KR101150263B1 - Human interleukin-11 mutants having improved biological activity - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to human interleukin-11 (IL-11) variants with increased biological activity, wherein the removal and specific sites of the N-terminal portion in wild-type mature human interleukin-11 Interleukin-11 variants with increased biological activity by introducing substitution of amino acid residues, genes encoding them, expression vectors comprising them, microorganisms transformed with the expression vectors, and interleukin-11 variant proteins are produced using the microorganisms It is about how to. The interleukin-11 variant of the present invention has a higher biological activity than recombinant interleukin-11, which is conventionally used as a therapeutic agent, and thus may be usefully used for the prevention and treatment of thrombocytopenia.

Description

HUMAN INTERLEUKIN-11 MUTANTS HAVING IMPROVED BIOLOGICAL ACTIVITY

1 confirms the expression of the human IL-11-2 variant protein of the present invention in E. coli,

Lane 1: molecular weight size marker, lane 2: pGEX-IL11-2, (-) IPTG

Lane 3: pGEX-IL11-2, (+) IPTG

Figure 2 confirms the expression of the human IL-11-3 variant protein of the present invention in E. coli.

Lane 1: molecular weight size marker, lane 2: pGEX-IL11-3, (-) IPTG

Lane 3: pGEX-IL11-3, (+) IPTG

The present invention provides an interleukin-11 variant with increased biological activity by introducing a removal of a portion of the N-terminal residue and substitution of a specific position amino acid residue in a wild-type mature human interleukin-11, a gene encoding the same, an expression vector comprising the same, and the expression vector. And a method for producing interleukin-11 variant protein using the microorganism.

Interleukin-11 (abbreviated as "IL-11") is a cell growth factor that was first discovered in primate bone marrow stromal cells, PU-34 (Paul et al . , Proc. Natl. Acad. Sci. USA 8: 7512, 1990). The IL-11 protein is made up of 199 amino acids. When secreted from the cell, the first 21 amino acids are truncated, resulting in a mature protein of 178 amino acids.

IL-11 has a variety of activities that affect the liver, digestive system, lungs, heart, central nervous system, bones, joints, and immune system, including the hematopoietic system. For example, IL-11 interacts with other cytokines such as IL-3, IL-4, IL-7, IL-12, IL-13, SCF, FLT3L, GM-CSF, etc. to inhibit the activity of hematopoietic progenitor cells. By promoting red blood cells or leukocyte production (Quesniaux et al., Blood 80: 1218, 1992; Hirayama et al . , Proc. Natl. Acad. Sci. USA 89: 5907, 1992; Cairo et al . , Pediatr. Res. 34: 56, 1993). In particular, IL-11 is known to be able to increase the production of megakaryocytes and improve platelet levels in peripheral blood, and has been used as a treatment for thrombocytopenia (Bruno et al . , Exp. Hematol. 19: 378, 1991; Tsukada et al., Blood 81: 866, 1993). In addition to hematopoiesis, IL-11 has been reported to promote repair of damaged digestive organs, stimulate osteoclasts to induce bone resorption, and increase neuronal proliferation (Du et al., Blood 83: 33, 1994; Hughes et al. , Calcif. Tissue Int . 53: 362, 1993; Du et al., J. Cell Physiol . 16: 3300, 1996).

Recombinant human IL-11 is currently marketed under the brand name Newmega (Nemega, Genetics Institute) as a biological agent for preventing or treating thrombocytopenia occurring in chemotherapy cancer patients with FDA approval in 1997. However, the new mega products are known to cause various side effects such as edema, dyspnea, conjunctival hyperemia, arrhythmia, and their use is limited.

Accordingly, the present inventors have made intensive studies to develop IL-11 variants with increased biological activity without causing various side effects previously found in recombinant human IL-11. The present invention has been accomplished by introducing mutations through removal of some and substitution of specific amino acid residues to develop IL-11 variants with improved toxicity and low toxicity.

Accordingly, it is an object of the present invention to provide an IL-11 variant which can be usefully used as a preventive and therapeutic agent for thrombocytopenia due to its high biological activity without causing side effects or toxicity to the human body.

In order to achieve the above object, the present invention provides IL-11 variants with increased biological activity by the introduction of the removal of the N-terminal part and substitution of specific amino acid residues in wild-type mature IL-11.

The present invention also provides a gene encoding the IL-11 variant.

In addition, the present invention provides an expression vector comprising the gene and a microorganism transformed with the expression vector.

Finally, the present invention provides a method for producing IL-11 variant protein using the transgenic microorganism.

Hereinafter, the present invention will be described in detail.

The present invention provides IL-11 variants with increased biological activity by removing a portion of the N-terminal part of the wild-type mature IL-11 and replacing a amino acid residue at a specific position with another amino acid to introduce a mutation.

Specifically, the IL-11 variant in which the mutations are introduced in accordance with the present invention is characterized by the presence of nine N-terminal amino acid residues in the amino acid sequence of known wild type mature IL-11 (Ohsumi et al., FEBS Lett . 288: 13-6, 1991). The 10th amino acid residue, valine (Val), was replaced with alanine (Ala). The IL-11 variant into which the mutation has been introduced has an amino acid sequence set forth in SEQ ID NO: 1 , which is preferably encoded by a gene having a nucleotide sequence set forth in SEQ ID NO: 2 .

In addition, the IL-11 variant according to the present invention may further include a substitution of aspartic acid (Asp), aspartic acid (Asp), asparagine (Asn) in the amino acid sequence set forth in SEQ ID NO: 1 above. The IL-11 variant to which the mutation is further introduced has an amino acid sequence as set out in SEQ ID NO: 3 , which is preferably encoded from a gene having a nucleotide sequence as set out in SEQ ID NO: 4 .

A summary of IL-11 variants prepared according to the present invention is shown in Table 1 below.

There are several prolines (Pro) at the N-terminus of wild type IL-11, which generally interferes with the expression and purification of the protein. In the variant of the present invention, nine amino acids, including proline residues, are removed from the N-terminus so that the protein can be expressed and purified very efficiently.

In addition, the variant of the present invention has the advantage that the biological activity is increased by the substitution of alanine (Ala) in valin (Val) 10th amino acid. On the other hand, when IL-11 is placed in an acidic solution, it is cleaved between 134th amino acid aspartic acid (Asp) and 135th amino acid proline (Kenley RA and Warne NW, Pharm Res . 11: 72, 1994), IL of the present invention In the -11 variant, the 134th aspartic acid has a similar structure, but is substituted with asparagine (Asn), which has a different functional group, so that the biological activity can be maintained more stably without being cleaved even in an acidic solution. Gene mutations for the production of the IL-11 variant of the present invention can be produced by site-directed mutagenesis, but in addition to the known method used for gene mutations in consideration of codon bias of the host cell. You can.

In addition, according to the present invention, the IL-11 variant into which the mutation is introduced and the gene encoding the same may be synthesized according to a known DNA or peptide synthesis method. In addition, the thus prepared gene may be inserted into a microorganism expression vector known in the art to prepare an expression vector and then introduced into an appropriate host cell, for example, E. coli or yeast cell.

As a known microorganism expression vector usable in the present invention, pGEX4T-1 (Amersham Biosciences, USA) or pRSET (Invitrogen, The Netherlands) can be exemplified. -S-transferase (GST) is a vector configured to be expressed in a fused form, the GST gene immediately after the tac promoter, multi-cloning site (MCS) so that foreign genes can be inserted after it ) Exists. The GST fusion system consists of a pGEX plasmid vector, a GST purification module, and a GST detection module. This configuration not only expresses a large amount of foreign genes in E. coli, but also expresses foreign genes in the form of fusion proteins with GST. Therefore, foreign gene products can be separated easily and simply by using GST purification module and detection module. In addition, since pRSET contains a T7 promoter site that regulates the expression of foreign genes and has six histidine-tags, it enables partial purification of the recombinant protein produced after expression, thereby allowing for desired recombinant protein. There is an advantage that can be purified quickly with high efficiency.

In the production of the vector, expression control sequences such as promoters and terminators, self-replicating sequences, and secretion signals may be appropriately selected and combined according to the type of the host cells to produce the IL-11 variant or the gene encoding the same. .

Using a transformant into which an expression vector containing the IL-11 variant coding gene prepared as described above is introduced, DNA of the IL-11 variant gene of the present invention can be replicated in large quantities or a large amount of IL-11 variant protein can be produced. . For example, in a preferred embodiment of the present invention, the E. coli BL21 is transformed after inserting the IL-11 variant coding gene of the present invention into the expression vector pGEX4T-1 and transformed into E. coli BL21 / pGEX-hIL-. On March 24, 2005, 11-3 was deposited with the Korea Culture Center of Microorganisms (KCCM) as Accession No. KCCM 10649P.

The IL-11 variant protein expressed from the E. coli transformant can be purified using affinity binding chromatography, preferably column chromatography using affinity with GST or histidine residues present in the fusion protein. The purified fusion protein is human IL having a variant amino acid sequence by cleaving with a suitable protease, preferably thrombin, factor X or enterokinase, which recognizes a recognition site present in the vector. -11 variant proteins can be produced in large quantities.

As described above, the human IL-11 mutant protein produced in large quantities from E. coli transformants has increased biological activity, so that the same therapeutic effect can be obtained even when using a smaller amount than the conventional recombinant human IL-11. The toxicity can be reduced, making it more suitable for use as a therapeutic.

Accordingly, the present invention provides a pharmaceutical agent having a prophylactic and therapeutic effect of thrombocytopenia prepared by mixing the human IL-11 mutant protein as an active ingredient and mixing or diluting with a suitable pharmaceutically acceptable carrier or excipient according to a conventional method. Composition. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. The pharmaceutical compositions of the invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of the human IL-11 variant of the invention range from 1 to 1000 μg / kg body weight, preferably 3 to 100 μg / kg body weight, and can be administered once or in several doses. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore the dosage should in any way be regarded as the present invention. It does not limit the scope of.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Comparative Example Isolation, Purification and Activity Measurement of IL-11-1 Variants

<1-1> IL-11-1 Variant Preparation

Human HepG2 cells (ATCC HB 8065) to prepare variants in which the N-terminal seven amino acids were removed from a known wild type mature human IL-11 amino acid sequence (Ohsumi et al., FEBS Lett . 288: 13-6, 1991). After isolation of RNA at RT-PCR was performed to obtain the IL-11 variant cDNA with seven N-terminal amino acid residues removed. Specifically, RNA was isolated from HepG2 cells using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions, and then reacted for 5 minutes in an ice bath for 5 minutes at 65 ℃ 5 ㎍ RNA. According to the manufacturer's instructions, an appropriate amount of enzyme and reagent of the first strand cDNA synthesis kit (RT-PCR, Roche, IN, USA) for RT-PCR was added to the RNF. The mixture was reacted at 25 ° C. for 10 minutes, at 42 ° C. for 60 minutes, at 99 ° C. for 5 minutes, and at 4 ° C. for 5 minutes to synthesize cDNA. 5 μl of the obtained cDNA, 5 μl of 2.5 mM dNTP mixture, 5 μl of PCR buffer, 0.5 μl of Taq polymerase (High Fidelity PCR System, Roche), 10 pmol / μl of SEQ ID NO: 5 forward primer IL11-PR-5 and 2 μl of reverse primer IL11-full-3 of SEQ ID NO: 6 and 30.5 μl of distilled water were mixed, followed by PCR reactor (PCR thermal cycler MP, model TP3000, TaKaRa) for 1 minute at 94 ° C and 1 ° C at 55 ° C. The reaction for 1 minute at min and 72 ° C. was repeated 40 times. At this time, the forward primer IL11-PR-5 of SEQ ID NO: 5 used for PCR was designed to include a BamHI recognition site and the reverse primer IL11-full-3 of SEQ ID NO: 6 to include a SalI recognition site.

The cDNA obtained therefrom was cloned into pBluescript SK vector (Stratagene, USA) and subjected to sequencing to confirm the nucleotide sequence of the IL-11 variant gene from which 7 N-terminal amino acids were removed. The IL-11 variant gene was digested with BamHI and SalI and inserted into the same BamHI / SalI site of pGEX4T-1 (Amersham Biosciences, USA), a glutathione S-transferase (GST) binding protein expression vector. To prepare plasmid pGEX-hIL-11-1. As such, the variant from which the seven amino acid terminus was removed from the wild-type mature IL-11 was named an IL-11-1 variant, and the variant has an amino acid sequence set forth in SEQ ID NO: 7 .

<1-2> Selection of IL-11-1 Variant Expressing Transformants

Transform the E. coli BL21 (Novagen, USA) with the plasmid pGEX-hIL-11-1 comprising the IL-11-1 variant from the seven N-terminal amino acids prepared in Example <1-1> After inoculation into the LB liquid medium and incubated at 37 ℃ until the OD ₆₀₀ reached 0.5. When the desired absorbance was reached, IPTG (isopropyl-β-o-thiogalactopyranoside) was added to the culture solution to a final concentration of 1 mM and further incubated for 3 hours. The E. coli cultures were recovered by centrifugation, and then subjected to SDS-PAGE to select E. coli transformants that were confirmed to express GST-IL11-1 variant fusion protein.

<1-3> Purification of the IL-11-1 Variant Protein

In order to produce and purify the IL-11-1 variant protein from the E. coli transformant selected in Example <1-2>, the following procedure was performed.

1) E. coli crushing

E. coli transformants were cultured in LB liquid medium and centrifuged to E. coli cells obtained by adding a buffer solution (25 mM Tris, pH 8.0) to prepare a cell suspension. The supernatant was separated from the supernatant by pulverizing E. coli by treating the suspension with ultrasonic waves.

2) Glutathione agarose chromatography

The protein expressed from the E. coli transformant has a property of being adsorbed to glutathione agarose resin because GST protein is coupled to the N-terminus. In order to purify the fusion protein by affinity column chromatography using the same, the glutathione agarose column (Sigma) was equilibrated with 25 mM Tris-HCl (pH 8.0) and the supernatant prepared in step 1) was flowed. The column was washed with 25 mM Tris (pH 8.0) and then again eluted (25 mM Tris-HCl, 150 mM NaCl, 10 mM reduced glutathione, pH 8.0) to obtain GST-IL11-1 variant. The fusion protein was eluted.

3) thrombin treatment

In order to separate only the IL-11-1 variant protein from which the GST part was removed from the GST-IL11-1 variant fusion protein, the thrombin site between the GST protein part and the IL-11-1 variant protein part was cleaved as follows. Thrombin was treated. Ten units of thrombin per mg of GST-IL11-1 variant fusion protein were added and reacted at 25 ° C. for 1 hour. 12% SDS-PAGE was performed to confirm that the GST moiety and the IL-11-1 variant protein moiety were separated. The thrombin-treated protein solution was passed through a glutathione agarose column and received a flow-through that was not adsorbed to the resin to recover only the IL-11-1 variant protein from which the GST portion was removed.

4) Cation Exchange Chromatography

After diluting the passage solution containing the IL-11-1 variant protein obtained by the thrombin treatment with 20 mM phosphate buffer (pH 7.0), the CM Sepharose Fast Flow column (Amersham Biosciences) )). After washing the column with 20 mM phosphate buffer (pH 7.0), 20 mM phosphate buffer (containing 0.25 M NaCl, pH 7.0) was flowed to obtain the IL-11-1 variant protein.

5) Anion Exchange Chromatography

The IL-11-1 variant protein solution obtained by cation exchange chromatography was diluted three-fold with 20 mM phosphate buffer (pH 7.0) and then loaded onto a DEAE Sepharose Fast Flow column (Amersham Biosciences). It was. IL-11 protein was not adsorbed to the anion exchange resin and flowed out through the flow.

6) Purity Check

The purity of IL-11-1 variant protein purified as described above was confirmed by 12% SDS-PAGE and reversed phase HPLC (Hypersil C ₄ column), and it was confirmed that the purity was 95% or more.

Example 1 Preparation and Activity Measurement of IL-11-2 Variants

<1-1> IL-11-2 Variant Preparation

The plasmid pGEX-hIL-11-1 produced in Comparative Example as a template and SEQ ID NO: 8 in the hIL-11 (V_A) -1, and SEQ ID NO: 9 of the hIL-11 (V_A) region using primer pair -2 Site-directed mutagenesis was performed to replace the 10th amino acid Val in the amino acid sequence of known wild type mature IL-11 with Ala to prepare the IL-11-2 variant. The prepared IL-11-2 variant gene has seven N-terminal amino acid residues removed from the base sequence, but after thrombin cleavage during protein production, nine N-terminal amino acid residues are removed. Sequencing of the amino acid residues was confirmed by sequencing, and the IL-11-2 variant in which 9 N-terminal amino acid residues were removed and Val, the 10th amino acid of wild-type mature IL-11, was substituted with Ala was SEQ ID NO: It was confirmed that it had an amino acid sequence described as 1 . The gene encoding the IL-11-2 variant of SEQ ID NO: 1 has a nucleotide sequence represented by SEQ ID NO: 2 , and the plasmid containing this gene was named pGEX-hIL-11-2.

<1-2> Selection of IL-11-2 Variant Expressing Transformants

E. coli BL21 with plasmid pGEX-hIL-11-2 comprising an IL-11-2 variant in which the nine N-terminal amino acids prepared in Example <1-1> were removed and the tenth amino acid was substituted with Ala (Novagen) was transformed and inoculated into LB liquid medium and incubated at 37 ° C until OD ₆₀₀ reached 0.5. When the desired absorbance was reached, IPTG was added to the culture to a final concentration of 1 mM and further incubated for 3 hours. The cultured E. coli was recovered by centrifugation, and then subjected to 12% SDS-PAGE to select E. coli transformants which confirmed the expression of the GST-IL11-2 variant fusion protein ( FIG. 1 ).

<1-3> Purification of the IL-11-2 Variant Protein

The E. coli transformant expressing the GST-IL11-2 variant fusion protein was disrupted in the same manner as in <1-3> of the comparative example, and the GST-IL11-2 variant fusion protein was isolated from the lysate by glutathione agarose chromatography. After that, it was again thrombin-treated and subjected to cation exchange chromatography and anion exchange chromatography to purify only the IL-11-2 variant protein from which GST was removed.

The purity of the thus purified IL-11-2 variant protein was confirmed by SDS-PAGE and HPLC.

Example 2 Preparation and Activity Measurement of IL-11-3 Variants

<2-1> IL-11-3 Variant Preparation

Example plasmid pGEX-hIL-11-2 obtained in the mold 1 and SEQ ID NO: 10 of hIL-11 (D_N) and -1 of SEQ ID NO: 11 of hIL-11 (D_N) region using primer pair -2 Designated mutagenesis was further performed to prepare an IL-11-3 variant in which Asp, the 134th amino acid, was substituted with Asn in the amino acid sequence of wild-type mature IL-11. Sequencing of the amino acid residues was confirmed by sequencing, and nine N-terminal amino acid residues were removed, and IL- with 10 and 134 amino acids Val and Asp substituted with Ala and Asn, respectively, of wild-type mature IL-11. It was confirmed that the 11-3 variant had the amino acid sequence set forth in SEQ ID NO: 3 . The gene encoding the IL-11-3 variant of SEQ ID NO: 3 has a nucleotide sequence set forth in SEQ ID NO: 4 , and the plasmid containing the gene was named pGEX-hIL-11-3.

<2-2> Selection of IL-11-3 Variant Expressing Transformants

Plasmid pGEX-hIL-11- containing an IL-11-3 variant in which the nine N-terminal amino acids prepared in Example <2-1> were removed and the 10th and 134th amino acids were substituted with Ala and Asn, respectively E. coli BL21 (Novagen) was transformed into 3 and then inoculated in LB liquid medium and incubated at 37 ° C until OD ₆₀₀ reached 0.5. When the desired absorbance was reached, IPTG was added to the culture to a final concentration of 1 mM and further incubated for 3 hours. The cultured E. coli was recovered by centrifugation, and then subjected to 12% SDS-PAGE to select E. coli transformants, which confirmed the expression of the GST-IL11-3 variant fusion protein ( FIG. 2 ).

The Escherichia coli transformant BL21 / pGEX-hIL-11-3 selected therefrom was deposited on March 24, 2005 as Korea Accession No. KCCM 10649P to Korea Culture Center of Microorganisms (KCCM).

<2-3> Purification of the IL-11-3 Variant Protein

The E. coli transformant expressing the GST-IL11-3 variant fusion protein was disrupted in the same manner as in <1-3> of the comparative example, and the GST-IL11-3 variant fusion protein was isolated from the lysate via glutathione agarose chromatography. After that, it was again thrombined and subjected to cation exchange chromatography and anion exchange chromatography to purify only the IL-11-3 variant protein from which GST was removed.

The purity of the thus purified IL-11-3 variant protein was confirmed by SDS-PAGE and HPLC.

Example 3 Confirmation of Biological Activity of IL-11 Variant Protein

In vitro activity of the IL-11 variant proteins prepared in Comparative Examples and Examples 1 and 2 was measured in B9-11 cells (Lu, ZY, et al. , J. Immunol. Methods 173: 19-26, 1994). It was. Specifically, 1 × 10 ⁴ B9-11 cells were plated in a 96-well plate and 10 μl of 0.1, 1, 10. 10 ² , 10 ³ , 10 ⁴ and 10 ⁵ ng / ml IL-11 variant protein was added. Added and incubated for 3 days. Thereafter, 50 µl of XTT solution was added to each well and reacted for 4 hours, and then the absorbance was measured at 490 nm with an ELISA reader. At this time, a commercial recombinant human IL-11 (New Mega, Genetics Institute) was used as a control, the results are shown in Table 2 below.

As a result, the activity of the control Newmega was measured at 6.2 × 10 ⁶ units / mg, whereas the IL-11-2 and IL-11-3 variants were 8.8 × 10 ⁶ units / mg and 8.6 × 10 ⁶ units / mg, respectively. Mg showed higher biological activity than the control. However, IL-11-1 variant showed lower activity than the control group at 5.3 × 10 ⁶ units / mg.

From this, the IL-11-2 and IL-11-3 variants according to the present invention can be used as a novel thrombocytopenia treatment agent that is expected to have an excellent therapeutic effect due to its high biological activity in place of the existing recombinant human IL-11 New Mega. It was confirmed that there is.

As described above, the IL-11 variant according to the present invention can be used as a prophylactic and therapeutic agent of thrombocytopenia because the high biological activity can be expected to have excellent therapeutic effect even if a small amount is used and the toxicity can be reduced as the dosage is reduced. Can be.

<110> VIROMED CO., LTD. <120> HUMAN INTERLEUKIN-11 MUTANTS HAVING IMPROVED BIOLOGICAL ACTIVITY <130> FPD / 200503-0061 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> IL-11-2 mutant <400> 1 Ala Ser Pro Asp Pro Arg Ala Glu Leu Asp Ser Thr Val Leu Leu Thr   1 5 10 15 Arg Ser Leu Leu Ala Asp Thr Arg Gln Leu Ala Ala Gln Leu Arg Asp              20 25 30 Lys Phe Pro Ala Asp Gly Asp His Asn Leu Asp Ser Leu Pro Thr Leu          35 40 45 Ala Met Ser Ala Gly Ala Leu Gly Ala Leu Gln Leu Pro Gly Val Leu      50 55 60 Thr Arg Leu Arg Ala Asp Leu Leu Ser Tyr Leu Arg His Val Gln Trp  65 70 75 80 Leu Arg Arg Ala Gly Gly Ser Ser Leu Lys Thr Leu Glu Pro Glu Leu                  85 90 95 Gly Thr Leu Gln Ala Arg Leu Asp Arg Leu Leu Arg Arg Leu Gln Leu             100 105 110 Leu Met Ser Arg Leu Ala Leu Pro Gln Pro Pro Pro Asp Pro Pro Ala         115 120 125 Pro Pro Leu Ala Pro Pro Ser Ser Ala Trp Gly Gly Ile Arg Ala Ala     130 135 140 His Ala Ile Leu Gly Gly Leu His Leu Thr Leu Asp Trp Ala Val Arg 145 150 155 160 Gly Leu Leu Leu Leu Lys Thr Arg Leu                 165 <210> 2 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> IL-11-2 mutant <400> 2 gcttccccag accctcgggc cgagctggac agcaccgtgc tcctgacccg ctctctcctg 60 gcggacacgc ggcagctggc tgcacagctg agggacaaat tcccagctga cggggaccac 120 aacctggatt ccctgcccac cctggccatg agtgcggggg cactgggagc tctacagctc 180 ccaggtgtgc tgacaaggct gcgagcggac ctactgtcct acctgcggca cgtgcagtgg 240 ctgcgccggg caggtggctc ttccctgaag accctggagc ccgagctggg caccctgcag 300 gcccgactgg accggctgct gcgccggctg cagctcctga tgtcccgcct ggccctgccc 360 cagccacccc cggacccgcc ggcgcccccg ctggcgcccc cctcctcagc ctgggggggc 420 atcagggccg cccacgccat cctggggggg ctgcacctga cacttgactg ggccgtgagg 480 gggctactgc tgctgaagac tcggctgtga 510 <210> 3 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> IL-11-3 mutant <400> 3 Ala Ser Pro Asp Pro Arg Ala Glu Leu Asp Ser Thr Val Leu Leu Thr   1 5 10 15 Arg Ser Leu Leu Ala Asp Thr Arg Gln Leu Ala Ala Gln Leu Arg Asp              20 25 30 Lys Phe Pro Ala Asp Gly Asp His Asn Leu Asp Ser Leu Pro Thr Leu          35 40 45 Ala Met Ser Ala Gly Ala Leu Gly Ala Leu Gln Leu Pro Gly Val Leu      50 55 60 Thr Arg Leu Arg Ala Asp Leu Leu Ser Tyr Leu Arg His Val Gln Trp  65 70 75 80 Leu Arg Arg Ala Gly Gly Ser Ser Leu Lys Thr Leu Glu Pro Glu Leu                  85 90 95 Gly Thr Leu Gln Ala Arg Leu Asp Arg Leu Leu Arg Arg Leu Gln Leu             100 105 110 Leu Met Ser Arg Leu Ala Leu Pro Gln Pro Pro Pro Asn Pro Pro Ala         115 120 125 Pro Pro Leu Ala Pro Pro Ser Ser Ala Trp Gly Gly Ile Arg Ala Ala     130 135 140 His Ala Ile Leu Gly Gly Leu His Leu Thr Leu Asp Trp Ala Val Arg 145 150 155 160 Gly Leu Leu Leu Leu Lys Thr Arg Leu                 165 <210> 4 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> IL-11-3 mutant <400> 4 gcttccccag accctcgggc cgagctggac agcaccgtgc tcctgacccg ctctctcctg 60 gcggacacgc ggcagctggc tgcacagctg agggacaaat tcccagctga cggggaccac 120 aacctggatt ccctgcccac cctggccatg agtgcggggg cactgggagc tctacagctc 180 ccaggtgtgc tgacaaggct gcgagcggac ctactgtcct acctgcggca cgtgcagtgg 240 ctgcgccggg caggtggctc ttccctgaag accctggagc ccgagctggg caccctgcag 300 gcccgactgg accggctgct gcgccggctg cagctcctga tgtcccgcct ggccctgccc 360 cagccacccc cgaacccgcc ggcgcccccg ctggcgcccc cctcctcagc ctgggggggc 420 atcagggccg cccacgccat cctggggggg ctgcacctga cacttgactg ggccgtgagg 480 gggctactgc tgctgaagac tcggctgtga 510 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer IL11-PR-5 <400> 5 ggattccctc gagtttcccc agaccct 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer IL11-full-3 <400> 6 gtcgactcac agccgagtct tcagcag 27 <210> 7 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> IL-11-1 mutant <400> 7 Pro Arg Val Ser Pro Asp Pro Arg Ala Glu Leu Asp Ser Thr Val Leu   1 5 10 15 Leu Thr Arg Ser Leu Leu Ala Asp Thr Arg Gln Leu Ala Ala Gln Leu              20 25 30 Arg Asp Lys Phe Pro Ala Asp Gly Asp His Asn Leu Asp Ser Leu Pro          35 40 45 Thr Leu Ala Met Ser Ala Gly Ala Leu Gly Ala Leu Gln Leu Pro Gly      50 55 60 Val Leu Thr Arg Leu Arg Ala Asp Leu Leu Ser Tyr Leu Arg His Val  65 70 75 80 Gln Trp Leu Arg Arg Ala Gly Gly Ser Ser Leu Lys Thr Leu Glu Pro                  85 90 95 Glu Leu Gly Thr Leu Gln Ala Arg Leu Asp Arg Leu Leu Arg Arg Leu             100 105 110 Gln Leu Leu Met Ser Arg Leu Ala Leu Pro Gln Pro Pro Pro Asp Pro         115 120 125 Pro Ala Pro Pro Leu Ala Pro Pro Ser Ser Ala Trp Gly Gly Ile Arg     130 135 140 Ala Ala His Ala Ile Leu Gly Gly Leu His Leu Thr Leu Asp Trp Ala 145 150 155 160 Val Arg Gly Leu Leu Leu Leu Lys Thr Arg Leu                 165 170 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer hIL-11 (V_A) -1 <400> 8 ggatcccctc gagcttcccc agaccct 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer hIL-11 (V_A) -2 <400> 9 agggtctggg gaagctcgag gggatcc 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer hIL-11 (D_N) -1 <400> 10 ccagccaccc ccgaacccgc cggcgcc 27 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer hIL-11 (D_N) -2 <400> 11 ggcgccggcg ggttcggggg tggctgg 27  

Claims (9)

The amino acid of SEQ ID NO: 1 in which wild-type human mature interleukin-11 (IL-11) removes nine N-terminal amino acid residues and replaces the tenth amino acid with alanine (Ala). Human IL-11 variant protein having a sequence. SEQ ID NO: amino acid sequence represented by 1125th amino acid is replaced with asparagine (Asn) of SEQ ID NO: 3 Human IL-11 mutant protein comprising the amino acid sequence as described. A polynucleotide encoding the IL-11 variant protein of claim 1. The method of claim 3, A polynucleotide having the nucleotide sequence set forth in SEQ ID NO : 2 or SEQ ID NO: 4 . An expression vector containing the polynucleotide of claim 3. delete Microorganism transformed with the expression vector of claim 5. delete A pharmaceutical composition for preventing or treating thrombocytopenia, comprising the IL-11 variant protein of claim 1 or 2 as an active ingredient and a pharmaceutically acceptable carrier.

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