TW201815817A - Molecularly altered adiponectin, and pharmaceutical composition including molecularly altered adiponectin - Google Patents

Abstract

The purpose of the present invention is to provide molecularly altered adiponectin that can be produced as a uniform product, and has the same or greater activity compared to natural adiponectin. This molecularly altered adiponectin is prepared by obtaining a fusion protein that includes: a trimerized domain such as the coiled-coil domain of a matrilin family protein; and the globular region of adiponectin.

Description

Molecularly modified adiponectin and pharmaceutical composition containing molecularly modified adiponectin

The present invention relates to a molecularly modified adiponectin. More specifically, the present invention relates to a molecularly modified adiponectin comprising a C-terminal side globular region of adiponectin and a coiled coil region of a parent protein.

It is known that obesity caused by high-fat food or insufficient exercise not only promotes the onset and deterioration of metabolic syndrome or diabetes, but also increases the risk of cardiovascular disease. Especially in Japan, heart disease and cerebrovascular disease account for about 27% of the causes of death, and cardiovascular disease has become one of the three major causes of death. In recent years, cardiovascular diseases have increased with obesity caused by changes in dietary habits or lifestyles and insufficient exercise. For example, in Japan, the number of patients with acute myocardial infarction is estimated to be about 250,000 people per year, and about 30% of them are estimated to die. In addition, the number of patients receiving continuous medical treatment for ischemic heart disease (stenosis and myocardial infarction) is estimated to be about 900,000. In the treatment of ischemic heart disease, cardiac catheterization can be performed, but for patients with obesity or diabetes more than 6 hours after the onset of the disease, the shrinkage of myocardial infarction can not be predicted in many cases. Therefore, in addition to the currently used cardiac catheterization, the industry is also looking forward to the development of medicaments for adjuvant therapy. Adiponectin is a fat cell hormone that was specifically expressed and secreted by adipocytes and was discovered in 1996. Compared to other hormones, adiponectin is present in extremely large amounts in plasma. Adiponectin is known to have a variety of functions, in addition to controlling glucose levels or controlling metabolic processes including fatty acid breakdown, it is also associated with arteriosclerosis inhibition, cardioprotective effects, reduction of lesions of myocardial infarction, repair of damaged blood vessels, macrophages It is related to the adhesion of blood vessel walls and the inhibition of phagocytosis by low density lipoprotein (LDL). The decrease in the blood concentration of adiponectin is one of the causes of the onset and deterioration of lifestyle-related diseases such as obesity-related metabolic syndrome, diabetes, cardiovascular disease, and cancer. It is generally considered that adiponectin is effective for inhibiting smooth muscle proliferation and treating arteriosclerosis (Patent Document 1). Adiponectin is a peptide of about 30 kDa, which exists in the range of 2-30 mg / L in the circulation of healthy people. Adiponectin includes a N-terminal collagen-like region (collagen-like region, cAd), and a C-terminal spherical region (gAd). Among them, it is known that a spherical region (gAd) on the C-terminal side can be used for the prevention and treatment of arteriosclerosis (Patent Document 2). Adiponectin in plasma is a mixture of trimer (low molecular weight trimer), hexamer (medium molecular weight hexamer), and twelve to eighteen polymers (high molecular weight polymer). Under there. It is known that compared with the total amount of adiponectin, the level of hexamer or higher polymer polymers is strongly related to obesity, coronary artery disease, etc., and it is generally considered that the polymer polymers have the highest physiological activity (non-patented Reference 1). Most of the mechanisms of cardioprotective signals caused by adiponectin are mediated by activation of AMPK (adenosine monophosphate-activated protein kinase). Adiponectin has anti-inflammatory, anti-oxidant, and anti-apoptotic effects, and it has been confirmed that these effects bring protective effects on ischemia-reperfusion. It is revealed that there is a treatment method for supplementing reduced adiponectin, or activating AMPK or PPAR (peroxisome proliferator-activated receptor) to become a metabolic syndrome accompanied by obesity, diabetes, cardiovascular disease, and cancer. (Non-patent documents 2 to 4). [Prior Art Literature] [Patent Literature] [Patent Literature 1] Japanese Patent No. 3538711 [Patent Literature 2] International Publication No. 2003/099319 (Japanese Patent No. 4147220) [Non-Patent Literature] [Non-Patent Literature 1 ] Am. J. Cardiovasc Dis 2012; 2 (4): 253-366 [Non-Patent Document 2] Nature, 503, 493-499 (2013) [Non-Patent Document 3] Nature Medicine, 11, 1096-1103 (2005) [Non-Patent Document 4] Cardiovascular Res. 74 (2007) 471-479

[Problems to be Solved by the Invention] Natural adiponectin exists in a state in which trimers, hexamers, twelve to octamers, and the like are mixed together via a collagen-like region. Therefore, in the manufacture of pharmaceuticals using natural adiponectin, it is impossible to control the formation of polymers and it is difficult to obtain uniform adiponectin. Therefore, there are problems in terms of specifications such as manufacturing quality and it is difficult to handle. . An object of the present invention is to provide a molecularly modified adiponectin and a pharmaceutical composition containing the molecularly modified adiponectin, which can produce a uniform product and have an activity equal to or higher than that of natural adiponectin. [Technical means to solve the problem] In the present invention, a molecularly modified adiponectin is produced by making a fusion protein including a trimerization region and a spherical region of adiponectin. The molecularly modified adiponectin of the present invention naturally associates to form a homotrimer. It is believed that polymers such as twelve to eighteen polymers of adiponectin are active, but surprisingly, it has been found that the molecularly modified adiponectin of the present invention, which forms a trimer, has the same or more than natural adiponectin Effect. Therefore, in the present invention, there is provided a molecularly modified adiponectin comprising a C-terminal side spherical region of adiponectin and a coiled coil region of a parent protein. The molecularly modified body adiponectin can effectively utilize the coiled-coil region of the parent protein to efficiently form a trimer. The molecularly modified adiponectin of the present invention may use a coiled coil region of a parent protein located at the N-terminal side and a spherical region of adiponectin located at the C-terminal side. The molecularly modified adiponectin of the present invention may further include at least one ZZ region of protein A of staphylococcus, and the ZZ region of protein A may also be located at the N-terminal side of the coiled-coil region of the parent protein. As the maternal protein included in the molecularly modified adiponectin, maternal protein-2 can be used. For the molecularly modified adiponectin of the present invention, a polypeptide containing an amino acid sequence of amino acids 114 to 244 of sequence number 1, a polypeptide containing an amino acid sequence of amino acids 912 to 952 of sequence number 2, And a polypeptide comprising an amino acid sequence of SEQ ID NO: 3. In addition, as the molecularly modified body adiponectin of the present invention, an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, and 11 can be used. As another aspect of the present invention, a terpolymer of the aforementioned molecularly modified adiponectin is provided. In another aspect of the present invention, a nucleic acid encoding the molecularly modified adiponectin is provided. As the nucleic acid of the present invention, a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8 and 10 is preferred. Furthermore, as an embodiment of the present invention, a vector comprising the nucleic acid is provided. As another aspect of the present invention, a host cell comprising the nucleic acid or the vector is provided. As another aspect of the present invention, there is provided a pharmaceutical composition comprising a trimer of a molecularly modified body adiponectin and a carrier acceptable in formulation. The pharmaceutical composition of the present invention can be used for treating or preventing arteriosclerosis, treating or preventing diabetes, or treating or preventing obesity. Arteriosclerosis includes stenosis, myocardial infarction, or cerebral infarction. [Effects of the Invention] According to the present invention, it is possible to provide a homogeneous trimer as compared with the natural adiponectin, which forms a diversified polymer, and has a physiology equivalent to or exceeding that of the natural adiponectin Molecularly modified body adiponectin. For example, by using about one third of the concentration of natural adiponectin to modify the body adiponectin, the same effect as natural adiponectin can be exhibited. Furthermore, since the molecularly modified adiponectin of the present invention activates a signal transmission mechanism mediated by natural adiponectin, the disease caused by the lack of adiponectin, such as cardiovascular disease such as arteriosclerosis, Prevention and treatment of heart diseases including heart failure, cardiac hypertrophy, fatal arrhythmia, cerebral infarction, obesity, diabetes, hypertension, hyperlipidemia and other so-called lifestyle diseases and malignant tumors (cancer) effective.

(Molecular Modified Adiponectin) The molecularly modified adiponectin of the present invention is a fusion protein comprising a spherical region of adiponectin and a coiled coil region of a parent protein, preferably a coiled coil region of a parent protein fused to the N-terminal side. A fusion of adiponectin globular regions at the C-terminal side. By fusing the coiled coil region and the adiponectin spherical region, a trimer can be formed extremely efficiently. The molecularly modified adiponectin may further include a ZZ region as an IgG-binding region of Staphylococcus protein A. The ZZ region may be linked to the N-terminus or C-terminus, or both the N-terminus and the C-terminus of the molecular modification body adiponectin, which is a fusion protein. Preferably, the Z region is connected to the N-terminus. It is known that the expression of proteins in soluble components is generally promoted by linking the Z regions when performing expression from a host cell. The molecular modified body adiponectin in this case exhibits the same physiological activity as adiponectin, such as AMPK activation effect, anti-apoptotic effect, anti-inflammatory effect, and myocardial infarction lesion reduction effect, even if it does not contain the ZZ region, but It was surprisingly confirmed that the introduction of the ZZ region not only promoted the performance in soluble components, but also exhibited the same physiological activity as the natural adiponectin even at a lower concentration of protein. The molecularly modified body adiponectin of the present invention includes, for example, a polypeptide comprising an amino acid sequence of amino acids 114 to 244 of sequence number 1, a polypeptide comprising an amino acid sequence of amino acids 912 to 952 of sequence number 2, and A polypeptide comprising an amino acid sequence of SEQ ID NO: 3. The molecularly modified adiponectin of the present invention includes, for example, amino acid sequences of SEQ ID NOs: 5, 7, 9, and 11. The molecularly modified adiponectin of the present invention can be produced by a common genetic engineering method. More specifically, the molecularly modified adiponectin of the present invention can be produced by introducing a gene encoding a molecularly modified adiponectin into a expression vector and introducing it into an appropriate host cell for expression. (Adiponectin) The spherical region on the C-terminal side of adiponectin contained in the molecularly modified adiponectin of the present invention can be derived from natural adiponectin. The amino acid sequence of natural adiponectin has been determined and can be obtained by known methods. In the present invention, human adiponectin is preferably used. For example, adiponectin having an amino acid sequence of SEQ ID NO: 1 (Fig. 1) can be used. As described above, adiponectin includes an N-terminal collagen-like region (collagen-like region, cAd), and a C-terminal side spherical region (gAd). The amino acid sequence of the spherical region at the C-terminus of adiponectin is a polypeptide comprising amino acid sequences of amino acids 114 to 244 of sequence number 1. As long as it has adiponectin activity, it is not limited to polypeptides containing amino acid sequences of amino acids 114 to 244 of SEQ ID NO. 1, and one or more amino acid sequences having such amino acid sequences may also be used. A variant polypeptide that is substituted, deleted, or added to an amino acid sequence. Examples of such a variant polypeptide include those having a preservation substitution of an amino acid sequence to the amino acid sequences of amino acids 114 to 244 of SEQ ID NO: 1. Examples of the variant polypeptide include those having amino acid sequences of amino acids 114 to 244 of SEQ ID NO: 1 that have homology of 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more. (Trimerization region) A polymerized region is connected to the N-terminal side of the spherical region of the C-terminal side of adiponectin in the molecularly modified adiponectin of the present invention. The multimerization zone promotes multimerization of the polypeptide bound to the multimerization zone. The polymerized region is preferably a terpolymerized region, and generally has a coiled spiral region. Examples of such a trimer region include a coiled coil region such as an isoleucine zipper, a protein of the matrilin family, GCN4, and the like. Particularly preferred is the coiled-coil region of the parent protein family protein. Maternal protein family proteins are secreted extracellular matrix proteins, including maternal protein-1 to maternal protein-4. In the present specification, the maternal protein family proteins including maternal protein-1 to maternal protein-4 are also simply referred to as maternal proteins. Maternal protein-1 is a component of the extracellular matrix of non-articular cartilage and is mainly secreted by chondrocytes. Maternal protein-2 is a secreted protein associated with matrix association. Maternal protein-3 is a secreted protein expressed in cartilage tissue. Maternal protein-4 is expressed in the fetal kidney, embryo and placenta, and is an important secreted protein for the extracellular matrix of cartilage. Among them, especially in the present invention, it is preferable to use a human parent protein as a trimerization region, and it is particularly suitable to use a human parent protein-2 coiled coil region. The amino acid sequence of the parent protein-2 is represented by SEQ ID NO: 2 (Fig. 1), and the coiled coil region is a polypeptide comprising the amino acid sequences of amino acids 912-952 of SEQ ID NO: 2. As long as it has trimer-forming activity and does not impair the activity of the molecularly modified body adiponectin, it is not limited to polypeptides containing the amino acid sequences of amino acids 912 to 952 of SEQ ID NO: 2, and these amino acids can also be used A variant polypeptide in which one or more amino acids of the sequence are substituted, deleted, or added. Examples of such a variant polypeptide include those having a preservation substitution of an amino acid sequence to the amino acid sequence of amino acids 912 to 952 of SEQ ID NO: 2. Examples of the variant polypeptide include those having an amino acid sequence of amino acids 912 to 952 of SEQ ID NO: 2 with homology of 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more. By fusing the spherical region of adiponectin with the trimerization region, the resulting fusion protein forms a trimer. Natural adiponectin is a mixture of trimer, hexamer, and a combination of polymers. In contrast, regarding the molecularly modified adiponectin of this case, at least about 60% of the expressed polypeptides Preferably, at least about 70%, and most preferably at least about 75%, is present as a trimer. (ZZ region) The molecularly modified adiponectin of the present invention may further include the Z region of protein A of Staphylococcus. The Z region is a region derived from Staphylococcus protein A that binds to the Fc region of an antibody. The Z region is generally used to solubilize unfolded or misfolded proteins (see, for example, Biochemistry 1994, 33, 4207-4211). It is preferably used in the form of a ZZ zone in which two Z zones are arranged in series. As the ZZ region, a polypeptide having the amino acid sequence described in SEQ ID NO: 3 (FIG. 1) can be used. As the nucleotide sequence encoding the ZZ region, for example, a commercially available vector such as pEZZ18 (GE Healthcare Japan Co., Ltd.) can be used. As long as the solubilization of the protein is promoted and the activity of the molecularly modified body adiponectin is not impaired, it is not limited to the polypeptide comprising the amino acid sequence of SEQ ID NO. A variant polypeptide in which an acid is substituted, deleted, or appended to an amino acid sequence. Examples of such a variant polypeptide include those having a conservative substitution with an amino acid to the amino acid sequence of SEQ ID NO: 3. Examples of the variant polypeptide include those having an amino acid sequence of amino acids 114 to 244 of SEQ ID NO: 3 that have a homology of 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more. (Tag for purification) In order to facilitate purification, the molecularly modified adiponectin may include a tag for purification. Purification tags are well known to the industry and are not particularly limited. For example, His (Histidine) tag, GST (glutathione S-transferase) tag, MBP (Maltose binding protein, maltose) can be used. Binding protein) tag, Strep (II) tag, etc. After purification, it is not necessary to cut the area containing the tag. In the case where the area containing the tag is cut, a recognition site such as a protease can be introduced into an adjacent portion of the purification tag, and the protease can be used to cut the area. Any linker may be included between the adiponectin globular region and the coiled coil region of the parent protein. The linker is not particularly limited, and a coiled coil region derived from a parent protein or a region adjacent to a spherical region of adiponectin may be used. (Nucleic acid) The nucleic acid usable in the present invention includes a nucleic acid sequence encoding the C-terminal side globular region of adiponectin and the coiled coil region of the parent protein, respectively. As such a nucleic acid sequence, for example, a nucleic acid sequence including a polypeptide encoding an amino acid sequence including amino acids 114 to 244 of sequence number 1 and an amino acid sequence of amino acids 912 to 952 of sequence number 2 can be exemplified. By. Furthermore, a nucleic acid sequence encoding the ZZ region of protein A of Staphylococcus may be included. As the nucleic acid encoding the ZZ region of protein A, a commercially available vector derived from pEZZ18 (GE Healthcare Japan Co., Ltd.) can be used. As the nucleic acid encoding the ZZ region of the protein A, for example, a nucleic acid sequence encoding a ZZ region including the amino acid sequence of SEQ ID NO: 3 can be used. Furthermore, in order to facilitate purification, the nucleic acid usable in the present invention may include a nucleic acid sequence encoding the aforementioned purification tag. It may also include a nucleic acid sequence encoding an arbitrary linker sequence. The nucleic acid usable in the present invention is not particularly limited as long as it is an amino acid sequence encoding the molecularly modified adiponectin. For example, nucleic acid sequences with sequence numbers 4, 6, 8 and 10 or nucleic acid sequences encoding amino acid sequences with sequence numbers 5, 7, 9 and 11 can be used. (Vector) Expression vectors include plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like. For example, pET-24a-d, pUC, pBI, pSMA, pBluscript, λgt11, λZAP can be used. Any vector, such as pUB110, pTP5, YEp13, YCp50, baculovirus, adenovirus, adeno-associated virus, retrovirus, etc., is used in the host cell at the same time, and it is not particularly limited. As the expression vector, a promoter, a promoter, a splicing signal, a polyadenylation site, a terminator, a selection marker, an origin of replication, and the like can be used. The host can use any of prokaryotes and eukaryotes. As the prokaryotic host, E. coli, Bacillus subtilis, and the like can be used, and as the eukaryotic host, yeast, mammalian cells, insect cells, and the like can be used. As a method for introducing the expression vector, any known method such as a calcium phosphate method, a lipofection method, an electroporation method, or the like can be used. The molecularly modified adiponectin can be recovered from a host cell culture medium by a known method and purified. The expressed molecularly modified adiponectin uses a trimerization region to form a trimer, and is accumulated in a cell or secreted outside the cell. The recovery and purification can be performed by a general method. For example, various chromatography methods such as affinity chromatography, gel filtration chromatography, and ion exchange chromatography can be used. In addition, whether a terpolymer is formed can be analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) under non-heating and non-reducing conditions and western ink Easy analysis by point analysis, etc. (Pharmaceutical composition) The pharmaceutical composition of the present invention can be made into the pharmaceutical composition of various administration forms by using the molecularly modified adiponectin of the present invention as an active ingredient, and further adding a pharmaceutically acceptable carrier. In addition, a gene therapy carrier encoding a nucleic acid encoding the molecularly modified adiponectin of the present invention may be used as an active ingredient, and a pharmaceutically acceptable carrier may be added to prepare a pharmaceutical composition in various administration forms. As an administration form of a pharmaceutical composition, oral administration and parenteral administration are possible. In the case of parenteral administration, various forms such as intravenous, intraarterial, subcutaneous, intramuscular, and intraperitoneal administration can be used. Examples of the pharmaceutical composition for oral administration include lozenges, capsules, granules, fine granules, syrups, enteric solvents, sustained-release capsules, and the like. Carriers such as lactose, crystalline cellulose, starch, and lubricants such as magnesium stearate, talc, and calcium stearate can be used in the pharmaceutical composition for oral administration. Furthermore, it can also apply with coating agents, such as ethyl cellulose, hydroxypropyl cellulose, cellulose acetate phthalate, and hypromellose phthalate. Moreover, when a capsule is used, an enteric capsule can be formed using an enteric coating agent and the like. Intravenous, intraarterial, subcutaneous, intramuscular, and intraperitoneal administration agents may include carriers such as distilled water, stabilizers, emulsifiers, and buffers. The operator may choose a carrier in consideration of storage stability and the like. The pharmaceutical composition of the present invention can be administered with a molecularly modified body adiponectin at a dosage of, for example, 0.01 to 0.05 μg / day, but the dosage can be changed according to the administration route, sex, age, weight, and disease state, etc. . The pharmaceutical composition containing the molecularly modified adiponectin of the present invention activates AMPK, which is activated by adiponectin binding to the adiponectin receptor. Therefore, the therapeutic and preventive effects of natural adiponectin are expected. Diseases can be expected to have the same high therapeutic and preventive effects. For example, a pharmaceutical composition comprising the molecularly modified adiponectin of the present invention is useful for cardiovascular diseases such as arteriosclerosis, heart diseases including heart failure, cardiac hypertrophy, fatal arrhythmia, cerebral infarction, obesity, diabetes, The so-called lifestyle diseases such as high blood pressure and hyperlipidemia, and malignant tumors (cancers), etc. exhibit preventive and therapeutic effects. Prevention and treatment include not only the prevention and treatment of the disease, but also the prevention and treatment of the symptoms accompanying the disease, and the suppression of the recurrence of the disease. Treatment includes not only radical treatment, but also conditions that improve symptoms compared to before treatment. Hereinafter, the present invention will be described based on examples, but the following examples are intended to illustrate the present inventors, and are not intended to limit the present inventors. [Example] <Example 1: Preparation of hMat2cc-gAd expressing E. coli strain> In order to express the spherical region (114 residues to 244 residues) of adiponectin (SEQ ID NO: 1) in humans in E. coli Polypeptide (hereinafter referred to as hMat2cc-gAd) (sequence number 5) (Figure 2) of a polypeptide (hereinafter referred to as hMat2cc-gAd) with a coiled coil region (912 residues to 952 residues) of human maternal protein 2 (SEQ ID NO: 2) linked to an amino group The DNA sequence (sequence number 4) (Figure 2) encoding the polypeptide was artificially synthesized with DNA added to the 5 'end of the NdeI recognition sequence and the 3' end of the XhoI recognition sequence for selection in the expression vector. This synthetic DNA was inserted downstream of the T7 promoter of plasmid pET-24a (+) (Merck Co., Ltd.), and the obtained plasmid was introduced into E. coli BL21 (DE3) strain (Merck Co., Ltd.) to obtain hMat2cc-gAd represents an E. coli strain. <Example 2: Production of ZZ-hMat2cc-gAd expressing Escherichia coli strain> The ZZ region of protein A derived from Staphylococcus aureus was linked to the amino group terminal of hMat2cc-gAd (SEQ ID NO: 5) in E. coli Polypeptide (SEQ ID NO: 3) (hereinafter referred to as ZZ-hMat2cc-gAd) (SEQ ID NO: 7) (FIG. 3), a DNA sequence encoding the polypeptide (SEQ ID NO: 6) (FIG. 3) is designed, and artificially synthesized is useful DNAs selected from the NcoI recognition sequence to the 5 'end and the XhoI recognition sequence to the 3' end selected from the expression vector. This synthetic DNA was inserted downstream of the T7 promoter of plasmid pET-24d (+) (Merck Co., Ltd.), and the obtained plasmid was introduced into E. coli BL21 (DE3) strain (Merck Co., Ltd.) to obtain ZZ-hMat2cc-gAd expresses an E. coli strain. <Example 3: Preparation of hMat2cc-gAd-ZZ-expressing E. coli strain> In order to express in E. coli a polypeptide having the ZZ region (sequence number 3) linked to the carboxy terminus of hMat2cc-gAd (sequence number 5) (hereinafter, referred to as For hMat2cc-gAd-ZZ) (SEQ ID NO: 9) (Figure 4), the DNA sequence encoding the polypeptide (SEQ ID NO: 8) (Figure 4) was designed, and it was artificially synthesized and added to the 5 'end of the expression vector. DNA with the NcoI recognition sequence and the 3 'end XhoI recognition sequence. This synthetic DNA was inserted downstream of the T7 promoter of plasmid pET-24d (+) (Merck Co., Ltd.), and the obtained plasmid was introduced into E. coli BL21 (DE3) strain (Merck Co., Ltd.) to obtain hMat2cc-gAd-ZZ represents an E. coli strain. <Example 4: Production of E. coli strain expressing ZZ-hMat2cc-gAd-ZZ> In order to express E. coli in hMat2cc-gAd (SEQ ID NO: 5), both the amino group terminal and the carboxyl terminal are linked to a ZZ region (sequence number 3) Polypeptide (hereinafter referred to as ZZ-hMat2cc-gAd-ZZ) (sequence number 11) (Figure 6), designing the DNA sequence (sequence number 10) (Figure 5) encoding the polypeptide, and artificially synthesize additional useful DNA cloned from the 5 'end NcoI recognition sequence and the 3' end XhoI recognition sequence of the expression vector. This synthetic DNA was inserted downstream of the T7 promoter of plasmid pET-24d (+) (Merck Co., Ltd.), and the obtained plasmid was introduced into E. coli BL21 (DE3) strain (Merck Co., Ltd.) to obtain ZZ-hMat2cc-gAd-ZZ represents an E. coli strain. <Example 5: Preparation and character analysis of hMat2cc-gAd> The hMat2cc-gAd-expressing E. coli strain was cultured in a 2xYT medium containing conomycin and isopropyl-β-thiogalactopyranoside, and was centrifuged. The separated and recovered bacterial cells were resuspended in a buffer solution containing 20 mM Tris-HCl (pH 7.5) and subjected to ultrasonic disruption. The obtained cell disrupted matter was separated into a soluble component and an insoluble component by centrifugation, and the soluble component was subjected to a HiTrap Q FF column (GE Healthcare Japan Co., Ltd.) and a HiTrap TALON crude column (GE Healthcare Japan). Co., Ltd.) was continuously purified. A protein concentration measurement was performed on the purified product using a BCA protein analysis kit (Thermo Scientific). Furthermore, SDS-PAGE analysis and Western blot analysis were performed under non-heating and non-reducing conditions, and it was confirmed that hMat2cc-gAd forms a trimer and has high purity. <Example 6: Preparation and character analysis of ZZ fusion hMat2cc-gAd> For ZZ-hMat2cc-gAd expressing E. coli strain, hMat2cc-gAd-ZZ expressing E. coli strain, and ZZ-hMat2cc-gAd-ZZ expressing E. coli strain, The cells were cultured in 2xYT medium containing concomycin and isopropyl-β-thiogalactoside, and the cells recovered by centrifugation were resuspended in 20 mM Tris-HCl (pH 7.5). Buffer and perform ultrasonic disruption. The obtained cell disrupted matter was separated into a soluble component and an insoluble component by centrifugation, and the soluble component was subjected to a HiTrap TALON crude column (GE Healthcare) and a HiLoad 16/60 Superdex 200 pg (GE Healthcare) column. Purification was performed continuously. A protein concentration measurement was performed on the purified product using a BCA protein analysis kit (Thermo Scientific). In addition, SDS-PAGE analysis and Western blot analysis were performed under non-heating and non-reducing conditions, and it was confirmed that the three types of ZZ fusion hMat2cc-gAd form a trimer and have high purity. <Example 7: Study of AMPK and Akt activity effects of hMat2cc-gAd using rat cardiomyocytes> Using rat cardiomyocytes, it was confirmed that the molecular protective body adiponectin hMat2cc-gAd was used to activate the cardioprotective signal . The cardiomyocytes of the rats were cultured in DMEM (Dulbecco's modified Eagle medium) and treated with 10 μg / ml hMat2cc-gAd for 18 hours. The cardiomyocytes were homogenized in a lysis buffer, and a 30% protein was separated by SDS-PAGE under denaturing conditions using a 10% polypropylene ammonium gel. After electrophoresis, the separated proteins were transferred to a membrane for Western blot analysis. Specifically, a primary antibody is diluted at a ratio of 1: 1,000, and a secondary antibody compounded with HRP (Horseradish Peroxidase, horseradish peroxidase) is used at a ratio of 1: 15,000. The detection system used ECL-PLUS Western Ink Dot Detection Kit (manufactured by Amersham Pharmacia Biotec). The primary antibody system used was an antibody against p-AMPK (phosphorylated AMPK) and p-Akt (phosphorylated Akt) (Cell Signaling Technology). An anti-tubulin antibody (Oncogene) is used to measure the amount of tubulin as an internal standard for the amount of protein. Here, AMPK is a core medium in adiponectin signal transmission mechanism, and is a protein that is phosphorylated by activating adiponectin receptor. Phosphorylation and activation of AMPK are known to activate the cardioprotective signals that act on the inhibition of cardiac hypertrophy, diabetic cardiomyopathy, and myocardial infarction (Am. J. Cardiovasc Dis 2012; 2 (4): 253-366 ), And activate signals that cause sugar intake or fatty acid burning (Nature Medicine 2002: Vol. 8, 1288-1295). Phosphorylated AMPK (p-AMPK) in turn phosphorylates Akt downstream. The results are shown in FIG. 7. In the figure, the left side is P-AMPK and P-Akt, and the right side is the result of performing Western blot method on P-AMPK again for the sake of caution. + Is treated by hMat2cc-gAd,-is untreated. For those treated with hMat2cc-gAd, it was confirmed that the amounts of p-AMPK and p-Akt were increased compared with the control, and the adiponectin signal was activated by the molecular modification of adiponectin. 〈Example 8: Study of AMPK and Akt activity caused by ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, and ZZ-hMat2cc-gAd-ZZ using rat cardiomyocytes〉 Rat cardiomyocytes were used to compare In the same manner as in Example 7, it was confirmed that the cardioprotective signal was activated even when adiponectin was modified by other molecules. Rat cardiomyocytes were treated in the same manner as in Example 7 using ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, and ZZ-hMat2cc-gAd-ZZ. The cardiomyocytes were homogenized in a lysis buffer, and a 30% protein was separated by SDS-PAGE under denaturing conditions using a 10% polypropylene ammonium gel. After electrophoresis, the separated proteins were transferred to a membrane for Western blot analysis. Specifically, a primary antibody is diluted at a ratio of 1: 1,000 and a secondary antibody complexed with HRP is used at a ratio of 1: 15,000. The detection system used ECL-PLUS Western Ink Dot Detection Kit (manufactured by Amersham Pharmacia Biotec). The primary antibodies used were directed against antibodies against p-AMPK (phosphorylated AMPK), T-AMPK (all AMPK), p-Akt (phosphorylated Akt), and T-Akt (all Akt). An anti-tubulin antibody (Oncogene) is used to measure the amount of tubulin as an internal standard for proteins. The results are shown in FIG. 8. The pictures on the right and left are for the sake of prudence and the Western blotting method was performed twice. It was confirmed that the amounts of p-AMPK and p-Akt in the molecularly modified adiponectin were increased compared with the control, and the molecularly modified adiponectin was used to activate the adiponectin signal. Especially for ZZ-hMat2cc-gAd, the activation of AMPK and Akt is more obvious. 〈Example 9: Study of AMPK and Akt activity caused by ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, and ZZ-hMat2cc-gAd-ZZ using rat fibroblasts ＞ It was confirmed that even rat fibroblasts The use of other types of molecular modification of adiponectin also activates adiponectin signals. Rat fibroblasts were treated with a molecularly modified adiponectin, ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, or ZZ-hMat2cc-gAd-ZZ, at a concentration of 10 μg / ml for 18 hours. In the same manner as in Example 8, p-AMPK (phosphorylated AMPK) (Cell Signaling Technology), T-AMPK (all AMPK) (Cell Signaling Technology), p-Akt (phosphate Akt), T-Akt (all Akt). The results are shown in FIG. 9. It was confirmed that the adiponectin signal was activated by the molecular modification of adiponectin even in fibroblasts. Especially for ZZ-hMat2cc-gAd, the activation of AMPK and Akt is more obvious. <Example 10: Study on anti-apoptotic effect of hMat2cc-gAd using rat cardiomyocytes> It was confirmed that rat cardiomyocytes inhibited apoptosis due to hypoxia by using hMat2cc-gAd. Rat cardiomyocytes were treated in a serum-free medium under normal oxygen for 48 hours, or under hypoxia (<1% O ₂ and 5% CO ₂ at 37 ° C) for 12 hours, and then at 10 μg / ml of hMat2cc-gAd in the presence or absence of oxygen (<21% O ₂ and 5% CO ₂ at 37 ° C.) was left to stand for 24 hours. TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining (green) according to the usual method (green) will stain cells that cause apoptosis. All dead cells were stained by DAPI (4 ', 6-diamidino-2-phenylindole, 4', 6-diamidino-2-phenylindole) staining (blue). Representative photographs of the results of TUNEL staining and DAPI staining are shown in the upper section of FIG. 10, and the ratio of dead cells due to TUNEL-positive apoptosis that is quantitatively analyzed is shown in the lower section of the graph. It was shown that the ratio of TUNEL-positive cells under hypoxia, that is, the ratio of dead cells due to apoptosis, was significantly reduced compared with the control by hMat2cc-gAd, and hMat2cc-gAd exerted an anti-apoptotic effect. 〈Example 11: Study of anti-apoptotic effects of rat cardiomyocytes due to ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, and ZZ-hMat2cc-gAd-ZZ ＞ It was confirmed that rat cardiomyocytes were used Other types of molecularly modified adiponectin also inhibit apoptosis due to hypoxia. In the same manner as in Example 10, rat cardiomyocytes were treated with ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, or ZZ-hMat2cc-gAd-ZZ, and the ratio of dead cells due to apoptosis was measured. A representative photograph of the results of TUNEL staining is shown in the upper part of FIG. 11, and the ratio of dead cells due to TUNEL-positive apoptosis that is quantitatively analyzed is shown in the lower part of the graph. It shows that the ratio of TUNEL positive cells under hypoxia, that is, the ratio of dead cells due to apoptosis, even when other types of molecular modification body adiponectin are used, it is significantly reduced compared with the control, and other types of molecular modification Body adiponectin exerts an anti-apoptotic effect. <Example 12: Study of anti-inflammatory effects (inhibition of mRNA expression levels of TNFα and IL-1β) by hMat2cc-gAd in rat cardiomyocytes> It was confirmed that inflammation of cardiomyocytes was suppressed by hMat2cc-gAd. If cardiomyocytes are stimulated by lipopolysaccharide (LPS), they will produce TNFα and IL-1β, which will cause inflammation. Based on the mRNA amounts of TNFα and IL-1β, it was confirmed that hMat2cc-gAd inhibited inflammation. Neonatal rat cardiomyocytes (NRVM) were seeded into 24-well culture dishes at a concentration of 250 μl / well (2.5 × 10 ⁵ cells / well). After treatment with hMat2cc-gAd at a concentration of 10 μg / ml for 1 hour, treatment with LPS at 100 ng / ml for 6 hours induced the expression of TNFα and IL-1β as inflammatory cytokines. The amount of mRNA of TNFα and IL-1β was measured by RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The control system used PBS (phosphate buffer saline, phosphate buffer solution) instead of molecularly modified body adiponectin. The results are shown in FIG. 12. Using hMat2cc-gAd, the mRNA expression levels of TNFα and IL-1β were reduced compared to the control, and it was confirmed that inflammation was suppressed. When the molecularly modified body adiponectin was used in this way, an anti-inflammatory effect was confirmed at a concentration of 10 μg / ml. On the other hand, in the case of natural adiponectin, when TNF-α production due to LPS stimulation was measured, it was confirmed that TNFα production was not inhibited at a concentration of 10 μg / ml, but at a concentration of 30 μg / ml Inhibition (Nature Medicine, 11, 1096-1103 (2005)). This shows that the molecularly modified adiponectin of the present invention exerts the same effect even if it is about one third of the concentration of natural adiponectin. <Example 13: Study of anti-inflammatory effect (TNF inhibitory effect) using rat cardiomyocytes> It was confirmed that inflammation of cardiomyocytes was suppressed even when adiponectin was modified by another type of molecular modification. Neonatal rat cardiomyocytes (NRVM) were seeded into 24-well culture dishes at a concentration of 250 μl / well (2.5 × 10 ⁵ cells / well). After 1 hour of treatment with ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ or ZZ-hMat2cc-gAd-ZZ at a concentration of 10 μg / ml, inflammation was induced by treatment with 100 ng / ml of LPS for 6 hours. The amount of TNFα mRNA was determined by RT-PCR. The control used PBS instead of the molecularly modified adiponectin. The results are shown in FIG. 13. Using ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, or ZZ-hMat2cc-gAd-ZZ, the expression of TNFα was reduced compared to the control, and inhibition of inflammation was confirmed. <Example 14: Effect of infarct size after ischemia-reperfusion injury due to molecular modification of adiponectin treatment> It was confirmed that the size of infarction after ischemia-reperfusion injury by molecular modification of adiponectin hMat2cc-gAd Reduced compared to wild-type untreated mice. 1.0 μg / g of the molecularly modified adiponectin hMat2cc-gAd was administered into the carotid artery of a plurality of C57BL / 6 mice. For control mice, PBS was administered. LAD (Left Anterior Descending) vessels were ligated for 30 minutes 30 minutes after administration, followed by reperfusion for 48 hours. After LAD arteries were re-sutured, 1 ml of 1.0% Ivan's Blue (Sigma Chemilal Co.) was injected from the jugular vein, and non-infarcted tissue was stained. Thereafter, the heart was taken out, washed with PBS, and a cross-sectional section was prepared, and then stained with 1.0 ml of 1.5% chlorinated 2,3,5-triphenyltetrazole (Sigma Chemilal Co.) at 23 ° C. 5 minutes while staining the infarcted area. The photograph in FIG. 14 shows the heart tissue stained with Ivan's blue showing the ischemic area (AAR) and 2,3,5-triphenyltetrazole chloride showing the infarcted area (IA). photo. The blue part indicates healthy heart muscle that is not affected by ischemia, and the white area indicates dead tissue. Furthermore, the area measurement of the left ventricle area (LV), ischemic area (AAR), and infarcted area (IA) was determined by performing area measurement using a computer using the software ImageJ. The ratio of ischemic area (AAR) to left ventricular area (LV) (AAR / LV), the ratio of infarcted area (IA) to ischemic area (AAR) (IA / AAR), and the infarcted area (IA) The ratio (IA / LV) to the left ventricular area (LV) is shown in the graph of FIG. 14. Compared with the control group, the IA / AAR and IA / LV of the group administered hMat2cc-gAd were reduced. Therefore, hMat2cc-gAd has been shown to be effective in reducing myocardial infarction. <Example 15: Effect of infarct size after ischemia-reperfusion injury due to molecular modification of adiponectin treatment> It was confirmed that infarction size after ischemia-reperfusion injury was reduced even when other molecular modifications of adiponectin were used . 1.0 μg / g of ZZ-hMat2cc-gAd or ZZ-hMat2cc-gAd-ZZ was administered in the same manner as in Example 14. For control mice, PBS was administered. In the same manner as in Example 14, Ivan's blue staining and 2,3,5-triphenyltetrazolium chloride staining were performed after ischemia-reperfusion injury, and the ischemic area (AAR) and infarction were measured. The area of area (IA) and left ventricular area (LV) are graphed. The results are shown in FIG. 15. Compared with the control, the IA / AAR and IA / LV of the group administered the molecularly modified adiponectin were reduced. Therefore, it has been shown that ZZ-hMat2cc-gAd or ZZ-hMat2cc-gAd-ZZ is effective for reducing myocardial infarction.

FIG. 1 is a diagram showing the amino acid sequence (sequence number 1), the parent protein-2 amino acid sequence (sequence number 2), and the ZZ region amino acid sequence (sequence number 3). In the amino acid sequence of the adiponectin and the amino acid sequence of the parent protein-2, the amino acid sequences of the adiponectin globular region and the coiled coil region are indicated in bold type, respectively. FIG. 2 is a diagram showing the DNA sequence (sequence number 4) of hMat2cc-gAd and the amino acid sequence (sequence number 5) of hMat2cc-gAd. FIG. 3 is a diagram showing the DNA sequence (sequence number 6) of ZZ-hMat2cc-gAd and the amino acid sequence (sequence number 7) of ZZ-hMat2cc-gAd. FIG. 4 is a diagram showing the DNA sequence (sequence number 8) of hMat2cc-gAd-ZZ and the amino acid sequence (sequence number 9) of hMat2cc-gAd-ZZ. FIG. 5 is a diagram showing a DNA sequence (sequence number 10) of ZZ-hMat2cc-gAd-ZZ. Fig. 6 is a diagram showing a ZZ-hMat2cc-gAd-ZZ amino acid sequence (SEQ ID NO: 11). FIG. 7 is a photograph showing Western blot method of activation of AMPK and AKT caused by hMat2cc-gAd in rat cardiomyocytes. FIG. 8 is a photograph showing Western blotting methods for activation of AMPK and AKT caused by ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, ZZ-hMat2cc-gAd-ZZ using rat cardiomyocytes. FIG. 9 is a photograph showing Western blot method of activation of AMPK and AKT caused by ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ and ZZ-hMat2cc-gAd-ZZ using rat fibroblasts. FIG. 10 is a photograph and a graph showing an anti-apoptotic effect of hMat2cc-gAd using rat cardiomyocytes. The upper section is a representative photograph of the results of TUNEL staining and DAPI staining, and the lower section is a graph showing the quantitative analysis of the ratio of dead cells due to apoptosis of TUNEL-positive cells. FIG. 11 is a photograph and a graph showing the anti-apoptotic effect caused by ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, and ZZ-hMat2cc-gAd-ZZ using rat cardiomyocytes. The upper section is a representative photograph of the results of TUNEL staining, and the lower section is a graph showing quantitative analysis of the ratio of dead cells due to apoptosis of TUNEL-positive cells. Fig. 12 is a graph showing the anti-inflammatory effects of hMat2cc-gAd using rat cardiomyocytes. The graph on the right shows the expression of IL-1β, and the graph on the left shows the expression of TNFα. FIG. 13 is a graph showing the anti-inflammatory effects of rat cardiomyocytes due to ZZ-hMat2cc-gAd, hMat2cc-gAd-ZZ, and ZZ-hMat2cc-gAd-ZZ. The graph shows the expression level of TNFα. 14 is a photograph and a graph showing the effect of infarct size after ischemia-reperfusion injury due to hMat2cc-gAd. The photographs are of Ivan's blue staining showing the ischemic area (AAR) and sections of the heart tissue stained with 2,3,5-triphenyltetrazole chloride showing the infarcted area (IA). The graph shows the ratio of ischemic area (AAR) to left ventricular area (LV) (AAR / LV), the ratio of infarcted area (IA) to ischemic area (AAR) (IA / AAR), and the infarcted area ( IA) ratio to left ventricular area (LV) (IA / LV). 15 is a photograph and a graph showing the effect of infarct size after ischemia-reperfusion injury due to ZZ-hMat2cc-gAd and ZZ-hMat2cc-gAd-ZZ. The photograph is a photograph of a section of heart tissue stained with Ivan's Blue and 2,3,5-triphenyltetrazolium chloride. The diagram shows AAR / LV, IA / AAR, and IA / LV.

Claims (15)

A molecularly modified body adiponectin comprises a C-terminal side spherical region of adiponectin and a coiled coil region of a parent protein. For example, the molecularly modified adiponectin of claim 1, wherein the coiled-coil region of the parent protein is located at the N-terminal side, and the adiponectin spherical region is located at the C-terminal side. The molecularly modified adiponectin of claim 1 or 2, further comprising at least one ZZ region of protein A of Staphylococcus. For example, the molecularly modified adiponectin of claim 3, wherein the ZZ region of protein A is located on the N-terminal side of the coiled-coil region of the parent protein. The molecularly modified body adiponectin according to any one of claims 1 to 4, wherein the parent protein is parent protein-2. For example, the adiponectin of claim 3 or 4, which comprises a polypeptide comprising an amino acid sequence of amino acids 114 to 244 of sequence number 1, a polypeptide comprising an amino acid sequence of amino acids 912 to 952 of sequence number 2 And a polypeptide comprising an amino acid sequence of SEQ ID NO: 3. The molecularly modified adiponectin of claim 6, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9 and 11. A trimer which is a trimer of the molecularly modified body adiponectin according to any one of claims 1 to 7. A nucleic acid encoding a molecularly modified body adiponectin according to any one of claims 1 to 7. The nucleic acid of claim 9, comprising a nucleic acid sequence selected from the group consisting of sequence numbers 4, 6, 8, and 10. A vector comprising a nucleic acid as claimed in claim 9 or 10. A host cell comprising a nucleic acid as claimed in claim 9 or 10, or a vector as claimed in claim 11. A pharmaceutical composition comprising a pharmaceutically effective amount of a trimer of a modified adiponectin as claimed in claim 8 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 13, which is used for treating or preventing arteriosclerosis. The pharmaceutical composition according to claim 14, wherein the arteriosclerosis is stenosis, myocardial infarction or cerebral infarction.