KR100549167B1 - Production of biologically active interleukin-12 protein using plant cell suspension culture - Google Patents

Abstract

The present invention relates to a recombinant protein for biological activity using plant cell suspension culture, and a method for producing the same, more specifically, a method for producing human interleukin-12 in plant cell suspension culture, and a recombinant protein for human interleukin-12. The present invention can produce a large amount of high biological activity human interleukin-12 at a low cost by establishing a plant expression system that produces and secretes human interleukin-12 in a biologically active form in a plant cell culture system.

Human Interleukin-12, Plant Cell Culture, Secretion, Tobacco

Description

Production of biologically active interleukin-12 using plant cell suspension culture {PRODUCTION OF BIOLOGICALLY ACTIVE INTERLEUKIN-12 PROTEIN USING PLANT CELL SUSPENSION CULTURE}

1 is a photograph showing the results of amplifying a subunit p35 gene and a gene of p40 of IL-12 using PCR, respectively.

Figure 2 shows the schematic diagram of the production of the pMYL27 (Fig. 2a) and pMYL28 (Fig. 2b) of the present invention.

Figure 3a is a result of PCR amplification of the subunit p35 gene of IL-12 contained in the five transformed Nicothia Natacum and a sudden result of the amplified product, Figure 3b is five kinds of transformation The result of PCR amplification of the subunit p40 gene of IL-12 included in Nicothia natabacum was suddenly a result of the amplified product.

4A and 4B are photographs showing the results of Northern blot analysis of Nicothia natabacum transformed with the subunits p35 (FIG. 2A) and p40 (FIG. 2B) genes of IL-12, respectively.

5 shows Northern of four transformed Nicothia Natacum lines expressing both subunits of IL-12 by crossing Nicotia Natacum, transformed with subunits p35 and p40 genes of IL-12, respectively. It is a photograph showing the result of blot analysis.

FIG. 6 shows Northern blots of four transformed Nicothia Natacum lines expressing both subunits of IL-12 by crossing Nicotia Natacum, transformed with subunits p35 and p40 genes of IL-12, respectively. It is a photograph showing the analysis result.

7 is a photograph showing the results of Western blot analysis of L2728-1 and L2728-3 lineage suspension cell culture medium showing high gene expression in callus lines derived from the four L2728 transgenic plants of FIG. 5.

Figure 8 is a graph showing the growth of the suspension cells (white circles) by suspension culture of the L2728-3 strain, a transformed tobacco cell line according to Example 3, quantitative analysis of recombinant human IL-12 secreted from the cells ( Black circle).

Figure 9 is a graph showing the physiological activity of human IL-12 produced through suspension cell culture of the transformed tobacco cell line L2728-3 line according to Example 4.

10 is a graph showing the increase in production of human IL-12 by the addition of gelatin in plant cell culture according to Example 5. FIG.

The present invention relates to transformed plant cells expressing biologically active interleukin-12, a method for producing biologically active interleukin-12 by a plant cell suspension culture method using the same, and a recombinant interleukin-12 protein.

As a method for producing a large amount of foreign protein, microbial and animal cell culture systems are widely used. However, foreign protein production by microbial culture is not possible in the case of a protein whose activity depends on post-translational modification, because it is impossible to produce physiologically active protein because microbial modification system is different from eukaryotic cells. many. In this case, the protein must be produced by animal cell culture. Since such a system adds serum to the medium, the price of the medium is very high, and there is a concern that the medical protein may be infected by the animal virus infection of the host cell. Since a large amount of protein is present, it is very difficult and expensive to separate and purify the produced and secreted medical protein.

Recently developed plant cell culture systems have been proposed as the most suitable system for the production of small, medium and small quantities of specialized, expensive, and high purity proteins. Single polypeptide proteins can be produced with good yields in a relatively short time when using other lower eukaryotic systems such as bacteria or yeast, but these systems may require the correct post-translational modifications or combinations of multiple subunits for biological activity. Not applicable to the protein required. Thus, plant cell culture is very advantageous when the protein of interest is multimeric and when accurate post-translational modification is required.

Recently, antibodies and antibody fragments (Van Engelenet, F. et al, Plant Mol . Biol . Vol 26, pp. 1701-1710, 1994); LaCount, W. et al., Biotechnol . Lett . vol. 19, pp. 93-96, 1997; Wongsamuth, R. et al., Biotechnol . Bioeng . vol. 54, pp. 401-415, 1997; Fischer, R. et al . Eur . J. Biochem . vol. 262, pp. 810-816, 1999; Fischer, R. et al., Biotechnol . Appl . Biochem . vol. 30, pp. 109-112, 1999; Liu, F. et al., Biotechnol . Bioprocess Eng . vol. 4, pp. 259-263, 1999; Shrap, J. et al., Biotechnol . Prog . vol. 17 , pp. 979-992 , 2001), enzymes (Verdelhan, M. et al., J. Biosci . Bioeng . vol . 87, pp . 302-306, 1999), and therapeutically valuable proteins (Kwon , et al. Biotechnology and Bioengneering.vol . 81, pp . 870-875, 2003; Magnuson, N. et al . Protein Expr . Purif . vol . 13, pp . 45-52, 1998; Francisco, J. et al ... Bioconjugate Chem vol.8, pp.708-713 , 1997; Lee, J. et al Mol Cells vol.7, pp.783-787, 1997;.... Terashima, M. et al Appl Microb. Biotech . Vol. 52, pp. 516-523, 1999; Terashima, M. et al., Biochem . Eng . J. vol. 4, pp. 31-36, 1999; James, E. et al . Protein Expr . Plant cell culture systems are used to produce a variety of biologically active proteins, including Purif . Vol. 19, pp. 131-138, 2000). Potential concerns for the use of plant cell cultures for protein production include allergic reactions to plant protein glycans and other plant antigens, and products contaminated with mycotoxins, preparations, and pesticides (Doran, P , Curr . Opinion Biotechnol . Vol. 11, pp. 199-204, 2000).

Plant cell culture system It offers several advantages over existing microbial or animal cell culture systems. First, foreign protein produced by transformed plant cells is more stable since plant pathogens such as fungi and viruses can be easily monitored and they are usually not pathogenic to humans (Doran, PM supra). Second, many foreign genes can be easily introduced into one plant cell by sexual crossing. Third, since the culture medium is mainly composed of sucrose and some kinds of salts and does not contain macromolecules, the purification step is simple and economical after incubation. Fourth, plant cells can be mass cultured at low cost, and post-transitional modifications in plant cells are very similar to animal cells compared to prokaryotic cells. Plant expression systems have advantages over prokaryotic expression systems in the expression of useful foreign proteins (Miele, L. et al., Trends Biotechnol . Vol. 15, pp. 45-50, 1997; Herbers, K. et. al., Curr Opin Biotechnol.vol . 10, pp. 163-168, 1999; Doran, PM supra. Plant expression systems are also very economical and simple compared to other expression systems.

Currently, industrialization is mainly focused on secondary metabolites of plants such as Taxol, alkaloids, and natural pigments, which are mostly used for producing useful substances using industrialized plant cell cultures.

Proteins that are attracting much attention include antigenic proteins, cytokines, or monoclonal antibodies, which are highly economical, and plants that produce antigenic proteins can be used as a material for extracting recombinant antigenic proteins for use in vaccines. However, when the plant is used for food, it may exert the effects of oral vaccines. After the rats were treated with the synthetic LT-B gene expression for the heat-sensitive enterotoxin of E. coli expressed in potato orally conversion plant transformed the antigen to confirm that the protection against enterotoxin of E. coli via the oral administration It was confirmed that immunity was induced (Haq et al ., Science . Vol.268, pp. 714-716, 1995). There was also a report of the first oral immunity in humans by confirming the immune effect when the same potato was administered to humans (Tacket et al ., Nature Med. Vol. 4, pp. 607-609, 1998).

The expression of physiologically active proteins in plants was reported by De Zoeten et al. ( Virology. Vol. 172, pp. 213-222, 1989) after reporting the expression of human interferon genes in plant cells (enkephalin) (Takamatsu et al. , FEBS Lett . Vol. 269, pp. 73-76, 1990), angiotensin-I-converting enzyme inhibitor (ACEI) (Hamamoto et al., Bio / Technology vol. 11, pp 930-932, 1993), and small-sized peptide expression such as α-trichosanthin (Kumagai et a l., Proc. Natl. Acad. Sci. USA vol. 90. pp427-430, 1993), and the like. And expression of growth factors such as erythropoietin (Matsumoto et al ., Plant Mol Biol . Vol. 27, pp.1163-1172, 1995). The cytokines and related proteins reported so far have been expressed as partially active bioactive substances, but economic problems such as the amount of expression have not been examined. In particular, as shown in the experiments of erythropoietin (Matsumoto et al ., 1995), the production of protein using plant suspension cell culture has been very limited success, such as active in vitro but not in vivo . .

Since monoclonal antibodies are composed of heterotetramers, they cannot be produced using E. coli or yeast, and they can be produced only through animal or animal cell culture. The risk of secondary infections coming from is one of the many proteins. Given these problems, monoclonal antibodies have been considered as one of the most suitable targets for the production of useful proteins through plant cell culture. Fisher, etc. (Fischer, R., et al. , J. Immunolog. Methods vol.226, pp.1-10, 1999) are within the recombinant 155-kDa antibody obtained from tobacco suspension culture cells not being secreted by the plant cell wall Remaining and not found in culture media.

Although many attempts have been made to produce foreign proteins through the introduction of foreign genes into plants, plant cell culture systems have problems such as slow growth and low expression rate, and thus, plant cell culture methods for efficient expression system development have not been established. (Su, WW, Appl. Biochem. Biotech . Vol. 50, pp. 189-230, 1995; Yeoman, MM et al., New Phyto l. Vol. 134, pp. 553-569, 1996). In addition, most of them accumulate foreign proteins in plant cells, and the accumulation rate of foreign proteins does not exceed 1%. In particular, the protein, such as the antigen, cytokines, etc., since the subsequent purification step is complicated even if a large amount is accumulated in the cell, recently, to try to separate and purify by inducing to be secreted into the medium using the culture of plant suspension cells have.

Human interleukin-12 (hIL-12) to be produced in the present technology is a heterodimer cytokine in which 35-kDa subunit (p35) and 40-kDa subunit (p40) are disulfide bonds with each other (Stern). , A. et al . Proc . Natl . Acad . Sci . USA vol. 87, pp.6808-12, 1990). IL-12 was originally identified as a natural killer cell stimulator by cytotoxic lymphocyte maturation factor (Gately, M. et al., J. Immunol . Vol. 136, pp. 1274-82, 1986), or other groups. (Kobayashi, M. et al . J. Exp . Med . Vol. 170, pp. 827-45, 1989). IL-12 promotes Th1-type cytokine responses, enhances cellular lymphocyte responses, and induces IFN secretion from NK cells (Chan, S. et al . J. Exp . Med . Vol. 173, pp. 869-79). , 1991), and Pleiotropic biological activity including proliferation stimulation of activated T cells and NK cells.

Two different genes encode subunits and co-expression of each gene in the same cell and the correct assembly of heterodimers are required to produce biologically active IL-12.

The production of all IL-12 is currently adenovirus (Chen, L. et al . J. Immunol. Vol. 159, pp. 351-159, 1997), Semliki Forest virus (Zhang, J. et al . Gene Ther. Vol. 4, pp. 367-374, 1997) and retroviruses (Zitvogel, L. et al. Hum Gene Ther. Vol. 5, 1493-1506, 1994) are produced in animal cells. Some are only available for production in insect cells (Shen, H. et al. Chin. J. Biotechnol. Vol. 14, pp. 205-212, 1998). However, in the case of IL-12 production by animal cell culture, there is a possibility that the medical protein is infected by the animal virus infection of the host cell, and the isolation of the produced and secreted medical protein due to the large amount of various proteins in the medium. There is a problem that purification is very difficult and expensive.

Up to now, the co-expression of different genes in plant cells and the resulting proteins form the correct heterodimer and induce secretion out of the plant cell in a biologically active heterodimeric protein state. The technology development is the first in the world, and the above technology has been reported to the society by the claimants with the claims (Kwon, et al. Biotechnology and Bioengneering . Vol. 81, pp . 870-875, 2003). .

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a method for producing a biologically active human IL-12 as a suspension culture method of plant cells.

It is another object of the present invention to provide plant cells and plants that express and secrete biologically active human IL-12.

Another object of the present invention is to provide a recombinant human IL-12 protein produced by a plant cell suspension culture method of high biological activity, stable and secreted into the culture medium.

It is another object of the present invention to provide a technique for enhancing the production of human IL-12 protein by using gelatin, which is a protein stabilizer, when producing human IL-12 protein by plant cell suspension culture.

In order to achieve the above technical problem, the present invention provides a method for producing interleukin-12 using a plant suspension culture system comprising a transgenic plant cell line expressing both p35 subunit and p40 subunit of interleukin-12.

The present invention also relates to a transgenic plant expressing both the p35 subunit and p40 subunit of interleukin-12 (IL-12).

The present invention also relates to a transgenic plant cell expressing both the p35 subunit and p40 subunit of interleukin-12 (IL-12).

The present invention also relates to a biologically active recombinant interleukin-12 protein produced by a plant cell suspension culture method prepared according to the above method.

The present inventors consider the problems of the conventional production method of IL-12 using animal cells, using a plant cell culture system to express the interleukin-12 of a heterodimeric protein in a biologically active form and to culture medium. Based on the secretion, the transgenic plants expressing each subunit of hIL-12, respectively, are crossed to prepare transgenic plants expressing complete hIL-12, inducing callus from the transgenic plants, and A cell suspension culture system was established. As a result of confirming the biological activity and production of hIL-12 secreted into the culture medium, it was confirmed that the protein is secreted into the culture medium in an active state by the original signal peptide, thereby completing the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides a transgenic plant expressing a 35 kDa subunit of interleukin-12, differentiates a transgenic plant expressing a 40 kDa subunit of interleukin-12, and expresses each subunit. By producing transgenic plants expressing both subunits by crossing the transgenic plants, IL-12 is secreted into plant cell culture media for easy separation and purification, and is expressed and secreted in a biologically active form to separate them. No assembly steps are required for each subunit, resulting in low production costs and simple manufacturing processes. This step is usually a limiting step because in the case of producing bioactive proteins, purification to a high level of single protein state may be excessively costly or impossible. Plant cell culture systems are useful for purification because the culture medium consists of sucrose and some salts and does not contain any high molecular weight macromolecules.

(a) providing a transgenic plant into which a gene encoding a p35 subunit of interleukin-12 (IL-12) is introduced, and a transgenic plant into which a gene encoding a p40 sub unit is introduced,

(b) cross-transforming the two transgenic plants to produce a transgenic plant expressing both subunits of IL-12,

(c) preparing a plant cell line by inducing callus from the transformed plant of b),

(d) suspending the plant cell line to express IL-12.

IL-12 usable in the present invention is human (Genebank No. M65290 (p40), M65291 (p35)), mouse (Genebank No. M86671 (p40), M86672 (p35)) or porcine (Genebank No. AB025723 (p35) , AB025724 (p40)), in addition to IL-12, includes all IL-12 classified as IL-12. IL-12 gene of the present invention can be expressed only by the structural gene, but preferably can be secreted outside the plant cell, including the structural gene and the signal sequence. The signal sequence usable in the present invention is intended to include not only the signal gene of IL-12 itself, but also any signal gene which does not adversely affect the production, secretion and activity of IL-12.

The present invention provides a recombinant vector pMYL27 expressing p35, a subunit of human interleukin-12. The region introduced into the plant of the pMYL27 vector is shown in Table 1, which includes an expression site of the p35 subunit of interleukin-12 and a neomycin expression site that is a selection marker, and an exemplary nucleotide sequence thereof is shown in SEQ ID NO: 1 Indicated. In addition, pMYL27 is prepared by the method as shown in FIG. 2A and the T-DNA region (Right Bord to Left Bord) which is directly introduced into a plant includes the structure of Table 1 below.

gene location Right bord 1-24 Nopalin synthase promoter 72-384 Neomycin Force Transporters II 385-1179 Transcription Termination Signal of Nopaline Synthetase 1568-1821 CaMV35S promoter 2515-3294 hIL-12 p35 subunit gene 3302-4078 Transcription Termination Signal of Nopaline Synthetase 4093-4346 Left bord 4989-5013

The present invention also provides a recombinant vector pMYL28 expressing p40 which is a subunit of human interleukin-12. The region introduced into the plant of the pMYL28 vector is shown in Table 2, which includes an expression site of the p40 subunit of interleukin-12 and a hygromycin expression site that is a selection marker, and an exemplary nucleotide sequence thereof is shown in SEQ ID NO: 2 is shown. In addition, pMYL28 was prepared by the method as shown in Figure 2b and the T-DNA region (Right Bord to Left Bord) introduced directly to the plant includes the structure of Table 2 below.

gene location Right bord 1-24 CaMV35S promoter 265-1046 hIL-12 p40 subunit gene 1058-2044 Transcription Termination Signal of Nopaline Synthetase 2056-2321 CaMV35S promoter 2570-3350 Hygromycin Force Transporterase 3386-3536 CaMV35S transcription termination signal 4248-4575 Left bord 4703-4727

In the step (d), it is preferable to add the step of adding gelatin to the culture medium. The amount of gelatin to be added is preferably 0.5% to 1.0%, and gelatin is preferably added during passage of plant cells.

As a method for transforming plants usable in the present invention, Agrobacterium-mediated transformation, direct transformation of protoplasts, microinjection of foreign DNA, and delivery to microprojectiles can be used. Preferably, the Agrobacterium transformation method. As a variety of methods for transforming the plant tissue, the method for regenerating the plant from the plant tissue can be appropriately selected according to the type of plant used, preferably regenerated with callus tissue derived from plant explants. Regeneration of plants from tissues transformed with tumefaciens is very well known in many plant species.

The invention also relates to a transgenic plant or plant cell expressing both the p35 subunit and p40 subunit of IL-12, preferably the plant or plant cell producing biologically active IL-12.

In the present invention, the transformed plant or plant cell includes all plants capable of transformation, including Nicotiana tabacum , Arabidopsis, rice, and the like, and preferably tobacco plants. Preferred plant cell line according to the present invention is nicotiana tabacum cell line L2728-3 (pMYL27 X pMYL28) producing hIL-12, which was deposited as KCTC 1021BP in the Gene Bank on June 7, 2001.

The present invention will be described in more detail with reference to the following illustrative examples, but the protection scope of the present invention is not intended to be limited to the following examples.

EXAMPLE

Example 1 Preparation and Analysis of Transgenic Plants

1-1: Cloning of Genes

Genes of p35 and p40 fragments of interleukin-12 were isolated and identified by PCR, and the results are shown in FIG. 1.

The genes of p35 and p40 fragments of interleukin-12 were treated with 15μg / ml PHA (phytohemagglutinin) in human blood to induce the expression of interleukin-12, and then peripheral blood mononuclear cells were isolated from the cells. A) + mRNA was isolated. The cDNA (including signal peptide) of the p35 fragment of interleukin-12 was synthesized by performing RT-PCR using the above-described total RNA as a primer of p35-F and p35-R.

p35-F: 5'-AAG GTA CCA ATT ATA AAA ATG TGG-3 '(SEQ ID NO: 3)

p35-R: 5'-AAG AGC TCT TTA GGA AGC ATT CA-3 '(SEQ ID NO: 4)

PCR was performed at 94 ° C for 3 min pre-denaturation, 94 ° C for 30 seconds denaturation, 55 ° C for 30 seconds primer annealing, and 72 ° C for 1 minute extension. Was performed twice. The amplified PCR product is a 761 bp fragment containing an open reading frame (ORF, Genebank M65291) of the p35 fragment gene of human interleukin-12. The PCR product was inserted into a pGEM-T vector (Promega, WI, USA) to prepare a pMYL25 vector and analyzed by the didioxynucleotide chain termination method to confirm that it is the p35 fragment gene of human interleukin-12. .

The cDNA (including signal peptide) of the p40 fragment of interleukin-12 was synthesized by performing RT-PCR using the above pRNA-p and p40-R as primers.

p40-F: 5'-GCG GAT CCA TGT GTC ACC AGC AGT TGG TCA TCT-3 '(SEQ ID NO: 5)

p40-R: 5'-TTG GTA CCC TAA CTG CAG GGC ACA GAT GCC CAT-3 '(SEQ ID NO: 6)

 PCR was performed at 94 ° C for 3 min pre-denaturation, 94 ° C for 30 seconds denaturation, 55 ° C for 30 seconds primer annealing, and 72 ° C for 1 minute extension. Was performed twice. The amplified PCR product is a 986 bp fragment containing the open reading frame (ORF, Genebank M65290) of the p40 fragment gene of human interleukin-12. The PCR product was inserted into a pGEM-T vector (Promega, WI, USA) to prepare a pMYL26 vector, and analyzed by the didioxynucleotide chain termination method (dideoxynucleotide chain termination method) to confirm that the p40 fragment gene of human interleukin-12. .

1-2: Preparation of Plant Expression Recombinant Vector

P35 fragment cDNA of human interleukin-12 on pMYL25 vector was digested with restriction enzyme Kpn I / Sac I and the same restriction exists between CaMV35S promoter and transcription termination signal of tobacco mosaic virus of pMY27, a plant double vector containing the same restriction enzyme Inserted into the enzyme site to prepare a plant expression recombinant vector pMYL27 vector for plant transformation (Fig. 2a). P40 fragment cDNA of human interleukin-12 on pMYL26 vector was cleaved with restriction enzyme Bam HI / Kpn I and the same restriction between CaMV35S promoter and transcription termination signal of the tobacco mosaic virus of pMY27, a plant double vector containing the same restriction enzyme Inserted into the enzyme site to prepare a plant expression recombinant vector pMYL28 vector for plant transformation (Fig. 2b).

1-3: Transformation

PMYL27 and pMYL28 containing genes of p35 and p40 fragments of interleukin-12, respectively, by Agrobacterium (An, G., Watson, BD and Chiang, CC (1986) Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system.Plant Physiol. 81, 301-305). E. coli HB101 with pRK2013 as a helper to transform plants into plants by E. coli H. coli HB101 was transferred to A. tumefaciens LBA4404 (Figurski, JJ and Helinski, DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.Proc.Natl Acad.Sci. USA 76, 1648-1652). Tobacco (Nicotiana tabacum L. cv. Havana SR ) human interleukin -12 fragment of the p35 and p40 genes are Agrobacterium bacteria Tome paksi Enschede (A comprising the p35 fragment and the p40 gene through the page tree rental junction (triparental mating) in the in tumefaciens ) An Agrobacterium mediated transformation method using LBA4404 was used (An, G. et al., Plant Physiol . vol. 81, pp. 301-305, 1986). A. tumefaciens containing pMYL27 and pMYL28 To introduce p35 and p40 fragment genes into tobacco, the leaves of Nicotiana tabaccum cv Havana were first sterilized in 1% chlrolax and 0.01% triton X-100 solution, washed three times with sterile distilled water, and then sterilized. Cut to size about 0.5 cm and then each section was diluted with A. tumefaciens LBA4404 containing pMYL27 and pMYL28, respectively (Murashige, T. and Skoog, F., Physiol.Pl . 15, pp. 473-497, 1962) was inoculated in liquid medium for 30 minutes. Inoculated sections were inoculated in MS regeneration medium (MS104) containing 1.0 mg / l NAA and 0.1 mg / l BAP, and then cultured in a 25 ° C. growth chamber. Three days later, 1.0 mg / l NAA, 0.1 mg / l BAP, Cultures were transferred to MS selection medium containing 300mg / l kanamycin and 500mg / lcefotaxime. Three months later, when the re-differentiated plants grew 2-3 cm in antibiotic selection medium, they were cut and transferred to MS rooting medium containing no growth regulator. When roots were formed in MS rooting medium and stems were grown to about 8 cm, they were transplanted into pots and purified in the greenhouse.

1-4: Transformant Analysis

The whole DNA was extracted from Qiagen's DNeasy plant mini kit from the leaves of plants grown about 10 cm after culturing the re-differentiated plants in MS medium containing antibiotics. P40-F (5'-GCG GAT), which is a primer specific for the genes of p35 and p40 used in the cloning of the gene, was used to determine whether the genes of p35 and p40 were normally introduced into the chromosome using the DNA as a template. PCR was performed using CCA TGT GTC ACC AGC AGT TGG TCA TCT-3 ') and p40-R (5'-TTG GTA CCC TAA CTG CAG GGC ACA GAT GCC CAT-3'). DNA amplified by PCR was subjected to electrophoresis on 1.2% agarose gel according to the method of Sambrook et al. (Sambrook, J., et al., Molecular cloning: A Laboratory Manual. NY, Cold Spring Harbor Laboratory Press, 1989). Hybond N ⁺ membranes were transferred, and cDNAs of p35 and p40 were labeled with isotopes using [α- ³² P] dCTP, followed by PCR- Southern blot analysis. As a result, it was confirmed that the cDNA corresponding to the IL-12 p35 subunit was merged into the genome of the plant nucleus (FIG. 3A), and the cDNA corresponding to the IL-12 p40 subunit was merged into the genome of the plant nucleus (FIG. 3A). 3b).

To identify transcription from each cDNA clone into RNA, cDNA of p35 and p40 according to the method of Sambrook et al. (Sambrook, J., et al., Molecular cloning: A Laboratory Manual. NY, Cold Spring Harbor Laboratory Press, 1989). Was labeled with [α- ³² P] dCTP using an isotope followed by Northern blot analysis of regenerated tobacco plants using the probe and the results are shown in FIGS. 4A and 4B.

Figures 4a and 4b shows the results of the blunt blot analysis of regenerated tobacco plants transformed with subunits p35 (Fig. 4a) and p40 (Fig. 4b), lane C of Figs. 4a and 4b is transformed with only the plant double vector pMY27 Negative control; rRNA is an internal control. Lanes 1 to 5 of FIG. 4A are transgenic plants having pMYL27 introduced therein including the p35 subunit gene, and lanes 1 to 6 of FIG. 4B are transgenic plants having introduced pMYL28 including the p40 subunit gene present. As a result of the Nobleblot analysis, it was found that the p35 and p40 subunit genes merged into the genome of the plant nucleus were transcribed into mRNA, respectively (FIGS. 4A and 4B). As shown in FIGS. 4A and 4B, expression levels of the genes were very different. Among the plants into which the p35 subunit was introduced, the L27-3 strain (FIG. 4A) and the L28 among the plants into which the p40 subunit was introduced. The best gene expression was identified in the -3 line (FIG. 4B) and the plants were selected for cross pollination.

Example 2: Selection of p35 and p40 Gene Coexpressing Plants by Artificial Crossing

Among the transformants in which the genes of p35 and p40, the IL-12 subunits, were introduced, the L27-3 and L28-3 strains, which had the highest expression levels, were crossed (Wernsman and Matzinger DF. 1980. Artificial Hybridization and self-pollination pp). 661-664). In order to prevent pollination with other plants after pollen was collected from flowering L27-3 plants, each artificially polluted plant was isolated and grown in artificial culture room. Seeds were harvested from the formed plants. In order to confirm whether p27 gene of L27-3 was introduced into the plant of L28-3 through breeding, a medium containing kanamycin (300mg / L) and hygromycin (50ml / L) normally obtained by artificial breeding Germinated at, the plants growing normally in a medium containing kanamycin and hygromycin were selected as plants containing both p35 and p40 genes (FIG. 5).

Example 3: Induction of suspension cells and expression of genes from mating plants

3-1: Induction of Suspension Cells and Suspension Cell Culture

Callus is 1 mg / L after cutting leaves of plants normally growing after 10 weeks of germination on MS medium containing kanamycin (300 mg / L) and hygromycin (50 ml / L) to a size of about 1 centimeter by 1 centimeter. Callus was induced on Murashige-Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid, 0.05 mg / L kinetin, 3% sucrose and 0.8% agar, and 1 mg / L 8 weeks after callus induction. Suspension cells were induced by being suspended in Murashige-Skoog (MS) liquid medium containing 2,4-dichlorophenoxyacetic acid, 0.05mg / L kinetin, and 3% sucrose. Suspension culture of plant cells was incubated at 25 ° C. at 100 rpm in an orbital shaker with approximately 1 g of callus (live weight) at a final volume of 50 mL in a 250 mL shaker flask. Suspension cells were subcultured by adding 10 mL of the cell culture solution containing the suspension cells to 40 mL of the same liquid medium at intervals of 7 days.

3-2: Northern Blot Analysis

Figure 6 is a northern blot result confirming the expression patterns of p35 and p40 genes of callus derived from plants containing both p35 and p40 genes in a medium containing kanamycin and hygromycin. It was carried out by the method. Where C is a negative control transformed with only the plant double vector pMY27 and rRNA is an internal control. Lanes 1 to 4 represent the respective crossed plants and L2728-1 (lane 3 in FIG. 6) and L2728-3 (FIG. 6) which simultaneously express the highest levels of lines p35 and p40 genes as seen in FIG. Lane 4) The callus of the system was selected to perform the following experiment.

3-3: Western blot analysis

Production of hIL-12 from the suspension cells of the L2728-1 and L2728-3 strains selected through Northern blot analysis was confirmed by Western blot analysis, and the experimental results are shown in FIG. 7.

Suspension cells of tobacco transformed with plant expression vectors alone, and L2728-1 and L2728-3 strains were suspended in about 2.5 g (live) of 1 mg / L of 2,4-dichlorophenoxyacetic acid, 0.05 mg / L kinetin, The cells were suspended in 50 mL of Murashige-Skoog (MS) liquid medium containing 3% sucrose and suspended at 25 ° C. at 100 rpm. After 5 days of culture, only the suspension cell culture solution was centrifuged at 4 ° C. at 13,000 rpm for 20 minutes. After lyophilization using a lyophilizer, it was dissolved in 1 ml of PBS solution, and the suspension cell culture solution was concentrated 50 times. 30 μl of the concentrate and 200 ng of recombinant human IL-12 (PharMingen) produced from insect cells were treated with 10% of Towbin et al. (Towbin et al., Biotechnology. Vol. 24, pp. 145-149, 1979). Western blot analysis was performed after SDS-PAGE on polyacrylamide gel. The antibody used for this Western blot analysis was used by diluting 1: 5,000 fold of monoclonal antibody (Pharmingen) specific for mouse-derived p35 and p40.

In Figure 7, PC is recombinant human IL-12 produced by insect cells (PharMingen), NC is a culture medium cultured suspension cells of tobacco transformed only with plant expression vectors, L2728-1 and L2728-3 is L2728-1 And cultured suspension cells of L2728-3 strain. As a result of Western blot analysis, it was confirmed that suspension cells of L2728-1 and L2728-3, tobaccos to which both p35 and p40 genes of hIL-12 were introduced, normally produced hIL-12 and secreted out of cells. , L2728-3 suspension cells were found to produce higher hIL-12 than L2728-1 suspension cells, L2728-3 suspension cell line was finally selected as a cell line for the production of hIL-12, the hIL-12 of the present invention Nicotiana tabacum cell line L2728-3 (pMYL27 X pMYL28) producing KCTC 1021BP was deposited in the Gene Bank on June 7, 2001. All subsequent experiments were performed using the L2728-3 suspension cell line.

3-4: Plant Cell Growth Analysis

To check the growth of cells according to the culture time of L2728-3 suspension cell, suspension culture cells after 5 days of subculture were removed by using a vacuum filter, and then the suspension cells were only about 2.5 g (live weight) of 1 mg. / L of 2,4-dichlorophenoxyacetic acid, 0.05mg / L kinetin, 50mL Murashige-Skoog (MS) liquid medium containing 3% sucrose was incubated at 25 ℃ at 100rpm. The growth of plant suspension cells according to the culture time was confirmed by removing the liquid medium by vacuum filtration of the suspension culture liquid to confirm the fresh cell weight (FCW), and drying the cell weight after drying the cells at 60 ° C. for 72 hours. (dry cell weight, DCW) was confirmed to check the growth of the cells.

The growth of suspended cultured transgenic tobacco cells is shown based on the dry weight of the suspended cells. Suspension cells of L2728-3 producing hIL-12 showed a growth curve of S-shape, which is a general culture curve, and after 3 days of induction, showed rapid growth from 3 days to 7 days (logistic period). The suspension showed slow growth of suspension cells (FIG. 8).

Example 4: Quantitative Analysis of hIL-12 and Confirmation of Biological Activity

4-1: Quantitative Analysis of hIL-12 Using ELISA

The production of hIL-12 secreted into the culture medium of L2728-3 suspension cells was quantitatively analyzed by the sandwich ELISA method described in Abrams et al . (Abrams JS, et al ., Current protocols in immunology, New York, Wiley, 1995). It was.

In summary, approximately 2.5 g (weight) of L2728-3 suspension cells were weighed in 50 mL of Murashige-Skoog (MS) liquid containing 1 mg / L of 2,4-dichlorophenoxyacetic acid, 0.05 mg / L kinetin, and 3% sucrose. Suspension culture was carried out at 25 ° C. at 100 rpm on the medium. After 1, 3, 5, 7, 9 and 11 days from the start of the culture, only the suspension cell culture solution was centrifuged at 13,000 rpm for 20 minutes at 4 ° C., and the supernatant 1 mL was overnight. 40 μl was used for analysis after dialysis of the protein sample with phosphate-buffer saline (PBS). In addition, the standard hIL-12 (PharMingen) produced through the known insect cell culture was used as a standard. The capture antibody used for sandwich ELISA analysis was derived from mouse-specific hIL-12 (p70) antibody (clone 20C2, BD PharMingen, San Diego, CA), and a mouse-derived hIL-12 (p40 / p70) specific antibody (clone C8.6, BD PharMingen, San Diego, CA) was used as an antibody for color development. Indicated.

As a result of quantitative analysis of the production of hIL-12 secreted into the culture medium of L2728-3 suspension cells, the production of hIL-12 of about 45 ㎍ / L level was confirmed after 1 day of culture. The production of -12 increased sharply after 3 days of culture and after 5 days of culture, the maximum production of hIL-12 was about 180 μg / L. However, after 5 days of cultivation, the production of hIL-12 began to decrease rapidly, and on day 9 of cultivation, the production of hIL-12 decreased to about 53 μg / L and on day 11 of culturing, the production of hIL-12 was about 25 μg / L. This rapid decrease in hIL-12 is presumed to be due to degradation by protease secreted by suspension cultured cells.

4-2: Analysis of Biological Activity of hIL-12 from L2728-3 Suspension Cells

Biological activity of proteins secreted into culture media of L2728-3 suspension cells was measured by Stern's method (Stern, AS et al ., 1990. Proc. Natl. Acad. Sci. USA. 87: 6808-6812).

In summary, approximately 2.5 g (weight) of L2728-3 suspension cells were weighed in 50 mL of Murashige-Skoog (MS) liquid containing 1 mg / L of 2,4-dichlorophenoxyacetic acid, 0.05 mg / L kinetin, and 3% sucrose. The suspension was cultured at 25 ° C. at 100 rpm after being incubated in the medium. After 5 days of culture, only the suspension cell culture medium was centrifuged at 13,000 rpm for 20 minutes at 4 ° C., and then the protein samples were diluted with phosphate-buffer saline (PBS) for 1 mL of the supernatant overnight. After dialysis, physiological activity was measured. Treatment of plant hemagglutinin (PHA) to suspend activated peripheral blood mononuclear cells (PBMC) 5 × 10 ⁵ in 100 μl RPMI medium containing 15 μg / mL PHA (phytohemagglutinin) and microtied with test sample The wells of the ter plate were aliquoted. After 72 hours of incubation, only PMBC was recovered by centrifugation, and then suspended in 100 μl of RPMI medium containing 10% FBS (HyClone Laboratories Inc., Logan, UT, USA), followed by standard hIL-12 and L2728-. HIL-12 and 1 μCi [methyl-3H] thymidine (Amersham Lifescience, NJ, USA) from 3 suspension cell cultures were added to each well and further incubated for 24 hours. Cells were obtained with a cell harvester (Inotech, Awitzerland) and the content of tritium was measured in a liquid scintillation counter (Packard, USA). The standard hIL-12 (PharMingen) produced through the culture of insect cells of known quantity was tested as a positive control and suspension cell culture medium of tobacco transformed with the plant expression vector pMY27 as a negative control.

Figure 9 shows the physiological activity of hIL-12 derived from the transformed plant L2728-3 suspension cells relative to the activity of hIL-12 derived from insect cells measured according to the method specified in Example 4-2 above. will be. In Figure 9, the white bar is a recombinant human IL-12 (PharMingen) produced from insect cells as a positive control, the black bar is a culture medium of suspension cultured tobacco cells transformed with the plant expression vector pMY27 as a negative control, gray Bars represent L2728-3 suspension cell cultures, which are transformed plants containing both the p35 and p40 genes of hIL-12. As a result, the physiological activity of prhIL-12 protein derived from L2728-3 suspension cells achieved in the present invention was about 2.0 times higher than that of hIL-12 derived from insect cells. That is, the activity of the hIL-12 protein of the present invention is superior to that produced in the existing insect cell expression system, and proves that Nicotiana Tabacum L2728-3 (KCTC 0670BP) is an excellent producer.

Example 5 Effect of Gelatin on the Production of hIL-12

As confirmed in Example 4-1, it was confirmed that physiologically active hIL-12 could be produced through culture of L2728-3 suspension cell, a tobacco in which p35 and p40 genes, which were subunits of hIL-12, were simultaneously introduced. However, after 5 days of culture, the production of hIL-12 began to decrease rapidly. This rapid decrease of hIL-12 was determined by the degradation of protease secreted by suspension cultured cells.

In Example 5 was performed to confirm the effect of the protein stabilizer on the production of hIL-12. The final concentration of gelatin in 50 mL Murashige-Skoog (MS) liquid medium containing 1 mg / L 2,4-dichlorophenoxyacetic acid, 0.05 mg / L kinetin, 3% sucrose, 0.5%, 1.0%, 2.0 After adding to about 2.5g of L2728-3 suspension cells (live weight) and suspending culture at 25 ℃ at 100rpm, the suspension cell culture solution 4, 1, 3, 5, 7, 9, 11 days after the start of culture After centrifugation at 13,000 rpm for 20 minutes at 20 ℃ 1mL of the supernatant was dialyzed protein samples with phosphate-buffered saline (PBS) overnight, ELISA analysis was performed by the method shown in Example 4 to quantify hIL-12.

FIG. 10 is a graph showing the amount of hIL-12 secreted into the culture medium when various concentrations of gelatin were added according to Example 5. FIG. All treatments with 0.5%, 1.0%, and 2.0% gelatin added showed an increase in hIL-12. When 0.5% gelatin was added, 580 µg / L of hIL-12 was increased after 5 days of culture. In the case of no addition of gelatin, the yield increased by 3.2 times compared to the production of about 180 ㎍ / L after 5 days of culture, and about 710 µg / L of hIL- after 5 days of culture when 1.0% of gelatin was added. The production of 12 showed no increase in the production of gelatin, compared with the production of about 180 μg / L after 5 days of culture. On the other hand, the difference in yield between treatments treated with 1.0% and 2.0% of gelatin was very small and did not show a significant difference. Therefore, when producing hIL-12 through plant cell cultures in which the p35 and p40 genes of hIL-12 were introduced, it was confirmed that 0.5% of gelatin was added to increase production. As shown in Examples 4-1 and 5, it was determined that the rapid decrease in the production of hIL-12 was due to the degradation of hIL-12 by protease secreted from suspension cells, and gelatin secreted protein secreted out of protein plant cells. It acts as a suitable substrate for degrading enzymes and is present at a higher concentration than hIL-12 produced by plant cells, thus reducing the contact frequency of hIL-12 with proteolytic enzymes. .

The present invention provides a transformed plant cell expressing a biologically active interleukin-12, and a plant cell suspension culture method using the same, thereby secreting a biologically active heterodimer, interleukin-12, out of plant cells in large quantities. It provides a method of production and can produce a large amount of safe and biologically active IL-12 at low cost.

<110> YANG, MOON-SIK          KWON, TAE-HO <120> PRODUCTION OF BIOLOGICALLY ACTIVE INTERLEUKIN-12 PROTEIN USING          PLANT CELL SUSPENSION CULTURE <130> DPP20020907KR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 5013 <212> DNA <213> Artificial Sequence <220> <223> T-DNA region of pMYL27 <400> 1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60 aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120 acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180 gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240 agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300 tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360 tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900 aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320 gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380 ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440 tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500 tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560 tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620 gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680 tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740 tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800 tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860 tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920 gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980 aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040 aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100 gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160 caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220 ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460 tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagagagat 2520 agatttgtag agagagactg gtgatttcag cgtgtcctct ccaaatgaaa tgaacttcct 2580 tatatagagg aaggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg 2640 agatatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 2700 tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga 2760 tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc 2820 ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc 2880 tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg 2940 agtagacgag agtgtcgtgc tccaccatgt tatcacatca atccacttgc tttgaagacg 3000 tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac 3060 cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt 3120 aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga 3180 atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt 3240 ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc accattctag 3300 aggatcccgg gtaccatgtg gccccctggg tcagcctccc agccaccgcc ctcacctgcc 3360 gcggccacag gtctgcatcc agcggctcgc cctgtgtccc tgcagtgccg gctcagcatg 3420 tgtccagcgc gcagcctcct ccttgtcgct accctggtcc tcctggacca cctcagtttg 3480 gccagaaacc tccccgtggc cactccagac ccaggaatgt tcccatgcct tcaccactcc 3540 caaaacctgc tgagggccgt cagcaacatg ctccagaagg ccagacaaac tctagaattt 3600 tacccttgca cttctgaaga gattgatcat gaagatatca caaaagataa aaccagcaca 3660 gtggaggcct gtttaccatt ggaattaacc aagaatgaga gttgcctaaa ttccagagag 3720 acctctttca taactaatgg gagttgcctg gcctccagaa agacctcttt tatgatggcc 3780 ctgtgcctta gtagtattta tgaagacttg aagatgtacc aggtggagtt caagaccatg 3840 aatgcaaagc ttctgatgga tcctaagagg cagatctttc tagatcaaaa catgctggca 3900 gttattgatg agctgatgca ggccctgaat ttcaacagtg agactgtgcc acaaaaatcc 3960 tcccttgaag aaccggattt ttataaaact aaaatcaagc tctgcatact tcttcatgct 4020 ttcagaattc gggcagtgac tattgataga gtgatgagct atctgaatgc ttcctaagag 4080 ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 4140 tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 4200 taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 4260 atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 4320 cgcggtgtca tctatgttac tagatcggga attcactggc cgtcgtttta caacgtcgtg 4380 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 4440 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 4500 atggcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 4560 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 4620 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 4680 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 4740 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 4800 tcggaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc 4860 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga 4920 aaagaaaaac caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc 4980 gtcaatttgt ttacaccaca atatatcctg cca 5013 <210> 2 <211> 4727 <212> DNA <213> Artificial Sequence <220> <223> T-DNA region of pMYL28 <400> 2 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60 aatctgatcc aagctcaagc tgctctagca ttcgccattc aggctgcgca actgttggga 120 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 180 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc 240 cagtgccaag cttctgcagc atgcagagat agatttgtag agagagactg gtgatttcag 300 cgtgtcctct ccaaatgaaa tgaacttcct tatatagagg aaggtcttgc gaaggatagt 360 gggattgtgc gtcatccctt acgtcagtgg agatatcaca tcaatccact tgctttgaag 420 acgtggttgg aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg 480 gaccactgtc ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt 540 tgtaggtgcc accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat 600 ggaatccgag gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt 660 cttctgagac tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt 720 tatcacatca atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc 780 tcctcgtggg tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag 840 cctttccttt atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt 900 tgatgaagtg acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt 960 gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt 1020 agacgagagt gtcgtgctcc accattctag aggatccatg tgtcaccagc agttggtcat 1080 ctcttggttt tccctggttt ttctggcatc tcccctcgtg gccatatggg aactgaagaa 1140 agatgtttat gtcgtagaat tggattggta tccggatgcc cctggagaaa tggtggtcct 1200 cacctgtgac acccctgaag aagatggtat cacctggacc ttggaccaga gcagtgaggt 1260 cttaggctct ggcaaaaccc tgaccatcca agtcaaagag tttggagatg ctggccagta 1320 cacctgtcac aaaggaggcg aggttctaag ccattcgctc ctgctgcttc acaaaaagga 1380 agatggaatt tggtccactg atattttaaa ggaccagaaa gaacccaaaa ataagacctt 1440 tctaagatgc gaggccaaga attattctgg acgtttcacc tgctggtggc tgacgacaat 1500 cagtactgat ttgacattca gtgtcaaaag cagcagaggc tcttctgacc cccaaggggt 1560 gacgtgcgga gctgctacac tctctgcaga gagagtcaga ggggacaaca aggagtatga 1620 gtactcagtg gagtgccagg aggacagtgc ctgcccagct gctgaggaga gtctgcccat 1680 tgaggtcatg gtggatgccg ttcacaagct caagtatgaa aactacacca gcagcttctt 1740 catcagggac atcatcaaac ctgacccacc caacaacttg cagctgaagc cattaaagaa 1800 ttctcggcag gtggaggtca gctgggagta ccctgacacc tggagtactc cacattccta 1860 cttctccctg acattctgcg ttcaggtcca gggcaagagc aagagagaaa agaaagatag 1920 agtcttcacc gacaagacct cagccacggt catctgccgc aaaaatgcca gcattagcgt 1980 gcgggcccag gaccgctact atagctcatc ttggagcgaa tgggcatctg tgccctgcag 2040 ttaggtaccg agctcgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag 2100 attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa 2160 gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag 2220 agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga 2280 taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattcgtaa tcatggtcat 2340 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 2400 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 2460 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 2520 aacgcgcggg gagaggcggt ttgcgtattg gctagagcag cttgccaaca tggtggagca 2580 cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc aaagggctat 2640 tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat 2700 ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg 2760 cgataaagga aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc 2820 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt 2880 ggattgatgt gataacatgg tggagcacga cactctcgtc tactccaaga atatcaaaga 2940 tacagtctca gaagaccaaa gggctattga gacttttcaa caaagggtaa tatcgggaaa 3000 cctcctcgga ttccattgcc cagctatctg tcacttcatc aaaaggacag tagaaaagga 3060 aggtggcacc tacaaatgcc atcattgcga taaaggaaag gctatcgttc aagatgcctc 3120 tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180 cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240 tgacgcacaa tcccactatc cttcgcaaga ccttcctcta tataaggaag ttcatttcat 3300 ttggagagga cacgctgaaa tcaccagtct ctctctacaa atctatctct ctcgagcttt 3360 cgcagatccc ggggggcaat gagatatgaa aaagcctgaa ctcaccgcga cgtctgtcga 3420 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga 3480 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag 3540 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct 3600 cccgattccg gaagtgcttg acattgggga gtttagcgag agcctgacct attgcatctc 3660 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct 3720 acaaccggtc gcggaggcta tggatgcgat cgctgcggcc gatcttagcc agacgagcgg 3780 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg 3840 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc 3900 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg 3960 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac 4020 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat 4080 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag 4140 gcatccggag cttgcaggat cgccacgact ccgggcgtat atgctccgca ttggtcttga 4200 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg 4260 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag 4320 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg 4380 ccccagcact cgtccgaggg caaagaaata gagtagatgc cgaccggatc tgtcgatcga 4440 caagctcgag tttctccata ataatgtgtg agtagttccc agataaggga attagggttc 4500 ctatagggtt tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg 4560 taaaatactt ctatcaataa aatttctaat tcctaaaacc aaaatccagt actaaaatcc 4620 agatcccccg aattaattcg gcgttaattc agtacattaa aaacgtccgc aatgtgttat 4680 taagttgtct aagcgtcaat ttgtttacac cacaatatat cctgcca 4727 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> Primer of p35-F <400> 3 aaggtaccaa ttataaaaat gtgg 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer of p35-R <400> 4 aagagctctt taggaagcat tca 23 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> Primer of p40-F <400> 5 gcggatccat gtgtcaccag cagttggtca tct 33 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer of p40-R <400> 6 ttggtaccct aactgcaggg cacagatgcc cat 33

Claims (9)

(a) providing a transgenic plant into which a gene encoding a p35 subunit of interleukin-12 (IL-12) is introduced, and a transgenic plant into which a gene encoding a p40 sub unit is introduced, (b) cross-transforming the two transgenic plants to produce a transgenic plant expressing both subunits of IL-12, (c) preparing a plant cell line by inducing callus from the transformed plant of (b), (d) suspending culture of said plant cell line to express IL-12. The method of claim 1, wherein the plant is Nicotiana tabacum. The method of claim 1, wherein the plant cell line is KCTC 1021BP. The method of producing IL-12 according to claim 1, wherein in step (d), gelatin is added to the culture medium to increase production of IL-12. The method of claim 1, wherein the IL-12 comprises a signal peptide. delete Transgenic plant cells that produce and secrete biologically active interleukin-12 (IL-12). 8. The plant cell of claim 7, wherein the plant cell is a tobacco cell. The plant cell of claim 7, wherein the plant cell is KCTC 1021BP.

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