KR20050112715A - Recombinant vector containing gene of hctla4-ig fusion protein and manufacturing process of the protein by using them - Google Patents

Abstract

The present invention relates to a recombinant vector pMYN409 comprising a hCTLA4-Ig fusion protein gene and a method for mass production of a hCTLA4-Ig fusion protein using the same, and more particularly, an expression vector comprising a gene encoding a recombinant hCTLA4-Ig fusion protein. pMYN409 and a method for producing a large amount of recombinant hCTLA4-Ig fusion protein through transformation in plant cells and suspension culture. As a result, the present invention enables the production of recombinant hCTLA4-Ig fusion protein having immunosuppressive activity equivalent to that of hCTLA4-Ig fusion protein expressed in existing animal cells using plant cell suspension culture, and is more economical than animal cell culture. It is effective to produce protein.

Description

Recombinant vector containing gene of hCTLA4-Ig fusion protein and manufacturing process of the protein by using them}

The present invention relates to a recombinant vector pMYN409 comprising a hCTLA4-Ig fusion protein gene and a method for mass production of a hCTLA4-Ig fusion protein using the same, and more particularly, an expression vector comprising a gene encoding a recombinant hCTLA4-Ig fusion protein. pMYN409 and a method for producing a large amount of recombinant hCTLA4-Ig fusion protein through transformation in plant cells and suspension culture.

In general, immunomodulators can be divided into immunopotentiators and immunosuppressants according to pharmacological actions that enhance or inhibit immune function. In recent years, as immunosuppressive agents have recently increased in transplantation of heart, liver and kidney, the necessity of drugs for suppressing the transplant rejection is rapidly increasing, and the immune function is enhanced to attack the tissues. The development of a therapeutic agent for autoimmune diseases, a type of inflammatory disease, has attracted much attention as it attracts commercial interest.

T cell activation plays an important role in initiating transplant rejection. Two signals are required for the full activity of T cells. One is intracellular activation signals due to the interaction between the major histocompatibility complex (MHC) of the antigen-specific antigen presenting cell (APC) and the T cell antigen receptor (TCR). It begins by delivering. The second is an antigen nonspecific costimulatory signal. Lack of synchronizing stimulatory signals after antigen recognition by TCR results in partial or failure of T cell activation resulting in T cell anergy, in which the T cells become unresponsive to subsequent antigenic attack. Cellular energy is most important for the induction of antigen-specific tolerence that prevents transplant rejection. The most important T cell synchronizing stimulatory signal is the binding between CD28 and CTLA4 of T cells and B7 receptors (CD80 and CD86) on the surface of APC. CTLA4 exhibits about 20-fold higher affinity with the B7 receptor than with CD28 (Linsley et al., J. Exp. Med. 561,1991, Linsley et al., Immunity, 1: 793, 1994). Unlike CD28, binding of CTLA4 to the B7 receptor carries a signal that inhibits or reduces T cell activation (Sebille et al., Philos. Trans. R. Soc. Lond., B Biol. Sci. 356: 649, 2001) .

Linsley et al., J. Exp. Med., 174: 561, 1991 also prepared a CTLA4-Ig fusion protein that artificially fused the Fc portion of an immunoglobulin IgG to the C-terminus of CTLA4. In addition, it has been reported that this fusion protein has an immunosuppressive effect. As a result, this immunoglobulin portion of CTLA4-Ig enables efficient purification, production of dimeric CTLA4 protein, and long half-life in vivo in an affinity chromatography column. Blocking of CD28 / B7 tuning signal by CTLA4-Ig was confirmed by rat cardiac transplantation (Guillot et al., J. Immunol. 164: 5228,2000; Hayashi et al., Transpl. Int. 13 (Suppl. 1). ), S329, 2000; Turka et al., Proc. Natl. Acad. Sci. USA 89: 11102, 1992), mouse eyelet xenograft (Feng et al., Transplantation 67: 1607, 1999; Lenschow et al., Science, 257: 789,2000), rat renal transplantation (Tomasoni et al., J. Am. Soc. Nephrol. 11, 747, 2000), monkey islet transplantation (Kirk et al., Proc. Natl. Acad. Sci. USA 94; 8789, 1997; Levisetti et al., J. Immunol. 159: 5187, 1997). This suggests the possibility of an important therapeutic agent in actual clinical practice. Indeed, clinical trials of CTLA4-Ig have shown convincing results as therapeutic agents (Abrams et al., J. Clin. Invest. 103: 1243, 1999, J. Exp. Med. 192: 681, 2000; Guinan et al. , N. Engl. Jmed, 340: 1704, 1999). While conventional immunosuppressive drugs such as steroid-based hormones, cyclophosphamide, cyclosporin, and FK506 have almost the non-specificity of suppressing the immune system, CTLA4-Ig has a disadvantage of T By specifically inhibiting only the activation process of the cell, it is expected to have relatively little side effects and excellent immunosuppressive effect.

However, these CTLA4-Ig fusion proteins are high in humans compared to protein preparations of up to 10 mg / kg / circuit cytokines (Abrams, JR et al., J. Clin. Invest., 103 (9)). : 1243, 1999: Greene JL et al., Arthritis Rheum. 46: 1470, 2002, Kremer, JM et al., N Engl J. Med., 349 (20): 1907, 2003), when using animal cell culture There is a problem of high production cost.

Recently, it has been reported that the production of CTLA4-Ig having biological activity in the milk of transgenic animals yields about five times higher yield than animal cells (Lui VCH, et al., J. Immuno., Meth. , 277: 171, 2003). In addition, with the development of plant biotechnology, attempts are being actively made to produce high value-added useful proteins through mass culture of plant cells, and economic superiority due to low-cost medium components and ease of protein production, isolation and purification Therefore, it is attracting attention as an alternative production system of pharmaceutical proteins such as cytokines, growth factors, and immunomodulators produced through existing microbial or animal cell cultures (Miele, L., Trends Biotechnol., 15: 45-50, 1997; Doran, PM, Curr. Opin. Biotechnol., 11: 199-204, 2000). In addition, when producing recombinant protein using plant cell culture, unlike prokaryotic cells such as Escherichia coli, a post-translational modification process is similar to that of animal cells, which not only maintains the biological activity of the protein, Compared to animal cell cultures using serum, there is less risk of incorporation of viruses or pathogens that are lethal to humans. However, the disadvantages of slow cell growth in plant cell culture systems and low protein expression rates compared to microorganisms or animal cells are still a problem to be overcome, and cost-effective and efficient mass production methods are urgently needed for commercialization.

Accordingly, the present inventors have made intensive efforts to improve the above problems. As a result, we have developed vectors and plant cells having a strong promoter system capable of inducing high expression. It was confirmed that mass culture is possible.

After all, the present invention is to provide a recombinant vector pMYN409 comprising the hCTLA4-Ig (human cytotoxic T lymphocyte antigen 4-Immunoglobulin) fusion protein-coding gene of the attached SEQ ID NO: 1.

In addition, the present invention has another object to provide a plant cell transformed with the vector.

Finally, an object of the present invention is to provide a method for producing a recombinant hCTLA4-Ig fusion protein comprising the step of suspension culture of the plant cells.

The present invention relates to a recombinant vector pMYN409 comprising a hCTLA4-Ig fusion protein-coding gene and a method for mass production of hCTLA4-Ig fusion protein using the same.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

First, the present invention relates to a recombinant vector pMYN409 comprising a hCTLA4-Ig (cytotoxic T lymphocyte antigen 4-Imunoglobulin) fusion protein-coding gene, wherein the recombinant vector pMYN409 is a rice amylase leader sequence of the attached SEQ ID NO: 1 (RAmy1A leader sequence) ) And recombinant hCTLA4-Ig fusion protein-coding gene sequences. In the attached Sequence Listing 1, 1 ~ 93bp is a signal peptide, 94 ~ 465bp is a CTLA4 extracellular region, and 466 ~ 1166bp is an IgG1 Fc fraction. In addition, this recombinant vector pMYN409 is a selection marker for transgenic plant cell lines, which utilizes resistance to antibiotic hygromycin, and secretes recombinant hCTLA4-Ig fusion protein to outside of plant cells during suspension culture by rice amylase leader sequence. It can be characterized in that (see Figure 1). In addition, the Ig in the fusion protein includes a hinge, CH2, and CH3 regions of the Cγ1 region of human IgG1.

The present invention also includes plant cells transformed with the recombinant vector pMYN409 comprising the hCTLA4-Ig fusion protein-coding gene. Such plant cells are preferably rice ( Oryza sativa L. cv. Dongjin), and in addition to tobacco (Nicotiana tabacum), corn (Zea mays), soybean (Glycine max), wheat (Triticum aestivum), tomato (Lycopersicon esculentum), Rapeseed (Brassica napus), potato (Solanum tuberosum) and the like can be used.

In the present invention, the recombinant vector pMYN409 is transduced into the plant cell by a microprojectile bombardment transformation method, and then only the transformed plant cell line is selected in a selection medium.

In addition, the present invention includes a method of mass-producing prhCTLA4-Ig (plant recombinant human CTLA4-Ig) by suspension culture of the transformed plant cells selected above. That is, only the plant cells transformed in the selection medium containing the antibiotic and resistant to the antibiotic are selected and suspended in culture. Suspension culture conditions are based on Chu N6 or AA medium or MS (Murashige and Skoog) medium, which is widely used in plant cell culture, and 10 ~ 60 g / L sucrose and growth regulator as a carbon source. 22-28 ° C., 80-150 rpm, cancer using medium containing L 2,4-D and 0.01-2.0 mg / L kinetin and 50 mg / L hygromycin for selection It is preferable to culture under conditions.

   As a result, the transformed plant cell line expresses and releases prhCTLA4-Ig into the medium during suspension culture. For example, in suspension culture of Oriza sativa pMYN409 (KCTC 10618BP), 30 g / L sucrose, 2 mg / L 2,4-D, 0.2 mg / L kinetin, 50 mg / L hygromycin ) Was cultured at 28 ° C., 120 rpm, in a dark condition using N6 liquid medium or AA rice suspension culture medium containing) to exchange recombinant culture with N6 medium without sucrose at 7 days to induce recombinant protein expression to obtain prhCTLA4-Ig. It is preferable to express. The prhCTLA4-Ig protein was obtained by centrifugation of Orissa sativa pMYN409 culture, purified by protein A affinity chromatography, followed by lyophilization, to separate the culture supernatant, oryza sativa pMYN409 of the present invention. For (KCTC 10618BP), the prhCTLA4-Ig production rate is about 10 mg / L per culture.

In addition, immunosuppressive activity of the prhCTLA4-Ig protein produced according to the present invention is characterized by lymphocyte mixed culture (MLR), ConA-induced T cell proliferation, in vitro T cell dependent antibody production. As a result of T cell proliferation inhibitory effect through [in vitro T-dependent Ab production] test, it was found that the recombinant hCTLA4-Ig equivalent or more produced in the conventional CHO cell expression system.

Accordingly, the present invention provides a recombinant vector comprising a hCTLA4-Ig fusion protein-coding gene and a plant cell line transformed using the same, and establishes a prhCTLA4-Ig expression system of the transformed plant cell, thereby having prhCTLA4 having immunosuppressive activity. Provide mass production of Ig As a result, the prhCTLA4-Ig can be produced in large quantities at low cost using a plant expression system, and immunosuppressive activity can also be confirmed and used for medical purposes.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

Example : Production of Recombinant CTLA4-Ig Fusion Protein in Transgenic Rice Suspension Cells

1. pMYN409 Vector Fabrication

A schematic diagram of the gene map of the recombinant expression vector expressing the hCTLA4-Ig gene is briefly shown in FIG. 1. First, in order to secrete the hCTLA4-Ig fusion protein into the medium, a synthetic fusion gene was prepared by binding a rice amylase leader sequence to the hCTLA-4 N-terminus using an overlapping polymerase chain reaction (PCR) technique. First, the forward primer CTLA4-F1 (5'-TCCAACTTGACAGCCGGGGCAA TGCACGTGGCCCAGCCTGC-3 ') containing the C-terminal and CTLA-4 N-terminal sequences of the rice amylase leader sequence (RAmy1A signal peptide) One 94-degree two-minute pre-denaturation step using reverse primer CTLA4-R1 (5'-CTCTGCAGAATCTGGG CACGGTTCTG-3 ') recognizing the -4 C-terminal site; PCR was performed under conditions of 94 ° C 45 sec denaturation, 55 ° C 45 sec annealing, 72 ° C 45 sec elongation, and 30 ° C 72 min 5 final elongation. It was.

An overlapping PCR using a rice amylase secretion signal gene obtained from the PCR product and rice cDNA as a template was carried out to prepare a synthetic fusion gene in which a high secretion leader sequence of rice amylase and a hCTLA4 gene were combined, and a pGEM-T easy vector. Vector pMYN407 was prepared by insertion into (Easy vector) (Promega, USA).

The vector pMYN408 was prepared by inserting the IgG1 Fc gene obtained by cleaving the pMYN406 vector containing the human immunoglobulin IgG1 Fc gene into the pMYN407 vector digested with the same restriction enzyme.

Finally, pMYN409, an expression vector, was prepared by cutting the hCTLA4-Ig fusion protein gene coupled with the rice amylase secretion signal on the pMYN408 vector with a restriction enzyme BamHI / SacI and inserting it into a pMYN75 expression vector containing the same restriction enzyme. It was. The pMYN409 vector confirmed that the hCTLA4-Ig gene was inserted between the RAmy3D promoter and the 3′-UTR through restriction enzyme mapping.

2. Preparation of Transgenic Plant Cell Lines

In order to introduce hCTLA4-Ig gene into rice ( Oryza sativa L. cv. Dongjin), the expression vector pMYN409 was transformed using a microprojectile bombardment.

First, the husks of rice seeds were removed, subjected to surface sterilization, washed with sterile distilled water, and placed on callus-inducing agar medium. After 7-10 days of incubation at 25 ° C for 16 hours in bright conditions and 8 hours in dark conditions, scutellum-derived calli were taken from the germinated seeds and transferred to new agar medium. Biolistic PDS-1000 / Bombardment was carried out using He (BioRad Laboratories, USA). After bombardment (Bombardment) was further incubated for 4 days at 25 ℃, dark conditions and the callus was cultured by transferring the callus to the selection medium (N6 SE) to which the antibiotic hygromycin B 50 mg / L was added. After culturing for 3 to 5 weeks, the transformed rice callus cell line was obtained by culturing until the callus showing resistance to the selection mark hygromycin grew to about 1 cm in diameter.

3. Confirmation of gene insertion through Southern blot analysis

Southern blotting was performed along with genomic DNA PCR to confirm whether the 11 kinds of transformed rice callus obtained through the Bombardment contain the hCTLA4-Ig gene.

First, genomic DNA was isolated from rice callus with DNAeasyplant mini kit (Qiage, CA, USA). PCR amplification was performed using the isolated genomic DNA as a template, and the forward primer was CTLA4-F1 (5'-TCCAACTTGACAGCCGGGGCAATGCACGTGGCCCAGCCTGC-3 '), and the reverse primer was hIgG-R1 (5'-CTCTAGACTCATTTACCCGGAGACAGGGAG-3'). . PCR products were electrophoresed on 1.0% agarose gel and stained with EtBr.

Figure 2A is a PCR amplified picture of the hCTLA4-Ig gene contained in 11 transformed rice callus cell line, M is the size marker, P is a positive control PCR PCR pMYN409 vector, N is the genome of the natural rice plant The gene was PCR-negative control group, lanes 1 to 11 PCR the transformed rice callus cell line of Example 2. The PCR amplified DNA fragment was found to be approximately 1167 bp, which is consistent with the expected size.

In addition, the PCR product was electrophoresed and then moved to a Hybond (TM) -N + nylon membrane (Amersham Pharmacia, USA) to perform a sudden blot. Southern blot probes were hybridized using a-32P-labeled hCTLA4 DNA. As shown in FIG. 2B, it was confirmed that the hCTLA4 DNA probe was bound to the PCR product amplified from the transformed cell line. From this, the hCTLA4-Ig gene was identified on the chromosome of 11 transformed rice callus cell lines of Example 2. It was confirmed that it was successfully inserted into the transgenic callus. As a result, the transgenic callus was normally introduced through PCR and Southern blotting and the highest expression rate was designated as Oryza sativa pMYN409 (Oriza sativa pMYN409). Oriza sativa pMYN409, which expresses prhCTLA4 -Ig of the present invention, was deposited with KCTC 10618BP on May 6, 2004, to the Gene Bank in the Biotechnology Research Institute.

4. Quantitative Analysis of Recombinant hCTLA4-Ig Fusion Protein Derived from Transgenic Rice Callus

An ELISA (enzyme linked immunosorbent assay) assay was performed to quantify the expression level of hCTLA4-Ig of 11 transformed rice callus cell lines obtained from Example 2 and to select high expressing cell lines.

First, rice callus was transferred onto N6 agar medium containing no sucrose to induce protein expression by the RAmy3D promoter. After crushing the rice callus maintained on the medium for 7 days, suspended in 500 μl of phosphate buffer solution, vortexing for 1 minute, centrifuged at 15,000 rpm, 4 ℃ for 30 minutes to separate only the supernatant for analysis Used.

For ELISA analysis, a 96-well plate was diluted 1: 1000 with goat anti-human IgG (KPL) in a coating buffer and dispensed 100ul per well. After overnight at 4 ° C., it was washed three times with washing buffer PBST (T 0.05% tween20-containing PBS). 200 ul of assay diluent per well (PBS buffer containing 2% FBS) was dispensed and reacted at room temperature for 1 hour, and then washed three times with washing buffer PBST. Callus extract samples were dispensed by 100 μl per well and diluted twice with analytical diluent to react at 37 ° C. for 1 hour. Again wash three times with the wash buffer solution and 100 μl per well of peroxidase-labeled goat anti-human IgG (KPL) diluted in a ratio of 1: 1000 to the assay diluent 37 The reaction was carried out at 1 ° C. for 1 hour. After washing three times with the washing buffer and reacted with 100ul of substrate for 15 minutes and the absorbance was measured at 405 nm.

Figure 3 graphically shows the results of ELISA quantitative analysis of recombinant hCTLA4-Ig fusion protein expressed in transformed rice callus cell line, about 54 μg hCTLA4- for BR-N002 showing the highest expression level among 11 cell lines. Recombinant protein production of Ig / g fresh calli was shown, the expression of hCTLA4-Ig per total protein was about 30 ㎍ CTLA4-Ig / mg total protein was confirmed to show a protein expression rate of about 3%.

5. Induction of suspension culture and production of hCTLA4-Ig in medium

The transformed rice callus cell line obtained from Example 2 was subjected to suspension culture. Suspension culture media include N6 liquid medium or AA rice suspension containing 30 g / L sucrose, 2 mg / L 2,4-D, 0.2 mg / L kinetin, 50 mg / L hygromycin Subcultured at intervals of two weeks while culturing at 28 ° C., 120 rpm and dark conditions using the culture medium.

To confirm the secretion of hCTLA4-Ig protein into the medium, the protein expression was induced by exchanging with a medium containing no sucrose after incubating for 10 days. After 2-3 days of medium exchange, the culture medium was recovered, and the hCTLA4-Ig was confirmed in the medium by ELISA.

Figure 4 graphically shows the cell growth and production of hCTLA4-Ig fusion protein in the suspension culture culture cells in suspension culture in N6 medium (open symbol) and AA medium (closed symbol). When recombinant protein expression was induced by exchanging with sucrose-free medium at 7 days of culture, hCTLA4-Ig production was rapidly increased in the medium after 2 to 3 days to produce hCTLA4-Ig fusion protein up to about 10 mg / L. Confirmed.

Experimental Example : Analysis of hCTLA4-Ig Fusion Protein

1. SDS-PAGE Electrophoresis and Western Blotting

After the suspension culture of Orissa sativa pMYN409, hCTLA4-Ig protein was purified from the culture using a Protein A column. Purity and molecular weight of the purified prhCTLA4-Ig were confirmed by 10% SDS-PAGE electrophoresis and Western blotting.

That is, Figure 6A is a SDS-PAGE electrophoresis picture of prhCTLA4-Ig, M is the molecular weight marker, lane 1 shows the modified molecular weight. As shown in the photo, the variable molecular weight was about 50kDa. FIG. 6B is a Western blotting picture of prhCTLA4-Ig, followed by electrophoresis, transfer to PVDF membrane, and reaction with a goat anti-human IgG antibody (KPL, USA) as a primary antibody. After washing the membrane, the membrane was washed after alkaline phosphatase attached rabbit anti-goat IgG antibody (KPL, USA). After the substrate reaction was moderated, the reaction was stopped by washing with tap water. As a result, it was confirmed that the prhCTLA4-Ig fusion protein having a molecular weight of about 50 kDa by reacting with a specific antibody as shown in the photo.

2. hCTLA4 / B7 Kinetic Analysis

Transformation suspension culture BR-N002 cell line expression hCTLA4Ig interaction and affinity with B7-1, B7-2 was confirmed by BIAcore 3000 (Biacore AB). A CM5 sensor chip was used as a chip to fix the ligand. B7-1 and B7-2 were used as ligands for fixing to the sensor chip, and the analysis group used CD28 (product of R & D system) and CHO cell expression hCTLA4Ig as a control. As the experimental group, hCTLA4-Ig derived from plant cells was used.

Immobilization of the ligand on the sensor chip was injected by diluting the ligand in a 10 mM sodium acetate buffer at pH 5.5 and injecting PBS into the running buffer. It was used and the flow rate was maintained at 5ul / ml.

Analytes were each reacted for 2 minutes at a flow rate of 30ul / ml at 25 ° C. After the injection of the analyte, the bound ligand and the analyte were artificially separated using 1 M NaCl / 50 mM NaOH.

TABLE 1

Mechanics obvious Constant Summary (Summary of apparent kinetic constants)

Ligand Analyte Ka ( ) ( ) Kd ( ) ( ) KD (M) ( ) T (Ka1) T (Kd1) Chi2 B7-1 B7-2 CD28AaPbCD28AP 131261562.4196316 1.115.815.020.9211.815.7 8.574.623.2338.46.054.97 59.814014910.762.461.4 13.535.633.711.468.894.2 5.152.723.812.584.032.51 Aa: CHO cell expression hCTLA4-IgPb: prhCTLA4Ig

As shown in Table 1, the binding force between each ligand and the analysis group (analyte) showed a similar pattern, in the case of B7-1 A and P showed about 10 times the association rate (association rate) compared to CD28 , B7-2 showed an association rate of about 100-fold. Also, the dissociation rate was higher in A and P than in CD28. In the case of B7-1, the affinity of A and P was twice as high as that of CD28, and in the case of B7-2, A was about 6 times and P was about 8 times higher. As a result, both CHO expressing hCTLA4-Ig and prhCTLA4-Ig had higher affinity for B7 family (B7-1, B7-2) than CD28, and prhCTLA4-Ig was similar to CHO expressing hCTLA4-Ig. It was confirmed to have affinity for (B7-1, B7-2).

3. Inhibition of ConA-induced T cell proliferation

To measure the proliferative capacity of T cells, spleens of BDF1 mice were extracted and the splenocytes were released to adjust the cell concentration to 1 × 10 6 cells / ml, each well in a 96-well plate. 200 μl of the cells were aliquoted and cultured. 5 μg / of mitogen ConA, a mitogen that induces C lymphocyte-derived hCTLA4-Ig and prhCTLA4-Ig at concentrations of 0.001, 0.01, 0.1, 1, 10 ug / ml and induces the proliferation of T lymphocytes Addition of ml and incubated for 3 days in 37 ℃, 5% CO2 incubator. After incubation, [3H] -thymidine was added, and the cells of each well were recovered using an automatic cell harvester, and then the degree of binding of [3H] -thymidine to DNA was measured. The proliferation of was examined.

As shown in FIG. 6, both CHO cell-derived hCTLA4-Ig and prhCTLA4-Ig inhibited T cell proliferation by Con A in a concentration-dependent manner, compared to the solvent control group (VH), and inhibited proliferation of 50% or more at a concentration of 1ug / ml or more The effect was shown. In addition, the degree of inhibition of T cell proliferation of prhCTLA4-Ig was almost the same as that of CHO cell-derived hCTLA4-Ig.

4. Inhibition of in vitro T-dependent antibody production (Ab production)

Activation of spleen cells using sRBC (sheep red blood cell), a T cell-dependent antigen, produces plasma cells that produce antibodies to sRBC. Spleen cells were adjusted to 1 X 107 cells / ml and then dispensed 0.5 ml / well into 48-well plates. Thereafter, 6.5 μl of sRBC diluted to 1 X 109 cells / ml with buffer (EBSS) was added to each well, followed by incubation for 5 days, and then the cultured cells were recovered and plaque assay was performed. ) Was used.

Plasma cells that produce antibodies to sRBC, sRBC, guinea pig complement and CHO cell-derived hCTLA4-Ig and prhCTLA4-Ig as 0.001, 0.01, 0.1, 1, Plaques were counted after incubation in a 5% CO2 incubator at 37 ° C for about 1 hour using 0.85% agar treated at a concentration of 10 ug / ml, and the count was calculated by PFC / 106 cells. It was set as. The more activated plasma cells, the more antibodies are generated and the guinea pig complement acts to dissolve the periplasmic sRBCs, thereby increasing the number of plaques.

As shown in FIG. 7, both CHO cell-derived CTLA4-Ig and prhCTLA4-Ig, both of which are concentration-dependent, have an antibody against sRBC, a T cell-dependent antigen of plasma cells. Production was inhibited and at least 80% at concentrations above 1 ug / ml. In addition, the inhibition of the production of the antibody against sRBC, which is a T cell-dependent antigen, of plasma cells of prhCTLA4-Ig was almost the same as that of CLA cell derived CTLA4-Ig.

As described and demonstrated in detail above, the present invention relates to a recombinant vector pMYN409 comprising a hCTLA4-Ig fusion protein-coding gene and a method for mass production of a hCTLA4-Ig fusion protein using the same, the method for culturing a transgenic plant cell according to the present invention Using this method to establish a recombinant hCTLA4-Ig fusion protein having immunosuppressive activity, thereby economically has the effect of mass production of recombinant hCTLA4-Ig fusion protein having immunosuppressive activity.

1 shows a schematic diagram of the gene map of the pMYN409 vector of the present invention.

2 is a photograph (A) obtained by PCR amplification of the hCTLA4-Ig gene included in 11 transgenic strains and a Southern blot of the amplified PCR product (B).

Figure 3 is a quantitative graph of prhCTLA4-Ig expressed in the callus of Orissa sativa pMYN409 (KCTC 10618BP).

4 is a quantitative graph of prhCTLA4-Ig expressed in suspension culture of Orissa sativa pMYN409 (KCTC 10618BP).

Figure 5 is a photograph (A) and Western blotting (B) of the electrophoresis of prhCTLA4-Ig protein of the present invention, respectively.

Figure 6 is a graph of the inhibitory effect of ConA-induced T cell proliferation of prhCTLA4-Ig protein of the present invention.

Figure 7 is a graph of the inhibitory effect of the production of in-vitro T cell-dependent antibody of the prhCTLA4-Ig protein of the present invention.

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Claims (6)

Recombinant vector pMYN409 comprising a human cytotoxic T lymphocyte antigen 4-Immunoglobulin (HCTLA4-Ig) fusion protein-coding gene of SEQ ID NO: 1. The recombinant vector pMYN409 according to claim 1, wherein the Ig in the hCTLA4-Ig is a hinge, CH2, or CH3 region of Cγ1 of human IgG1. Oryza sativa L., Tobacco (Nicotiana tabacum), Corn (Zea mays), Soybean (Glycine max), Wheat (Triticum), characterized in that transformed with the recombinant vector pMYN409 of claim 1 or 2. aestivum, tomato (Lycopersicon esculentum), rapeseed (Brassica napus) and potato (Solanum tuberosum) any one of the plant cells. The transformed plant cell (Accession No. KCTC 10618BP ) according to claim 3, wherein the plant cell is Oryza sativa L .. Method for producing a recombinant hCTLA4-Ig fusion protein comprising the step of suspending the plant cell of claim 3. According to claim 5, The suspension culture is Chu N6 or AA medium or MS (Murashige and Skoog) medium widely used in plant cell culture, such as 10 ~ 60 g / L sucrose, growth regulator 0.1 as a carbon source as a basic medium Method for producing a recombinant hCTLA4-Ig fusion protein characterized by using a medium containing ~ 2.0 mg / L 2,4-D and 0.01 to 2.0 mg / L kinetin, and hygromycin for selection .