-Glucuronic Acid), 1 mg / ml) is placed in a container with leaves and thoroughly vacuumed using a vacuum. The vacuum was maintained until it was. After that, the appearance of color was visually observed to confirm the introduction and expression of the gene.

When the gene of the leaf to which the gene is introduced is expressed, the beta-glucuronidase, a reporter gene, is produced, and the enzyme beta-glucuronidase is produced. When the X-gluc solution is added, the beta-glucuronidase is added. As the X-gluc is broken down, pigments are produced, causing the cells to appear blue. In the present invention, the leaf into which the gene was introduced was expressed by the reporter gene beta-glucuronidase expressed blue to confirm that the gene was introduced and expressed.

Example 5: Isolation and Purification of Recombinant GM-CSF Protein in Transgenic Plants

In a mortar containing an appropriate amount of liquid nitrogen, 1 g of tobacco leaves with the genes introduced were cut and crushed. Finely ground was added 2 ml of protease inhibitor-containing extract buffer (250 mM sucrose, 1 M Hepes, 1 mM MgCl ₂ and 1 mM DTT) per 0.5 g of plant weight. The extract was centrifuged at 12,000 rpm and 4 ° C. for at least 30 minutes, then the supernatant was removed and transferred to a new tube. The lysed cell solution was added to a Probond column (Quiagen GmbH, Germany) containing nickel resin, and gently shaken for 10 minutes at room temperature to allow the recombinant GM-CSF protein in the cell solution to adsorb well to the nickel resin.

The resin to which the recombinant GM-CSF protein was adsorbed was washed twice with the same amount of pH 7.8 adsorption buffer solution (20 mM sodium phosphate and 500 mM sodium chloride) to remove plant cell derived proteins adsorbed on the resin. An equal amount of pH 6.0 wash buffer solution (20 mM sodium phosphate, 500 mM sodium chloride) was added to the washed resin, and the mixture was gently shaken at room temperature to wash the resin again, and the same amount of pH 4.0 elution buffer solution (20 mM sodium) was added. Phosphate, 500 mM sodium chloride) was added to elute the recombinant GM-CSF protein.

Recombinant GM-CSF proteins expressed in transgenic plants have six histidine residues at the amino terminus and are easily isolated by binding to a cationically nickel nickel resin. Through this process, only recombinant GM-CSF protein from which all plant cell proteins were removed was purified. The eluted protein was concentrated using Centricon (Amicon Co., U.S.A.).

As shown in FIG. 1, when the recombinant GM-CSF protein was electrophoresed on an SDS-PAGE gel and stained with Coomassie blue, 19.3 kD of recombinant GM-CSF protein, which was not detected in wild-type tobacco, was detected in the cell eluate. In particular, when eluted with a nickel resin column, it was confirmed that pure recombinant GM-CSF protein from which all plant cell proteins were removed was detected.

A recombinant assay kit (Bio-Rad) was added to the recombinant GM-CSF protein purified by the above method, using a Bradford method, and the absorbance was measured at 595 nm using a UV-spectrophotometer, followed by BSA (Bovine Serum Albumin). Protein quantitation was performed as compared to the standard. The extracted protein was adjusted to the same amount and each sample was subjected to 8% polyacrylamide gel electrophoresis, and Western blot analysis was performed (see Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 108-121, CRC press). Western blot analysis was performed as follows. The protein in the SDS-PAGE gel was transferred to PVDF membrane using Semi-Dry Transfer Units (Hoefer, USA). After confirming that all proteins in the gel had migrated to the PVDF membrane, the PVDF membrane was dried. This PVDF membrane was reacted with 0.5% BSA / TBS (Tris-Buffered Saline) solution to which monoclonal antibody (Biosource Inc. Canada), which specifically reacts with the recombinant GM-CSF protein antigen, was reacted for 1 hour, followed by PVDF with TBS solution. The membrane was washed three times for 5 minutes. Then, after reacting with peroxidase labeled secondary antibody (peroxidase labeled-anti human IgG, KPL, USA) for 1 hour, the PVDF membrane was washed three times for 10 minutes with TBS solution. Finally, the PVDF membrane was added to the left buffer solution to which the ABTS coloring substrate solution (KPL, USA) was added, and the resultant was reacted for 30 minutes at room temperature and the result was read. As can be seen in Figure 2, Western blot analysis was carried out to confirm the same as the above results 19.3 kD recombinant GM-CSF protein.

Example 6: Determination of Expression of Recombinant GM-CSF Protein

In order to confirm the function of the recombinant GM-CSF protein isolated and purified from the leaves of tobacco expressing the gene introduced by agroinfiltration, the antigen-antibody response to the recombinant GM-CSF protein was 96-well microtiter. It was measured using a plate.

The recombinant GM-CSF protein purified in a 96-well microtiter plate was serially diluted in phosphate buffer, dispensed 100 μl per well, and left at 4 ° C. for at least 8 hours to fix the antigen in the wells. The protein-adsorbed plate was washed three times with washing buffer (0.05% Tween 20, 0.01 M Phosphate, pH 7.6) to remove the non-adsorbed antigen from the plate, and the monoclonal antibody against the recombinant GM-CSF protein antigen ( Biosource Inc.) was diluted in a dilution (0.1% BSA, 0.05% Tween 20, 20 mM Trisma base, 150 mM NaCl) with a certain dilution factor, and 100 μl per well was dispensed for reaction for 1 hour at room temperature. Induced. Then, each well was washed three times with washing buffer, and then the blocking buffer was dispensed into each well, and reacted at room temperature for 1 hour, followed by washing in the same manner as above. Peroxidase labeled secondary antibody (peroxidase labelled-anti human IgG, KPL, U.S.A.) was aliquoted and reacted and washed in the same manner as above. Finally, each well was reacted for 30 minutes at room temperature by adding ABTS color substrate (KPL, USA), followed by reading at 405 nm using an ELISA reader (ELISA Reader, Titertek, USA). 3 is shown. The vertical axis is O.D. Absorbance values at 405 nm are shown, the vertical axis (left) is the dilution factor when the initial titer of the antibody to the protein is 1, and the horizontal axis is the dilution factor of the purified recombinant GM-CSF. This is compared with the protein isolated from wild-type plants. As can be seen in Figure 3, the antigenicity of the recombinant GM-CSF protein separated and purified from the transgenic plants appeared to be excellent, compared to the protein isolated from the wild-type plants at all.

In addition, as shown in Figure 4 showing the ELISA results, it can be seen that the recombinant GM-CSF purified from the transgenic tobacco according to the present invention shows antigenicity to the antibody against the native GM-CSF, which is natural It can be seen that the physiological function similar to that of GM-CSF.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

As described in detail above, the present invention provides a novel polynucleotide encoding human GM-CSF and a plant expression vector of recombinant human GM-CSF comprising the same. The present invention also provides a transient transfected plant transiently expressing recombinant human GM-CSF and a transformed plant stably expressing recombinant human GM-CSF. On the other hand, the present invention provides a method for producing recombinant human GM-CSF. According to the present invention, not only can GM-CSF be produced in large quantities at low cost, but the produced GM-CSF exhibits the same biological properties and functions as native GM-CSF. Therefore, the GM-CSF of the present invention is useful as a therapeutic agent for various diseases such as aplastic anemia, congenital and idiopathic neutropenia, and Crohn's disease.

1 is a photograph showing the results of electrophoresis of GM-CSF protein expressed in transgenic plants, ie, transient transfected plants, in an SDS-polyacrylamide gel. M, molecular weight marker; Lane 1, protein of wild tobacco; And lane 2, GM-CSF expressed in the transgenic plant.

Figure 2 is a photograph showing the results of Western blot analysis of GM-CSF expressed in transgenic plants. M, molecular weight marker; Lane 1, protein of wild tobacco; And lane 2, GM-CSF expressed in transgenic tobacco.

3 is a graph showing the antigenicity of recombinant GM-CSF expressed in transgenic plants. Absorbance value at 405 nm on vertical axis; Horizontal axis, dilution factor of monoclonal antibody against GM-CSF; GM-CSF protein isolated and purified from transgenic tobacco; Rhombus, recombinant GM-CSF protein as positive control; Square, wild type tobacco protein.

4 is an ELISA plate photograph showing the antigenicity of recombinant GM-CSF expressed in transgenic plants. Landscape: (+), positive control expressed in E. coli ; (-), Wild type tobacco; 1-10, GM-CSF expressed in transgenic tobacco. Length: 1-7, antibody dilution factor; (-) PBS buffer.

<110> NEXGEN BIOTECHNOLOGIES, INC. <120> Human Granulocyte Macrophage Colony Stimulating Factor Expressed in plants <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> the nucleotide sequence of GM-CSF <220> <221> CDS (222) (1) .. (384) <400> 1 atg gca cca gct aga agc cct tcc cca tca act caa ccg tgg gag cac 48 Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 1 5 10 15 gtt aac gct att cag gaa gca cgc agg ctg cta aat ctc tcc agg gat 96 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 20 25 30 acc gcc gcg gaa atg aat gaa act gtc gag gtg ata tct gag atg ttc 144 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 35 40 45 gac ctt caa gag cct acg tgc ctt caa act cgt ctg gag ttg tac aag 192 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 50 55 60 caa ggt ctt aga ggc tca ttg acc aag ttg aag gga cca ctt acc atg 240 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 65 70 75 80 atg gcc agc cat tat aag cag cat tgc cct cca aca cca gag aca agt 288 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 85 90 95 tgt gct aca caa att atc act ttt gaa tct ttt aaa gaa aac ctc aaa 336 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 100 105 110 gac ttc ttg ctc gta atc ccc ttc gat tgt tgg gag ccc gtt cag gaa 384 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125 <210> 2 <211> 128 <212> PRT <213> Artificial Sequence <400> 2 Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 1 5 10 15 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 20 25 30 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 35 40 45 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 50 55 60 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 65 70 75 80 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 85 90 95 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 100 105 110 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125

Claims (9)

A polynucleotide encoding a human granulocyte macrophage colony promoter (GM-CSF) comprising the nucleotide sequence set forth in SEQ ID NO: 1. (Iii) the polynucleotide of claim 1; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule. A method of preparing a transient transfected plant that expresses recombinant human GM-CSF temporarily comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell of a plant; And (b) confirming whether the gene has been introduced into the plant cell. A transient transfected plant transiently expressing recombinant human GM-CSF prepared by the method of claim 3. Method for preparing a recombinant human GM-CSF comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) confirming whether the gene has been introduced into the plant cell; And (c) obtaining recombinant human GM-CSF from a plant comprising plant cells into which the gene has been introduced. A method for preparing a transgenic plant stably expressing recombinant human GM-CSF comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant. Transgenic plant stably expressing recombinant human GM-CSF prepared by the method of claim 6. Method for preparing a recombinant human GM-CSF comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining recombinant human GM-CSF from the transgenic plant. Recombinant human GM-CSF prepared by the method of claim 5 or 8.