KR100250829B1 - Mass production method of interleukin-13 - Google Patents

Abstract

PURPOSE: Provided is a method for manufacturing interleukin-13 in E. coli in a high yield. The interleukin-13 is useful in treating septic shock or rheumatoid arthritis and increases the activity of macrophage induced by IL-2. CONSTITUTION: A method for manufacturing interleukin-13 in E. coli is characterized by the following steps of: i) synthesizing cDNA using human T cells; ii) devising primers for PCR of IL-13 (5'primer having a site recognizing restriction enzyme while 3'primer having both a terminus codon and a site recognizing restriction enzyme); iii) amplifying IL-13 genes through PCR using cDNA as a template and the primers; iv) cutting the amplified IL-13 genes with restriction enzymes and making an expression vector pET IL-13 containing the gene segments; v) making primers for 5' terminal of IL-13 gene; vi) amplifying genes of IL-13 having different base sequences on 5' terminal using the primers and the expression vector pET IL-13; vii) cutting the amplified genes with restriction enzymes; viii) replacing the gene segments with UB A545 of vector pETUB A545 to make expression vector pET RIL-13; and ix) transforming E. coli with the expression vector to produce interleukin-13.

Description

Mass Production of Interleukin-13 in Escherichia Coli

The present invention relates to a mass production method of interleukin-13 (hereinafter referred to as IL-13) in Escherichia coli, and more particularly, to the 5 'terminal sequence of the IL-13 gene derived from activated human T cells. The present invention relates to a method for mass production of IL-13 protein by transforming E. coli into an E. coli preferred codon without changing the amino acid.

The production of blood cells is a process that occurs throughout the life of continuous production of short-lived blood cells from stem cells (Metcalf, D., Science, 254, 529 (1989)), the opposite of stimulation and inhibition. Maintain homeostasis by action. Hematopoietic growth factors involved in this process are a group of colony stimulation factor (CSF) growth factors such as G-CSF, M-CSF, CSF-1, GM-CSF, and IL-3, which alone proliferate and differentiate hematopoietic stem cells. To mature into hematopoietic cells (Metacalf, D., Science, 254, 529 (1989)). In contrast, the SCF-based hematopoietic growth factor group is ineffective or weak alone, but when combined with the CSF-based growth factor, it can synergistically enhance the proliferative response of hematopoietic stem cells by the CSF-based growth factor. Known (Ikebuchi, K. et al., Proc. Natl. Acad. USA, 84, 9035 (1987); Stanly, ER et al., Cell, 45, 667 (1986)). This synergistic growth factor is particularly important for the regulation of the first hematopoietic stem cells because synergism between hematopoietic growth factors can stimulate the proliferation of hematopoietic stem cells to optimal conditions (Williams, n. Et. Al. , Blood. 79, 58 (1992).

Cytokines are essential for the proliferation and differentiation of B cells involved in the production of antibodies. In addition to quantitatively determining the production of antibodies, cytokines are known to directly induce changes in the types of antibodies (Snaper, CM and Paul, WE, Science, 236, 944-947 (1987); Lutzker, SM et. Al., Cell, 53, 177-184 (1988)). Interleukin 4 (IL-4) is one of several cytokines produced by T cells activated by B7 and BB-1 antigens in B and mononuclear cells, and induces changes in IgG4 and IgE in B cells. Tumor growth factor-beta induces changes in IgA. This IL-4 stimulates B cells, T cells and Mast cells and further affects the growth of hematopoietic stem cells (Rennick, D. et al., Proc. Natl. Acad. USA, 84, 6889 (1987). ), And stimulates the proliferation of peripheral blood mononuclear cells (PBMC) activated by phytohemagglutinin (PHA) (Minty, A. et al., Nature, 362, 248 (1993)). IL-13, like IL-4, is a cytokine that is produced in T cells and has a molecular weight of about 10 kDa and is secreted without glycosylation with similarity to IL-4. IL-4 and IL-13 have significant similarities at the N- and C-terminal sites on their proteins, both of which are known to be important sites for binding to receptors (Morrison, BW et al., J. Biol. Chem., 267, 11957-11963 (1992). IL-13 has several functions on normal cells, and sometimes has the same function as IL-4. In addition, the subunits of the signaling receptors of the two cytokines acting and acting are similar to each other, but the subunits to which the two cytokines bind are different, so the two cytokines exhibit biological activity in different forms depending on the cell type. Zurawski, SM et al., EMBO J., 12, 2663 (1993). This fact was confirmed by the mutant of IL-4 acting as an antagonist of IL-4, and found to inhibit the proliferative effect on T cells of IL-4 and IL-13. IL-13, like IL-4, regulates inflammation by inhibiting the production of inflammatory cytokines induced by lipopolysaccharide in monocytes (PBMCs) of human peripheral blood. Unlike IL-4, IL-13 regulates inflammation. Working together to increase the synthesis of interferon-γ in large granular lymphocytes (LGL), it not only regulates the immune response but also increases the proliferation of B cells and the expression of CD23 surface antigens and is independent of IL-4 and IgG4 and It plays a wide variety of roles, including inducing the synthesis of IgE.

Therefore, if the mass production of IL-13 having various functions can be usefully used for the clinical treatment of inflammatory diseases such as septic shock, rheumatism and the like, the macronucleus induced by IL-2 It may be possible to apply the function to increase the killer activity of the cell.

Therefore, the present inventors synthesized cDNA by extracting mRNA from human T cells to obtain a large amount of IL-13 protein in Escherichia coli and expressing the IL-13 gene in E. coli by polymerase chain reaction. As a result of finding a very low degree and continuing to improve the research, the 5 'terminal sequence of the IL-13 gene was replaced with E. coli preferred codon without changing the amino acid and expressed in E. coli, resulting in a high expression rate of the IL-13 protein. It was found that it can be produced to complete the present invention.

An object of the present invention can be useful in the field of treatment of inflammatory diseases such as sepsis shock, rheumatism and the like, and the application of the function of IL-13 to increase the killer activity of megakaryocytes induced by IL-2. It is to provide a method for mass production of IL-13 protein.

In order to achieve the above object, the present invention comprises a recombinant IL-13 gene, wherein the 5'-terminal sequence of the polynucleotide encoding the IL-13 protein is transformed into a preferred sequence of E. coli without changing the amino acid, comprising the gene An expression vector and E. coli transformed with the expression vector are provided.

The present invention also provides a method for mass production of IL-13 protein comprising culturing the transformed E. coli in a medium suitable for expression of IL-13, and separating and purifying the produced IL-13.

1 shows the nucleotide sequence and the amino acid sequence of interleukin-13 derived from activated human T cells,

Figure 2 shows the process of cloning the interleukin-13 gene derived from activated human T cells with E. coli expression vector pET IL-13,

Figure 3 is cultured E. coli transformed with the respective expression vectors pET IL-13 and pET RIL-13 containing the interleukin-13 gene and the modified interleukin-13 gene of the present invention, the culture cell lysate is SDS-poly Acrylamide gel is the result of electrophoresis,

Figure 4 shows the procedure for cloning the interleukin-13 gene 5 'terminal sequence modified in accordance with the present invention E. coli expression vector pET RIL-13,

Figure 5 compares the 5 'terminal sequence of the native interleukin-13 gene derived from activated T cells with the 5' terminal sequence of the modified interleukin-13 gene of the present invention.

Hereinafter, the present invention will be described in detail.

First, human T cells are isolated, treated with Con-A, activated, RNA is extracted therefrom, and cDNA is synthesized by reverse transcriptase using oligo d (T) primer.

Meanwhile, the nucleotide sequence information of IL-13 (McKenzie, AN et al., Proc. Natl. Acad. USA, 90, 3735-3739 (1993)) corresponds to the 5 'and 3' ends of the IL-13 gene. The primers for the polymerase chain reaction (hereinafter, referred to as PCR) are each designed and synthesized. The 5 'primer has a restriction enzyme recognition site to facilitate cloning with an E. coli expression vector, and the corresponding 3' primer inserts an end codon and a restriction enzyme recognition site to synthesize a protein from the start codon and to artificially insert Allow protein synthesis to complete in the codon. IL-13 gene is amplified by PCR using human T cell cDNA synthesized above with these primers and templates.

The gene fragment obtained by cutting the amplified IL-13 gene with the restriction enzyme was replaced with the UB A545 gene of E. coli expression vector pETUB A545 (KCTC 0151BP), and the E. coli expression vector pET IL-13 containing the IL-13 gene. To produce.

On the other hand, a random primer for PCR for the 5 'end of the IL-13 gene is designed and synthesized. The random primer is designed to randomly modify only the base sequence without changing the amino acid sequence and to clone into an E. coli expression vector. Restriction enzyme recognition sites are inserted for ease. The 3 'primer and the random primer prepared above were used, and the expression vector pET IL-13 was used as a template to amplify a gene whose base sequence at the 5' end of IL-13 was modified by PCR. Gene fragment obtained by cleaving the IL-13 gene modified with the amplified 5 'end with a restriction enzyme was replaced with an appropriate E. coli expression vector, for example, the UB A545 gene of pETUB A545 (KCTC 0151BP), which is an E. coli expression vector. E. coli expression vector pET RIL-13 containing the modified IL-13 gene was constructed.

The IL-13 expression vector thus prepared is used to transform a suitable host microorganism, such as Escherichia coli strain BL21 (DE3) / pLysS. This transformed E. coli strain is cultured using a medium suitable for the expression of IL-13, such as empicillin and chloramphenicol-containing liquid luria medium, followed by mass culture using M9 medium. IL-13 expressed and secreted in the culture solution can be obtained in large quantities through an appropriate purification process.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are merely to illustrate the present invention, the present invention is not limited to these.

Preparation Example Amplification of the IL-13 Gene and Preparation of an E. Coli Expression Vector Containing the Same

(Step 1) cDNA synthesis from activated T cells

T cells were isolated from human blood and treated with Con-A (5 μg / ml) for 16 hours to activate the cells, followed by Korozinski et al. (Cholozynski, P. et al., Anal. Biochem., 162). , 156 (1987)), RNA was extracted as follows. 1 ml of RNA extract (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sacosyl, 0.1 M 2-mercaptoethanol) was added to 5x10 ⁶ activated T cells to disrupt the cell membrane. . To the cell lysate, 0.8 ml of 2 M sodium acetate (pH 4.0), 0.8 ml of phenol and 1.6 ml of chloroform-isoamyl alcohol (49: 1, v / v) were added and mixed well, followed by 12,000 g at 4 ° C. Centrifuged for 15 minutes. The aqueous solution of the upper layer was transferred to a new vessel containing isopropanol of eastern blood, left for about 1 hour at -20 ℃ and centrifuged for 20 minutes at 12,000 g at 4 ℃ to obtain an RNA precipitate. The precipitate was washed with 75% ethanol and then dried in vacuo and dissolved in 20 μl of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). To synthesize single stranded cDNA, 2 μl of 0.1 M methylmercury ((CH ₃ ) ₂ Hg) was added to 10 μl of the RNA solution, and the mixture was left at room temperature for 10 minutes to release the secondary structure of RNA and 2 μl of 1M 2. Mercaptoethanol was added and reacted for 5 minutes. 5 μl of 10-fold concentrated reverse transcriptase reaction solution (500 mM Tris-HCl, pH 8.3, 750 mM KCl, 30 mM MgCl ₂ , 10 mM dithiothreitol), 2.5 μl of 10 mM dNTP mixture (dGTP, dATP) , dTTP, dCTP each 10 mM), 24 μl diethylpyrocarbonate (DEPC) treated distilled water, 1.0 μl RNA degrading enzyme inhibitor (RNasin, 1 U / μl, Promega, USA), 1 μl oligo (dT) _12-18 primer (oligo (dT) _12-18 primer, Gibco BRL, USA) was added and allowed to stand at room temperature for 10 minutes to allow RNA template and primer to be well conjugated, followed by 2.5 μl reverse transcriptase (18 U / μl , Superscript RNase H-reverse transcriptase, BRL, USA) was added and reacted at 37 ° C. for 1 hour to synthesize human T cell cDNA strands.

(Step 2) Synthesis of the primer

To amplify the IL-13 gene by PCR from the cDNA prepared in step 1, the following primers were synthesized using a nucleic acid synthesizer (DNA Synthesizer, Applied Biosystems Inc., USA).

The primer NDEIL-13 [5'-GCCTTCATATGGGCCCTGTGCCTCCCTCTACAG-3 '] was designed to have a restriction enzyme NdeI recognition site (underlined) and to have a nucleotide sequence encoding the first to seventh amino acids of IL-13;

The primer IL-13L [5'-TTCTTGTCGACTTATCAGTTGAACCGTCCCTCGCGAA-3 '] was designed to have a restriction enzyme SalI recognition site (underlined) and an end codon of IL-13.

(Step 3) Amplification of the IL-13 Gene

To amplify the IL-13 gene, polymerase chain reaction was performed as follows. 2 μg of the primer NDEIL-13 obtained in step 2 and 2 μg of the primer IL-13L were added to the reaction tube, and 1 ng of activated human T cell cDNA obtained in step 1 was used as a template, followed by 10 μl of 10-fold polymerization buffer. Solution (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100, 2.5 mM MgCl ₂ ), 10 μl of 2 mM dNTP (2 mM dGTP, 2 mM dATP, 2 mM dCTP, 2 mM) dTTP), 2.5 units of Taq polymerase was added and distilled water was added to make the total volume 100 μl, followed by 1 minute at 95 ° C. (denature step), 40 seconds at 55 ° C. (immersion step), and 2 minutes at 72 ° C. (polymerization). PCR was performed 40 times under the conditions of step). PCR products obtained therefrom were separated from the 7% acrylamide gel, it was confirmed that the nucleic acid of about 340 base pairs was amplified, and purified from acrylamide gel under the same conditions. This nucleic acid fragment is hereinafter referred to as 'fragment IL-13'.

(Step 4) Gene Sequence Confirmation

The nucleotide sequence of the amplified gene was performed as follows by Sanger et al. (Sanger, F., PANS U.S.A., 74, 5463 (1977)).

First, 1 μg of the amplified 'fragment IL-13' was treated with Klenow under conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.9). A smooth end was prepared by treatment with a polymerase, which was then extracted with phenol / chloroform and dissolved in 20 µl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) solution. This is called 'fragment IL-13K'.

Meanwhile, 2 μg of plasmid M13mp18 was completely cleaved with restriction enzyme Smal under the conditions of NEB buffer 2, and then separated from the 0.7% agarose gel to isolate and purify the nucleic acid fragment of about 7 kb. This nucleic acid fragment is hereinafter referred to as "fragment M13-SM".

Subsequently, 100 ng of 'fragment IL-13K' and 100 ng of 'fragment M13-SM' were placed in a conjugation vessel, and 2 μl of 10-fold conjugation reaction solution (500 mM Tris-HCl, 100 mM MgCl ₂ , 100 mM dithio). Tethanol, 10 mM ATP, 250 μg / ml bovine serum albumin, pH 7.8) and 10 units of T4 DNA ligase were added, and distilled water was added to a total volume of 20 μl and reacted at 16 ° C. for 12 hours. Upon completion of the reaction, it was transformed into E. coli JM 105 (ATCC 47016) and transformed into 8 μl of 0.2 M IPTG (isopropyl-β-D-thiogalactopyranoside, USB cat. No. 17884, USA). 100 μl of pre-cultured helper cells (E. coli JM 105), 3 ml of 2XYT supernatant agar (soft agar, 16 g bactotrypton per liter, 10 g yeast extract, 5 g bactoagar) and 25 Mix the μL 4% X-gal solution and place a minimum plate A (min A plate, 10.5 g K ₂ HPO ₄ per 1 L, 1 g (NH ₄ ) ₂ SO ₄ , 0.5 g sodium citrate, 12 g Bactoa was applied evenly over 1 ml 20% MgSO ₄ , 0.5 ml 1% Vit. B, 10 ml 50% glucose). Transparent plaques produced by incubating at 37 ° C. for 12 hours were picked with a toothpick, incubated at 37 ° C. for 5 hours in 2 ml of 2XYT liquid medium, and centrifuged. Into the obtained supernatant, 1/4 volume of PEG / NaCl (20% florethylene glycol / 2.5 M sodium chloride) was added, M13 phages were collected, single-stranded nucleic acid was extracted, and the nucleotide sequence was analyzed. It was confirmed to have. Figure 1 shows the nucleotide sequence and amino acid sequence of the IL-13 gene.

(Step 5) Preparation of E. coli expression vector containing IL-13 gene

2 μg of plasmid pETUB A545 (Korean Patent Application No. 96-5096, KCTC 0151BP) was completely cleaved with restriction enzymes NdeI and SalI under conditions of NEB buffer 2, followed by electrophoresis on 1% agarose gel and subjected to 4.5 kb. Nucleic acid fragments were obtained. Hereinafter, this nucleic acid fragment is referred to as "fragment pET-N / L".

Meanwhile, 2 μg of 'fragment IL-13' obtained in step 3 was completely digested with restriction enzymes NdeI and SalI under the conditions of restriction enzyme NEB buffer solution 2, followed by electrophoresis separation with 7% acrylamide gel, followed by nucleic acid of about 340 base pairs. Obtained intercept. Hereinafter, this nucleic acid fragment is referred to as "fragment IL-13-N / L".

Put 100 ng of fragment IL-13-N / L and 100 ng of fragment pET-N / L into the conjugation vessel and add 2 μl of 10-fold conjugation solution and 10 units of T4 DNA ligase. Distilled water was added to a total volume of 20 μl and reacted at 16 ° C. for 12 hours. After the conjugation reaction, E. coli HB 101 (ATCC 33694) was transformed, transformed cells were selected on a solid Luria medium plate containing 50 μg / ml of empicillin, and plasmids were extracted therefrom to restriction enzymes and nucleotide sequences. The E. coli expression vector pET IL-13 containing the IL-13 gene was obtained by analysis. Figure 2 shows the manufacturing process of the vector for expressing the IL-1 gene in E. coli.

The plasmid pET IL-13 was transformed into an expression host E. coli BL21 (DE3) / pLysS (Novagen Inc., 53711 Madison, Wisconsin, USA).

(Step 6) Expression of IL-13 Gene in Escherichia Coli

Recombinant Escherichia coli cells obtained in step 5 were prepared using liquid Luria medium (6% bactotryptone, 0.5% yeast extract) containing 50 μg / ml of ampicillin and 25 μg / ml of chloramphenicol. 1% sodium chloride) was shaken for 12 hours. 3 ml of the culture was again added to 300 ml of M9 medium (40 mM K ₂ HP0 ₄ , 22 mM KH ₂ PO ₄ , 8.3 mM NaCl, 18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.4 % Casamino acid, 10 μg / ml Vit.B ₁ , 40 μg / ml empicillin, 25 μg / ml chloramphenicol) and shake incubated at 37 ° C. for about 4 hours so that the absorbance of the culture is about 0.3 at 650 nm. IPTG was added at a final concentration of 0.4 mM. About 4 hours after the addition of IPTG, the absorbance of the cell culture was measured, and E. coli cell precipitates were collected by centrifugation at 11,000 rpm for 25 minutes using a centrifuge (Beckman J2-21, JA14 rotor). The collected cell precipitates were electrophoresed on 12% polyacrylamide gels in the presence of SDS according to Lamley's method (Laemmli, Nature, 227, 680 (1970)). The results are shown in column C of FIG. 3, where it can be seen that the expression of IL-13 is very small.

<Example> Amplification and Expression of IL-13 Gene Having Modified Nucleotide Sequence at 5 ′ End

(Step 1) cDNA synthesis from activated T cells

CDNA was synthesized in the same manner as in Step 1 of Preparation.

(Step 2) Synthesis of the primer

Primer NRIL-13 [5'-GGCCGCATATGGGNCCNGTNCCNCCNTCNACNGCNCTNAG (A / G) GA (A / G) CTNAT (A / C / T) GA (A / G) GA (A / G) CTNGTNAA (C / T) ATCACCCAGAACCAGAAGGCT-3 ' ] Had a restriction enzyme NdeI recognition site (underlined) and randomly modified the nucleotide sequences encoding the 1st to 19th amino acids of IL-13 with no amino acid changes. It was designed to match the nucleotide sequence encoding the 26th amino acid.

(Step 3) Amplification of IL-13 gene having a modified nucleotide sequence at the 5 'end

2 μg of the primer NRIL-13 and 2 μg of the primer IL-13L obtained in the reaction vessel were added, and then 1 ng of the plasmid pET IL-13 obtained in step 5 of Preparation Example was added as a template, and 10 μl of 10-fold polymerization buffer solution ( 500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100, 2.5 mM MgCl ₂ ), 10 μl of 2 mM dNTP (2 mM dGTP, 2 mM dATP, 2 mM dCTP, 2 mM dTTP) After adding 2.5 units of Taq polymerase, distilled water was added to make the total volume 100 μl, followed by 1 minute at 95 ° C. (denature step), 55 seconds at 40 ° C. (immersion step), and 72 minutes at 2 ° C. (polymerization step). PCR was performed 25 times under the conditions of). PCR products obtained therefrom were separated from the 7% acrylamide gel was confirmed that the nucleic acid of about 340 base pairs were amplified, and purified from the acrylamide gel under the same conditions. This nucleic acid fragment is hereinafter referred to as "fragment RIL-13".

(Step 4) Preparation of an expression vector comprising an IL-13 gene having a modified nucleotide sequence at the 5 'end

2 μg of 'fragment RIL-13' obtained in step 3 was subjected to restriction enzyme NdeI under conditions of NEB buffer solution 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.9). After complete cleavage with SalI and electrophoresis separation with 7% acrylamide gel, nucleic acid fragments of about 340 base pairs were obtained. Hereinafter, this nucleic acid fragment is referred to as "fragment RIL-13-N / L".

Add 100 ng of 'fragment RIL-13-N / L' and 100 ng of 'fragment pET-N / L' to the conjugation vessel and add 2 μl of 10-fold conjugation reaction solution (50 mM Tris-HCl, pH 7.8, 10). mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP, 25 μg / ml bovine serum albumin) and 10 units of T4 DNA ligase were added, and distilled water was added to a total volume of 20 μl and reacted at 16 ° C. for 12 hours. I was. After the conjugation reaction, E. coli HB 101 (ATCC 33694) cells were transformed, transformed cells were selected on a solid Luria medium plate containing 50 µg / ml of empicillin, and plasmids were extracted therefrom to extract restriction enzymes and bases. Sequence analysis confirmed that the E. coli expression vector pET RIL-13 including the IL-13 gene modified at the 5 'end was produced. Figure 4 shows the construction of an E. coli expression vector comprising the IL-13 gene having a modified amino terminal in accordance with the present invention.

The plasmid pET RIL-13 was transformed into an expression host E. coli BL21 (DE3) / pLysS (Novagen Inc.).

(Step 5) Expression of IL-13 Gene Modified at 5 'End in Escherichia Coli

About 200 colonies of the recombinant E. coli obtained in step 4 were prepared in a liquid Luria medium (6% bactotriptone, 0.5% yeast extract, 1% sodium chloride) containing 50 μg / ml of empicillin and 25 μg / ml of chloramphenicol. Shake culture for hours. 3 ml of the culture was again added with 300 ml of M9 to (40 mM K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.3 mM NaCl, 18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.4 % Casamino acid, 10 μg / ml Vit.B ₁ , 40 μg / ml empicillin, 25 μg / ml chloramphenicol) and shake incubated at 37 ° C. for about 4 hours to obtain an absorbance of the culture of about 0.3 at 650 nm. IPTG was added at a final concentration of 0.4 mM. About 4 hours after the addition of IPTG, the absorbance of the cell culture was measured, and E. coli cell precipitates were collected by centrifugation at 11,000 rpm for 25 minutes using a centrifuge (Beckman J2-21, JA14 rotor). The collected cell precipitates were electrophoresed in 12% polyacrylamide gels in the presence of SDS according to the method of Ramley to confirm expression. The electrophoresis results are shown in FIG. 3. In Figure 3, the 19th column is E. coli cell lysate containing the expression vector pET RIL-13 # 19, the remaining columns are E. coli cell lysate containing the expression vector pET RIL-13 of the present invention, but the expression rate is weak, Column C is E. coli cell disruption containing pET IL-13 and column M is a standard molecular weight labeling protein. Here, clones with significantly increased expression compared to IL-13 gene with no amino terminus were selected and named pET RIL-13 # 19. The strain BL21 (DE3) / pLysS pET RIL-13 # 19 containing the expression vector was deposited in KCTC 8777P, Accession No. 29, 1997, to the Genetic Bank of Korea Institute of Science and Technology.

(Step 6) Confirming the base sequence of the IL-13 gene modified 5 'end

From the pET RIL-13 # 19 clone obtained in step 3, the nucleotide sequence of the 5 'end of IL-13 was confirmed by Sanger et al. Figure 5 compares the 5 'terminal sequence of the native IL-13 gene derived from activated T cells with the 5' terminal sequence of the modified IL-13 gene of the present invention.

As described above, when the 5 'terminal sequence of the natural IL-13 gene is transformed into E. coli preferred codon without changing the amino acid according to the present invention, the expression rate is significantly improved than that of the native IL-13. Therefore, according to the production method of the present invention, IL-13 can be mass-produced efficiently, and the mass-produced IL-13 is induced by IL-2 and clinical treatment of inflammatory diseases such as sepsis shock and rheumatism. It can be usefully used in research fields that apply the function of IL-13 to increase the killer activity of megakaryocytes.

Claims (7)

Recombinant interleukin-13 gene wherein the 5 'terminal sequence of the polynucleotide encoding interleukin-13 is modified to the preferred sequence of E. coli without changing the amino acid. The gene according to claim 1, wherein the base sequence at the 5 'end comprises the following sequence: ATG GGT CCA GTG CCT CCG TCA ACC GCG CTA AGA GAA CTG ATC GAA GAG CTC GTA AAT ATC. An expression vector comprising the gene of claim 1. The expression vector pET RIL-13. E. coli transformed with the expression vector of claim 3. 6. Escherichia coli BL21 (DE3) / pLysS pET RIL-13 # 19 (Accession No. KCTC 8777P). A method for producing interleukin-13 protein comprising culturing E. coli of claim 5 in a medium suitable for the expression of interleukin-13 and isolating and purifying the produced IL-13.

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