KR20170047557A

KR20170047557A - Composition for improving skin wrinkle

Info

Publication number: KR20170047557A
Application number: KR1020150147797A
Authority: KR
Inventors: 서주원; 최봉근; 성금화
Original assignee: 명지대학교 산학협력단
Priority date: 2015-10-23
Filing date: 2015-10-23
Publication date: 2017-05-08
Also published as: KR101773858B1

Abstract

The present invention provides a skin wrinkle alleviating composition including a wormwood extract and a predetermined fusion protein as active ingredients. The fusion protein includes a growth hormone or a fragment thereof and an Fc region of a modified immunoglobulin. The wormwood extract and the fusion protein which are the active ingredients of the composition according to the present invention effectively inhibit, through potentialization, generation of matrix metallo proteinase-1 (MMP-1) or elastase activity. Thus, decomposition of collagen or elastin in intradermic tissue can be minimized, and skin sagging phenomenon or skin wrinkle generation can be prevented or alleviated. Therefore, the composition according to the present invention can be utilized in a medical product, a cosmetic product, a personal care product, or the like for skin antiaging, skin wrinkle alleviation, skin elasticity maintenance and the like.

Description

[Composition for improving skin wrinkle]

The present invention relates to a composition for improving skin wrinkles, and more particularly, to a composition for improving skin wrinkles, which more effectively inhibits the production of collagenase MMP-1 (Matrix Metallo Proteinase-1) or elastinase The present invention relates to a composition for preventing or improving skin wrinkles.

The skin of the human skin consists largely of the epidermis, the dermis, and the subcutaneous fat layer. The basic role of the skin is to prevent water from evaporating from our body and to prevent harmful substances from invading from the outside. These skin are always exposed to external stimuli, so they have various defenses. In addition, it can be said that skin is healthy and clean is related to human beauty. In recent years, not only women but also men have been interested in various skin troubles such as skin aging, and a lot of related products are being developed.

As people get older, skin aging occurs. A typical symptom is wrinkle. The connective tissue involved in the wrinkling of the skin consists mainly of fiber components such as collagen fiber, reticular fiber and elastin fiber. Collagen and elastin in the dermis are important fiber structures that maintain the elasticity and elasticity of the skin by working closely with each other. Collagen is a fibrous tissue that gives skin flexibility and is a major component that accounts for about 70% of dermal tissue. The polypeptide chains of collagen fibers are synthesized in the fibroblast cell with the telopeptide at both ends in the granule cell wall, and then secreted out of the cell in the form of procollagen combined with a triple helical structure. The secreted pro-collagen is cleaved by specific enzymes to produce tropo-collagen, and these are combined with each other in the extracellular matrix to synthesize collagen fibers. The synthesis of such collagen fibers rapidly decreases as the skin ages, and as collagen fibers are deformed by the external environment, wrinkles are generated and deepened in the skin. This collagen degradation involves collagenase, MMP-1 (Matrix Metallo Proteinase-1, or Collagenase I). In addition, elastin, like collagen, is the main fiber structure that determines the elasticity of the skin. Its content is about 2%, which is a small amount compared to collagen, but plays a very important role in skin elasticity because it forms a network structure for supporting the epidermis. Elastin is degraded by enzymes called elastase and continues to be produced by fibroblasts until the age of 18-19 years, but after that, production is stopped and elastin, which is already produced, It is very difficult to recover its elasticity. Such collagen or elastin is a major factor affecting the wrinkling of the skin. In other words, the skin aging phenomenon such as skin wrinkle formation is caused by destruction of collagen, reduction of collagen synthesis, disappearance of elastin, etc., which occurs in skin epidermis but occurs more in dermis than in skin epidermis.

Since Collagenase and Elastase are involved in collagen and elastin degradation, which cause skin wrinkles, inhibitors of these enzymes have been used as cosmetic ingredients for improving skin wrinkles. Peptidylcarbamates, Peptidylthiocar-bamates, Hydroxamates, Cephalosporins, Sulphonate salts, and the like are examples of such components. Examples of conventional materials for promoting collagen synthesis include retinoids (RE36068), TGF-beta (transforming growth factor), betulinic acid (JP8-208424), and substances inhibiting the production of MMP- β (transforming growth factor), and various other natural products are known to inhibit MMP-1 biosynthesis.

Regarding the composition for improving wrinkles containing natural products, Korean Patent Registration No. 10-1292837 discloses a cosmetic composition for improving skin wrinkles containing Junglans mandshurica MAXIM as an active ingredient, Japanese Patent Application Laid-Open No. 10-0702461 discloses a cosmetic composition for improving skin wrinkles containing a dog dandelion extract, and Korean Patent Publication No. 10-0429590 discloses a composition for promoting collagen synthesis comprising a gangue extract.

Further, in recent years, attempts have been made to use a fusion peptide or a fusion protein as an effective ingredient of a composition for improving wrinkles. For example, Korean Patent Laid-Open Publication No. 10-2014-0138162 discloses a cosmetic composition for improving wrinkles containing a peptide that inhibits TRPV1 activity as an active ingredient. In Korean Patent Registration No. 10-1410897, A cosmetic composition for improving wrinkles comprising a PEG-peptide polymer (LAP-KTTKS, PEGylated Lipoic acid-peptide conjugate) is disclosed in Korean Patent Publication No. 10-0753472. Discloses a cosmetic composition for improving skin wrinkles comprising a peptide as an active ingredient and a fusion peptide (Tat-human type-I collagne DP) to which an autopositive Tat peptide is bound.

SUMMARY OF THE INVENTION The present invention has been made in view of the background of the prior art, and an object of the present invention is to provide a method of inhibiting the production of collagenase MMP-1 (Matrix Metallo Proteinase-1) or inhibiting the activity of elastinase Elastase And to provide a composition capable of effectively improving skin wrinkles.

In order to solve the above-mentioned problems, the present invention provides a composition for preventing or improving skin wrinkles comprising an extract of Mugwort and a predetermined fusion protein as an active ingredient. The fusion protein is characterized in that it comprises a growth hormone or a fragment thereof and an Fc region of a modified immunoglobulin.

The mugwort extract and the fusion protein, which are effective ingredients of the composition according to the present invention, effectively inhibit the production of MMP-1 (Matrix Metallo Proteinase-1) or the activity of elastase by synergistic action, Or the degradation of elastin can be minimized, thereby preventing or improving skin sagging phenomenon or skin wrinkle formation. Accordingly, the composition according to the present invention can be utilized as medicines, cosmetics, personal care products, etc. for preventing skin aging, improving skin wrinkles and maintaining skin elasticity.

Fig. 1 is a cleavage map of pAD11-hGH-hyFc, an expression vector of hGH-hyFC fusion protein.
FIG. 2 is a graph showing the effect of Artemisia officinalis extract on the expression level of MMP-1 (Matrix Metallo Proteinase) protein. FIG. 3 is a graph showing the effect of Artemisia sp. to be.
FIG. 4 is a graph showing the effect of Artemisia officinalis Extract on Elastase activity.
FIG. 5 is a graph showing the effect of the hGH-hyFC fusion protein on the elastase activity.
FIG. 6 is a graph showing the effect of the combination of the extract of Artemisia capillaris and the hGH-hyFC fusion protein on the elastase activity.

As used herein, the term "pharmaceutically acceptable" means not significantly stimulating an organism and not interfering with the biological activity and properties of the administered active substance.

The term "prophylactic, " as used herein, refers to any act that inhibits the symptoms of a particular disease or delays progression by administration of the composition of the present invention.

As used herein, the term "improvement" means all actions that at least reduce the degree of symptom associated with the condition being treated.

As used herein, the term "administering" means providing any of the compositions of the present invention to an individual by any suitable method. The term " individual " means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.

As used herein, the term "modified Fc region of immunoglobulin" refers to an Fc region of an immunoglobulin that has been modified such that the activity of antibody-dependent cell-mediated cytotoxicity (ADCC) or CDC (complement-dependent cytotoxicity) For this purpose, the Fc region of immunoglobulin IgG, IgA, IgM, IgD or IgE may be mutated or a part or all of the CH2 and CH3 regions of these immunoglobulins may be used in combination.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, including the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including simultaneously used drugs, and other factors well known in the medical arts.

The terms "protein "," polypeptide ", and "peptide ", as used herein, can be used interchangeably, unless otherwise stated.

Hereinafter, the present invention will be described in detail.

The composition for preventing or improving wrinkles of the skin according to the present invention contains the mugwort extract and a predetermined fusion protein as an active ingredient.

Mugwort extract

In the present invention, the mugwort extract can be prepared by various methods. In order to obtain the mugwort extract of the present invention, various organs or parts of mugwort such as leaves, stems, roots and the like may be used as the raw material, and it is preferable to use leaves or stems which are the ground part of mugwort. In addition, in the present invention, if the mugwort is a perennial herbaceous plant in Artemisia , its kind is not limited to a great extent. For example, Artemisia princeps There are Pampanini , Artemisia princeps. orientalis , Artemisia vulgaris L. , Artemisia capillaris , etc., and Artemisia capillaris ). On the other hand, in order to obtain an extract from mugwort, an extraction process is required. As the extraction method, a conventional extraction method known in the art can be used, for example, a solvent extraction method. The extraction solvent that can be used when preparing the mugwort extract by the solvent extraction method includes water, a lower alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol and butanol) or a mixture thereof, May be selected from the group consisting of water, alcohols, glycols, 1,3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane, It is preferred that the mixture is selected. When water is used as the extraction solvent, the water is preferably hot water. When an alcohol is used as an extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol. In addition, when the functional alcohol is used as the extraction solvent, the alcohol content is preferably 50 to 90%. It is obvious to those skilled in the art that extracts of the mugwort extract can be obtained not only by using the above-mentioned extraction solvent but also by using other extraction solvents, which exhibit substantially the same effect. For example, decompression by carbon dioxide, extraction by supercritical extraction with high temperature, extraction by extraction using ultrasound, separation by ultrafiltration membrane with constant molecular weight cutoff value, various chromatography (size, charge, hydrophobic or affinity The active fraction obtained through various purification and extraction methods, such as separation using the above-described method for separation according to sex, is also included in the extract of the present invention. The supercritical fluid extraction refers to supercritical fluid extraction. Generally, the supercritical fluid is extracted from the liquid and the liquid when the gas reaches the critical point at high temperature and high pressure, (J. Chromatogr. A. 1998; 479: 200-205). In addition, it has been reported that the supercritical fluid has a polarity similar to that of a nonpolar solvent. Carbon dioxide is a supercritical fluid with both liquid and gaseous nature, with the operation of supercritical fluid equipment reaching its critical pressure and temperature, resulting in increased solubility in fat-soluble solutes. When supercritical carbon dioxide passes through an extraction vessel containing a certain amount of sample, the lipophilic substance contained in the sample is extracted into supercritical carbon dioxide. When the supernatant carbon dioxide containing a small amount of cosolvent is passed through the sample remaining in the extraction vessel after extracting the lipid-soluble substance, components that were not extracted by pure supercritical carbon dioxide can be extracted. The supercritical fluid used in the supercritical extraction method of the present invention can effectively extract an active ingredient by using a mixed fluid in which a cosolvent is further mixed with supercritical carbon dioxide or carbon dioxide. These co-solvents may be used alone or in admixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether. Most of the extracted samples contain carbon dioxide. Since the carbon dioxide is volatilized into air at room temperature, the extract obtained by the above method can be used as a cosmetic composition, and the cosolvent can be removed by a reduced pressure evaporator. In addition, the ultrasonic extraction method is an extraction method using energy generated by ultrasonic vibration. Ultrasonic waves can destroy an insoluble solvent contained in a sample in a water-soluble solvent. Due to the high local temperature, Since the kinetic energy of the reactant particles is increased, sufficient energy required for the reaction is obtained. By inducing the high pressure by the shock effect of the ultrasonic energy, the mixing effect of the substance contained in the sample and the solvent is enhanced, thereby increasing the extraction efficiency. As the extraction solvent which can be used for the ultrasonic extraction method, one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether can be used. The extracted sample is recovered by vacuum filtration, and the filtrate is recovered, and the extract is removed by a vacuum evaporator and freeze-dried to obtain an extract.

Fusion protein

In the present invention, the fusion protein comprises a Fc region of a growth hormone or a fragment thereof and a modified immunoglobulin. In the present invention, a growth hormone is a peptide hormone capable of promoting growth of bone, cartilage and the like in humans and other animals. In addition, in the present invention, the fragment of the growth hormone is a peptide of the minimum length having a certain level of the function of the growth hormone. The growth hormone or fragment thereof constituting the fusion protein may be derived from a human or a mouse, and is preferably a human growth hormone (hGH) derived from a human. The growth hormone constituting the fusion protein may be, for example, a polypeptide having the amino acid sequence of SEQ ID NO: 1. The polypeptide having the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 3.

The fusion protein used as an active ingredient of the composition for preventing or improving skin wrinkles according to the present invention has a form in which a growth hormone or a fragment thereof is bound to an Fc region of a modified immunoglobulin, (I) < / RTI > Further, in the present invention, the fusion protein may preferably be a dimer in which two polypeptides represented by the following formula (I) are bound by respective IgFc domains.

X- (L) _w1- IgFc (I)

In the above formula (I), w1 is 0 or 1; X is a growth hormone or fragment thereof; L is a linker; IgFc is the Fc region of the modified immunoglobulin.

In the above formula (I), the growth hormone or a fragment thereof is preferably derived from a human and has, for example, the amino acid sequence of SEQ ID NO: 1. In addition, in the above formula (I), the growth hormone or a fragment thereof may be directly bound to the Fc region of the modified immunoglobulin or may be bound through a linker. The linker may be linked to the N-terminus, C-terminus or free radical of the Fc fragment and may also be linked to the N-terminus, C-terminus or free radical of the growth hormone. If the linker is a peptide linker, the linkage can occur at any linkage site. When the linker and the Fc are bound to each other after being separately expressed, the coupling may be accomplished using any of a variety of crosslinking agents known in the art. Examples of the cross-linking agent include 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, 4-azisosalicylic acid (4- such as N-hydroxysuccinimide ester, such as azidosalicylic acid, 3,3'-dithiobis (succinimidylpropionate) But are not limited to, imidoesters including disuccinimidyl esters, and bifunctional maleimides such as bis-N-maleimido-1,8-octane. In addition, the linker may be an albumin linker or a peptide linker. As an example, the peptide linker may be composed of 5 to 20 amino acids. The peptide linker may also be a polypeptide consisting of a glycine (Gly, G) residue and a serine (Ser, S) residue. The peptide linker may comprise an amino acid sequence of GSGGGS, GSGGGGSGGGS or GGGGSGGGSGGGSG.

Further, the Fc region of the immunoglobulin modified in the above formula (I) may be composed of a combination of the Fc region of IgD and the Fc region of IgG4. The Fc region is modified so that binding and / or complement binding does not occur with the Fc receptor. In particular, the Fc region of the modified immunoglobulin comprises a hinge region, a CH2 domain and a CH3 domain in the N-terminal to C-terminal direction. Wherein the hinge region comprises a human IgD hinge region, wherein the CH2 domain comprises a portion of an amino acid residue of a human IgD and a CH2 domain of a human IgG4, wherein the CH3 domain comprises a portion of an amino acid residue of the CH3 domain of human IgG4 Lt; / RTI > As used herein, the term "Fc region", "Fc fragment" or "Fc" refers to the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, Region and light chain constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region. In addition, the Fc region of the modified immunoglobulin has FcRn binding ability, so that the immunoglobulin can be recirculated and actively transported. FcRn is a protein found in the membranes of a number of cells. Immunoglobulins with increased binding activity for FcRn at neutral pH will bind to FcRn on the cell surface where the receptor is recirculated to the surface of the cell via internalization into the cell in the follicle along with the antibody. Hybrid Fc or hybrid Fc fragments are sometimes referred to herein as "hFc" or "hyFc ". As used herein, the term "Fc region variant" means that some amino acids in the Fc region are substituted or produced by combining different Fc regions. The Fc region variant may be modified to prevent cleavage at the hinge region. Specifically, the 144th amino acid and / or the 145th amino acid of SEQ ID NO: 8 can be modified. Preferably, the mutant may be a mutant in which K, which is the 144th amino acid of SEQ ID NO: 8, is substituted by G or S, and E in the 145th amino acid is substituted by G or S. In addition, the Fc region or Fc region variant of the modified immunoglobulin can be expressed by the following formula (II).

N '- (Z1) p-Y-Z2-Z3-Z4-C' (II)

In the above formula (II), N 'is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; p is an integer of 0 or 1; Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from the 98 position in the amino acid residues 90 to 98 of SEQ ID NO: 8; Y is an amino acid sequence having 5 to 64 consecutive amino acid residues in the N-terminal direction from the 162 position in the amino acid residues 99 to 162 of SEQ ID NO: 8; Z2 is an amino acid sequence having 4 to 37 contiguous amino acid residues in the C-terminal direction from the 163 position in the amino acid residue at positions 163 to 199 of SEQ ID NO: 8; Z3 is an amino acid sequence having 71 to 106 contiguous amino acid residues in the N-terminal direction from the 220 position in the amino acid residues 115 to 220 of SEQ ID NO: 9; Z4 is an amino acid sequence having 80 to 107 amino acid sequences from the 221 position to the C-terminal direction in the amino acid residues 221 to 327 of SEQ ID NO:

The modified Ig Fc domain may be as described in U.S. Patent No. 7,867,491 and the production of the modified Ig Fc domain may be performed with reference to what is described in U.S. Patent No. 7,867,491. In addition, the Fc fragment of the present invention may be a natural type sugar chain, an increased sugar chain compared to the native type, a reduced sugar chain compared to the native type, or a form in which the sugar chain is removed. Increase, decrease or elimination of immunoglobulin Fc sugar chains can be performed by conventional methods known in the art such as chemical methods, enzymatic methods and genetic engineering engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment rapidly reduces the binding affinity of the primary complement component C1 to C1q and results in a decrease or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) Thereby not inducing unnecessary immune responses in vivo. In this regard, immunoglobulin Fc fragments in the deglycosylated and aglycosylated form may be more suitable for the purposes of the present invention. As used herein, the term " deglycosylation "means enzymatically eliminating sugar from the Fc fragment and the term" aglycosylation "means that the Fc fragment is expressed in a prokaryote, preferably E. coli Which means that it is produced in an unglycosylated form. In addition, the Fc region of the modified immunoglobulin may include a polypeptide having an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: The polypeptide having the amino acid sequence of SEQ ID NO: 5 may be encoded by the nucleotide sequence of SEQ ID NO: In addition, the polypeptide having the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 can be encoded by the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: .

In addition, the fusion protein of the present invention may preferably be a polypeptide having the amino acid sequence of SEQ ID NO: 18.

The specific form of the composition

Since the composition of the present invention contains the mugwort extract and the fusion protein which can effectively inhibit the production of MMP-1 (Matrix Metallo Proteinase-1) or the activity of elastase by the synergistic action, Aging, sagging of the skin, wrinkles of the skin, and the like. In the composition of the present invention, the weight ratio of the active ingredient, mugwort extract to fusion protein is not particularly limited and is preferably 1: 1 to 1:20, more preferably 1: 5 to 1:15, desirable. The composition of the present invention may be formulated into a pharmaceutical composition, a cosmetic composition, a personal care product or the like depending on the intended use or aspect, and the content of the fusion protein, which is an active ingredient in the composition, And may be adjusted in various ranges depending on the intended use or aspect.

The content of the active ingredient in the pharmaceutical composition according to the present invention is not particularly limited and is, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, more preferably 1 to 30% by weight, Lt; / RTI > In addition, the pharmaceutical composition according to the present invention may further contain, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, excipient or diluent. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may further contain at least one known active ingredient (for example, hyaluronic acid, retinoic acid, etc.) having wrinkle-improving effect in addition to the mugwort extract and the fusion protein. The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration by a conventional method, and can be formulated into a pharmaceutical composition such as a filler, an extender, a binder, a wetting agent, a disintegrant, Diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be administered orally or parenterally to a mammal including a human according to a desired method. Examples of the parenteral administration method include external dermal application, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, Intravenous injection or intra-thoracic injection. The dosage of the pharmaceutical composition of the present invention is not limited as long as it is a pharmacologically effective amount and is not limited as long as it depends on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, Varies. In addition, the pharmaceutical composition of the present invention can be administered once a day or divided into several times. The pharmaceutical composition according to the present invention is preferably prepared in the form of a skin external preparation such as a cream, a gel, a patch, a spray, a research agent, a warning agent, a lotion, a liniment, a pasta agent or a cataplasma agent Do.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. Specifically, the cosmetic composition of the present invention can be prepared in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder. When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide Can be used. When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan. When the formulation of the cosmetic composition of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, Propellants such as carbon, propane / butane or dimethyl ether. When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester. The cosmetic composition according to the present invention is preferably prepared in the form of a lotion, a tonic, a conditioner, an essence, a lotion, a cream, an aerosol, a pack, a soap, a moisturizer and a spray in consideration of the mode of use.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

1. Human Growth Hormone and Immunoglobulin Fc Lt; / RTI > fusion protein hGH - hyFC Fusion proteins)

(1) Genetic constructing of hGH-hyFC fusion protein

The recombinant protein is fused to the human growth hormone (hGH) gene without the function of ADCC (antibody dependent cell-mediated cytotoxicity) and CDC (complement dependent cytotoxicity) .

Specifically, in order to prepare an expression vector containing a human growth hormone (hGH) gene, a known human growth hormone (hGH) having the amino acid sequence of SEQ ID NO: 1 and a signal peptide ) (GenBank Accession number: AAA98618.1) gene was used. On the other hand, the gene of human growth hormone (hGH) is composed of the nucleotide sequence of SEQ ID NO: 3. In addition, the signal peptide is located at the N-terminus of the secretory protein or membrane protein and has the amino acid sequence of SEQ ID NO: 7 as a peptide which becomes a signal when the protein passes through the membrane. Can be. The signal peptide is removed during transport of the target protein outside the cell. A construct containing human growth hormone (hGH) was prepared and fused with the modified Fc domain to obtain a recombinant protein expression vector pAD11-hGH-hyFc. Fig. 1 is a cleavage map of pAD11-hGH-hyFc, an expression vector of hGH-hyFC fusion protein. pAD11-hGH-hyFc, which is an expression vector of hGH-hyFC fusion protein, is composed of the nucleotide sequence of SEQ ID NO: 4.

To insert the fusion gene into the expression vector pAD11, an EcoR I site was generated at the 5 'end of the gene sequence of human growth hormone and an Xba I site was generated at the 3' end of the termination codon of hyFc. The expression vector pAD11 was obtained from the RcCMV backbone (available from Invitrogen, Carlsbad). pAD11 includes promoters derived from CMV (cytomegalovirus), poly (A) sequences derived from asthma growth hormone, gIVS (globin intervening sequence) derived from rabbit beta globin (Mol Cell Biol, 1988 : 4395) and other elements. To prepare the pAD11 vector, the RcCMV vector (Invitrogen) is modified in several places. First, the neomycin resistant site was removed by Xho I enzyme and gIVS was added 3 'to the CMV promoter site. In addition, a mouse DHFR (dihydrofolate reductase) gene (Pubmed, NM 010049) was added to the 5 'of the CMV promoter. The Nhe I site was generated at the 3 'end of the coding sequence and at the 5' end of the coding sequence of hyFc in order to produce a linkage between the 3 'end of human growth hormone and the 5' end of hyFc within the frame. After subcloning with each restriction enzyme site, the final expression vector was prepared.

For more detailed modification of the Fc domain, US Pat. No. 7,867,491 describes a hyFc (hybrid Fc) protein. The hyFc protein is a hybrid type of Fc of human IgD and Fc of human IgG4 and can exhibit an excellent half-life of the body when bound to a bioactive protein as compared with the Fc region of a previously modified immunoglobulin. In this experiment, a recombinant protein expression vector was prepared using the gene base sequence of the hyFc protein having the amino acid sequence of SEQ ID NO: 5 (SEQ ID NO: 6). The hyFc protein having the amino acid sequence of SEQ ID NO: 5 has 9 amino acids (90-98) at the C-terminal of IgD CH1 domain, 30 amino acids (133-162) of the hinge region of IgD, 8 Amino acids (shtqplgv 163-170), 100 amino acids (121-220) of the IgG4 CH2 domain, and 107 amino acids (221-327) of the IgG4 CH3 domain.

The present invention relates to hFc-2, hFc-3, hFc-4, hFc-5 and hFc-6 described in the examples of Korean Patent Registration No. 10-0897938 See all manufacturing methods. The hyFc protein used in the examples of the present invention is the same hybrid protein as hFc-5 described in the example of Korean Patent Registration No. 10-0897938. hFc-2 (SEQ ID NO: 10), hFc-3 (SEQ ID NO: 11), hFc-4 (SEQ ID NO: 12) and hFc-6 (SEQ ID NO: 13) have the same CH2 and CH3 sites as hyFc, . The hFc-2 (SEQ ID NO: 10), hFc-3 (SEQ ID NO: 11), hFc-4 (SEQ ID NO: 12) and hFc- 162), 10 amino acids (153-162), 20 amino acids (143-162), and 64 amino acids (99-162).

In addition, the present invention relates to a method for producing a recombinant protein expression vector pAD11-hGH-hyFc, comprising the steps of: pAD11 EPO-hFc-2, pAD11 EPO-hFc-3 described in the examples of Korean Patent Registration No. 10-0897938 , pAD11 EPO-hFc-4, pAD11 EPO-hFc-5, pAD11 G-CSF-hFc-2, pAD11 G-CSF-hFc-3, pAD11 EPO- See also all methods for producing expression vectors such as hFc-2, pAD11 p40N303Q-hFc-3, pAD11 p40N303Q-hFc-4, pAD11 p40N303Q-hFc-5, and pAD11 TNFR-hFc-5. In the example of Korean Patent Registration No. 10-0897938, a biologically active molecule such as human growth hormone (hGH) is hybridized with hFc-2, hFc-3, hFc-4, hFc-5 and hFc- A method for producing a vector for expressing a fusion protein having a form bound to a protein and a method for producing a fusion protein using the vector are described in detail.

PAD11-hGH-hyFc, a recombinant protein expression vector, was transfected into a CHO DG44 cell line (supplier: Columbia University, USA). Thereafter, the cells were cultured for 7 days, and the productivity of the cell line was confirmed from the culture.

(2) Securing hGH-hyFC fusion protein

The expression level of hGH-hyFC fusion protein produced using the CHO production system was evaluated by ELISA assay. In order to separate and purify the hGH-hyFC fusion protein from the culture, the protein purification process was monitored over time under UV 280 nm using protein A resin. Specifically, a peak appearing when an elution buffer (0.1 M glycine, pH 3.0) was passed through the purification column was collected and analyzed by SE-HPLC analysis to confirm that the target protein eluted. The size was also confirmed by SDS-PAGE analysis. As a result, the hGH-hyFC fusion protein could be obtained in high yield from the CHO cell line. The obtained hGH-hyFC fusion protein was dissolved in a predetermined buffer (20 mM Glycine, 5 mM Tris, pH 7.0) and used.

2. Artemisia spp. Artemisia capillaris ) Preparation of extract

Artemisia capillaris ; Hwasun Porphyry Company, Korea) Stems and leaves were washed twice with distilled water and then dried at room temperature for 2 days. 1500 g of 70% alcohol was added to 1000 g of dried aruminax, and the mixture was extracted at room temperature for about 24 hours. Thereafter, the extract was filtered with a filter paper having a pore size of 5 mu m or less, and the filtered extract was concentrated under reduced pressure. Thereafter, the concentrated extract was lyophilized and homogenized with a blender to obtain 96.7 g of Artemisia sp. The obtained powder of Artemisia sp. Extract was dissolved in dimethyl sulfoxide (DMSO).

3. In-vitro test for wrinkle-improving efficacy

(1) MMP-1 (Matrix Metallo Proteinase) protein expression level confirmation test

Human dermal fibroblasts were inoculated in a 6-well plate to a concentration of 2 × 10 ⁵ / ml per well using DMEM medium containing FBS. After 24 hours of inoculation, the medium was discarded, changed to DMEM medium containing no FBS, and cultured in starvation. IL-1α (Interlukin-1α) was treated to a concentration of 10 ng / ㎖ in the wells of the other groups except for the control group after 24 hours of incubation in starvation. IL-1α (Interlukin-1α), an inflammatory cytokine, stimulates the fibroblast of the dermal layer and promotes and activates the production of elastase or collagenase to produce elastin or collagen lt; RTI ID = 0.0 > collagen. < / RTI > Subsequently, drug samples such as retinoic acid and Artemisia princeps extract were treated to a predetermined concentration in the wells containing IL-1α (Interlukin-1α). After 48 hours of treatment, the medium was discarded in a 6-well plate and cold PBS was added to stop the reaction. The cells were lysed by adding 120 μl of lysis buffer to each well, discarding the cell lysate in each well using a cell scraper on ice, and transferring the cell lysate to the E-tube Transferred and placed on ice for 5 minutes. Then, the cell lysate in the E-tube was centrifuged at 12,000 rpm and 4 ° C for 20 minutes to take the supernatant, and the supernatant was transferred to a new E-tube. Proteins in the supernatant were quantified using the BCA protein assay kit. The quantified protein was mixed with 5 × sample buffer, heated at 100 ° C for 5 minutes in a heat block, and stored frozen.

Thereafter, protein levels of MMP-1 (Matrix Metallo Proteinase) protein were analyzed by SDS-PAGE from frozen-stored protein samples. Specifically, 20 to 30 μl of the protein sample was loaded on 10% acrylamide gel, subjected to electrophoresis, and transferred to a PVDF membrane for 30 minutes using Trans-Blot Turbo ™. Then, the transferred PVDF membrane was blocked with skim milk for 1 hour, and then the blocking buffer was removed and washed with TBS-T. After washing, primary antibody diluted with Can get signal solution was added to the PVDF membrane and incubated overnight at 4 ° C with slow shaking. Then, the PVDF membrane was washed with 1 × TBS-T for 30 minutes. After washing, the secondary antibody was added to the PVDF membrane and reacted at room temperature for 1 hour. After completion of the reaction, the PVDF membrane was washed with 1 × TBS-T for 30 minutes, the PVDF membrane was reacted with the ECL solution, and protein expression was confirmed using a chemiscope.

(2) Elastase activity confirmation test

Human dermal fibroblasts were inoculated in a 6-well plate to a concentration of 2 × 10 ⁵ / ml per well using DMEM medium containing FBS. After 24 hours of inoculation, the medium was discarded, changed to DMEM medium containing no FBS, and cultured in starvation. IL-1α (Interlukin-1α) was treated to a concentration of 10 ng / ㎖ in the wells of the other groups except for the control group after 24 hours of incubation in starvation. IL-1α (Interlukin-1α), an inflammatory cytokine, stimulates the fibroblast of the dermal layer and promotes and activates the production of elastase or collagenase to produce elastin or collagen lt; RTI ID = 0.0 > collagen. < / RTI > Then, drug samples such as retinoic acid, hGH-hyFC fusion protein, and Artemisia princeps extract were treated to a predetermined concentration in the wells containing IL-1α (Interlukin-1α). After 48 hours of treatment, the medium was discarded in a 6-well plate and cold PBS was added to stop the reaction. Then, PBS was discarded in a 6-well plate, and 150 μl of lysis buffer was added to each well. Cells in each well were lysed using a cell scraper on ice. After cell lysis, the cell lysate was transferred to an E-tube of 1.5 ml capacity and centrifuged at 13,000 rpm and 4 ° C for 15 minutes to take the supernatant. Subsequently, 90 상 of the supernatant was dispensed into each well of a 96-well plate, and 1.8 ㎕ of STANA, a specific substrate of elastase, was added to each well and reacted in an incubator at 37 캜 for 2 hours. Thereafter, the absorbance of the reaction solution was measured at a wavelength of 405 nm, and the elastase activity was calculated relative to a control (control) in which IL-1? (Interlukin-1?) And the drug sample were not treated. Then, protein was quantified using BCA protein assay kit, and elastase / protein graph was prepared.

(3) Test for confirming the efficacy of waxy extract for improving wrinkles

FIG. 2 is a graph showing the effect of Artemisia officinalis extract on the expression level of MMP-1 (Matrix Metallo Proteinase) protein. FIG. 3 is a graph showing the effect of Artemisia sp. to be. 4 is a graph showing the effect of Artemisia sp. Extract on the activity of elastase. In Figs. 2 and 4, "RA" represents retinoic acid. As shown in FIGS. 2 to 4, the extract of Artemisia cinerea effectively inhibited MMP-1 (Matrix Metallo Proteinase) protein expression level and elastase activity at a concentration of 30 μg / μl.

(4) Results of wrinkle-improving efficacy test of hGH-hyFC fusion protein

FIG. 5 is a graph showing the effect of the hGH-hyFC fusion protein on the elastase activity. In Fig. 5, "RA" represents retinoic acid. As shown in FIG. 5, the hGH-hyFC fusion protein effectively inhibited the elastase activity at a concentration of 90 μg / μl.

(5) Results of wrinkle-improving efficacy test according to the combination of Artemisia japonica extract and hGH-hyFC fusion protein

FIG. 6 is a graph showing the effect of the combination of the extract of Artemisia capillaris and the hGH-hyFC fusion protein on the elastase activity. In Fig. 6, "Artemisia sp." Represents Artemisia sp. In FIG. 6, "low concentration" represents the case where the treatment concentration of the extract of Artemisia officinalis is 1 μg / μl or the treatment concentration of the hGH-hyFC fusion protein is 10 μg / Mu] g / mu l or the treatment concentration of hGH-hyFC fusion protein is 30 [mu] g / mu l, and the "high concentration" . As shown in FIG. 6, when the extract of Artemisia japonica and the hGH-hyFC fusion protein was used in combination, the elastase activity was effectively inhibited at a much lower concentration than that of the extract of Artemisia japonica extract or hGH-hyFC fusion protein alone, This result is presumed to be due to the synergistic effect of the combination of the extract of Artemisia princeps and the hGH-hyFC fusion protein.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the scope of the present invention should be construed as including all embodiments falling within the scope of the appended claims.

<110> Myongji University Industry and Academia Cooperation Foundation <120> Composition for improving skin wrinkle <130> DP-15-915 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 191 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) (191) Human growth hormone (Genbank accession No. AIA66930.1) <400> 1 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 <210> 2 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> Gene for encoding signal peptide and human growth hormone (Genbank accession No. AAA98618.1) <400> 2 atggccaccg gcagccgcac cagcctgctg ctggccttcg gcctgctgtg cctgccctgg 60 ctgcaggagg gcagcgcctt ccccaccatc cccctgagcc gcctgttcga caacgccatg 120 ctgcgcgccc accgcctgca ccagctggcc ttcgacacct accaggagtt cgaggaggcc 180 tacatcccca aggagcagaa gtacagcttc ctgcagaacc cccagaccag cctgtgcttc 240 agcgagagca tccccacccc cagcaaccgc gaggagaccc agcagaagag caacctggag 300 ctgctgcgca tcagcctgct gctgatccag agctggctgg agcccgtgca gttcctgcgc 360 agcgtgttcg ccaacagcct ggtgtacggc gccagcgaca gcaacgtgta cgacctgctg 420 aaggacctgg aggagggcat ccagaccctg atgggccgcc tggaggacgg cagcccccgc 480 accggccaga tcttcaagca gacctacagc aagttcgaca ccaacagcca caacgacgac 540 gccctgctga agaactacgg cctgctgtac tgcttccgca aggacatgga caaggtggag 600 accttcctgc gcatcgtgca gtgccgcagc gtggagggca gctgcggctt c 651 <210> 3 <211> 573 <212> DNA <213> Artificial Sequence <220> <223> gene for encoding human growth hormone <400> 3 ttccccacca tccccctgag ccgcctgttc gacaacgcca tgctgcgcgc ccaccgcctg 60 caccagctgg ccttcgacac ctaccaggag ttcgaggagg cctacatccc caaggagcag 120 aagtacagct tcctgcagaa cccccagacc agcctgtgct tcagcgagag catccccacc 180 cccagcaacc gcgaggagac ccagcagaag agcaacctgg agctgctgcg catcagcctg 240 ctgctgatcc agagctggct ggagcccgtg cagttcctgc gcagcgtgtt cgccaacagc 300 ctggtgtacg gcgccagcga cagcaacgtg tacgacctgc tgaaggacct ggaggagggc 360 atccagaccc tgatgggccg cctggaggac ggcagccccc gcaccggcca gatcttcaag 420 cagacctaca gcaagttcga caccaacagc cacaacgacg acgccctgct gaagaactac 480 ggcctgctgt actgcttccg caaggacatg gacaaggtgg agaccttcct gcgcatcgtg 540 cagtgccgca gcgtggaggg cagctgcggc ttc 573 <210> 4 <211> 7082 <212> DNA <213> Artificial Sequence <220> <223> pAD11-hGH-hyFc <400> 4 gacggatcgg gactagagca ttgggggggg ggacagctca gggctgcgat ttcgcgccaa 60 acttgacggc aatcctagcg tgaaggctgg taggatttta tccccgctgc catcatggtt 120 cgaccattga actgcatcgt cgccgtgtcc caaaatatgg ggattggcaa gaacggagac 180 ctaccctggc ctccgctcag gaacgagttc aagtacttcc aaagaatgac cacaacctct 240 tcagtggaag gtaaacagaa tctggtgatt atgggtagga aaacctggtt ctccattcct 300 gagaagaatc gacctttaaa ggacagaatt aatatagttc tcagtagaga actcaaagaa 360 ccaccacgag gagctcattt tcttgccaaa agtttggatg atgccttaag acttattgaa 420 caaccggaat tggcaagtaa agtagacatg gtttggatag tcggaggcag ttctgtttac 480 caggaagcca tgaatcaacc aggccacctc agactctttg tgacaaggat catgcaggaa 540 tttgaaagtg acacgttttt cccagaaatt gatttgggga aatataaact tctcccagaa 600 tacccaggcg tcctctctga ggtccaggag gaaaaaggca tcaagtataa gtttgaagtc 660 tacgagaaga aagactaaca ggaagatgct ttcaagttct ctgctcccct cctaaagcta 720 tgcattttta taagaccatg ggacttttgc tggctttaga tctttgtgaa ggaaccttac 780 ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat 840 ataaaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag 900 attccaacct atggaactga tgaatgggag cagtggtgga atgcctttaa tgaggaaaac 960 ctgttttgct cagaagaaat gccatctagt gatgatgagg ctactgctga ctctcaacat 1020 tctactcctc caaaaaagaa gagaaaggta gaagacccca aggactttcc ttcagaattg 1080 ctaagttttt tgagtcatgc tgtgtttagt aatagaactc ttgcttgctt tgctatttac 1140 accacaaagg aaaaagctgc actgctatac aagaaaatta tggaaaaata ttctgtaacc 1200 tttataagta ggcataacag ttataatcat aacatactgt tttttcttac tccacacagg 1260 catagagtgt ctgctattaa taactatgct caaaaattgt gtacctttag ctttttaatt 1320 tgtaaagggg ttaataagga atatttgatg tatagtgcct tgactagaga tcataatcag 1380 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa 1440 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg 1500 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 1560 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct cccgatcccc 1620 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc 1680 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc 1740 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc 1800 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat 1860 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 1920 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 1980 atgggcct tccattgacg tcaatgggtg gagtatttac 2040 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 2100 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 2160 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 2220 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 2280 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 2340 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 2400 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata 2460 cgactcacta tagggagacc caagctggct agcgtgagtt tggggaccct tgattgttct 2520 ttctttttcg ctattgtaaa attcatgtta tatggagggg gcaaagtttt cagggtgttg 2580 tttagaacgg gaagatgtcc cttgtatcac catggaccct catgataatt ttgtttcttt 2640 cactttctac tctgttgaca accattgtct cctcttattt tcttttcatt ttctgtaact 2700 ttttcgttaa actttagctt gcatttgtaa cgaattttta aattcacttt tgtttatttg 2760 tcagattgta agtactttct ctaatcactt ttttttcaag gcaatcaggg tatattatat 2820 tgtacttcag cacagtttta gagaacaatt gttataatta aatgataagg tagaatattt 2880 ctgcatataa attctggctg gcgtggaaat attcttattg gtagaaacaa ctacatcctg 2940 gtcatcatcc tgcctttctc tttatggtta caatgatata cactgtttga gatgaggata 3000 aaatactctg agtccaaacc gggcccctct gctaaccatg ttcatgcctt cttctttttc 3060 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg 3120 taatacgact cactataggg cgaattgaag cttggtaccg agctcggatc cactagtcca 3180 gtgtggtgga attcatggcc accggcagcc gcaccagcct gctgctggcc ttcggcctgc 3240 tgtgcctgcc ctggctgcag gagggcagcg ccttccccac catccccctg agccgcctgt 3300 tcgacaacgc catgctgcgc gcccaccgcc tgcaccagct ggccttcgac acctaccagg 3360 agttcgagga ggcctacatc cccaaggagc agaagtacag cttcctgcag aacccccaga 3420 ccagcctgtg cttcagcgag agcatcccca cccccagcaa ccgcgaggag acccagcaga 3480 agagcaacct ggagctgctg cgcatcagcc tgctgctgat ccagagctgg ctggagcccg 3540 tgcagttcct gcgcagcgtg ttcgccaaca gcctggtgta cggcgccagc gacagcaacg 3600 tgtacgacct gctgaaggac ctggaggagg gcatccagac cctgatgggc cgcctggagg 3660 acggcagccc ccgcaccggc cagatcttca agcagaccta cagcaagttc gacaccaaca 3720 gccacaacga cgacgccctg ctgaagaact acggcctgct gtactgcttc cgcaaggaca 3780 tggacaaggt ggagaccttc ctgcgcatcg tgcagtgccg cagcgtggag ggcagctgcg 3840 gcttccgcaa caccggccgc ggcggcgagg agaagaagaa ggagaaggag aaggaggagc 3900 aggaggagcg cgagaccaag acccccgagt gccccagcca cacccagccc ctgggcgtgt 3960 tcctgttccc ccccaagccc aaggacaccc tgatgatcag ccgcaccccc gaggtgacct 4020 gcgtggtcgt ggatgtgagc caggaagatc ccgaagtgca gttcaactgg tacgtggatg 4080 gcgtggaagt gcacaacgcc aagaccaagc ccagagaaga gcagttcaac tccacctaca 4140 gagtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag gagtacaagt 4200 gcaaggtgtc caacaaaggc ctgcccagct ccatcgagaa gaccatcagc aaagccaaag 4260 gccagccag agaaccccag gtgtacaccc tgcctcccag ccaggaagag atgaccaaga 4320 accaggtgtc cctgacctgc ctggtgaaag gcttctaccc cagcgacatc gccgtggagt 4380 gggaaagcaa cggccagccc gagaacaatt acaagacaac ccctcccgtg ctggatagcg 4440 atggcagctt ctttctgtac agcagactga ccgtggacaa gagcagatgg caggaaggca 4500 acgtgttcag ctgcagcgtg atgcacgaag ccctgcacaa ccactacacc cagaagagcc 4560 tgtccctgag cctgggcaag tgactcgagt ctagagggcc ctattctata gtgtcaccta 4620 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 4680 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 4740 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 4800 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 4860 gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgagagc ttggcgtaat 4920 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 4980 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 5040 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 5100 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 5160 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 5220 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 5280 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5340 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5400 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5460 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5520 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5580 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5640 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5700 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5760 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5820 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5880 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5940 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 6000 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 6060 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 6120 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 6180 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 6240 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 6300 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 6360 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 6420 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 6480 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6540 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6600 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6660 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6720 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6780 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6840 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6900 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6960 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 7020 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 7080 tc 7082 <210> 5 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hyFc <400> 5 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 6 <211> 735 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for encoding hyFc <400> 6 cgcaacaccg gccgcggcgg cgaggagaag aagaaggaga aggagaagga ggagcaggag 60 gagcgcgaga ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg 120 ttccccccca agcccaagga caccctgatg atcagccgca cccccgaggt gacctgcgtg 180 gtcgtggatg tgagccagga agatcccgaa gtgcagttca actggtacgt ggatggcgtg 240 gaagtgcaca acgccaagac caagcccaga gaagagcagt tcaactccac ctacagagtg 300 gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaag 360 gtgtccaaca aaggcctgcc cagctccatc gagaagacca tcagcaaagc caaaggccag 420 cccagagaac cccaggtgta caccctgcct cccagccagg aagagatgac caagaaccag 480 gtgtccctga cctgcctggt gaaaggcttc taccccagcg acatcgccgt ggagtgggaa 540 agcaacggcc agcccgagaa caattacaag acaacccctc ccgtgctgga tagcgatggc 600 agcttctttc tgtacagcag actgaccgtg gacaagagca gatggcagga aggcaacgtg 660 ttcagctgca gcgtgatgca cgaagccctg cacaaccact acacccagaa gagcctgtcc 720 ctgagcctgg gcaag 735 <210> 7 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of signal peptide <400> 7 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala 20 25 <210> 8 <211> 383 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1). (383) Human IgD constant region (Genbank accession No. P01880) <400> 8 Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg 1 5 10 15 His Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly 20 25 30 Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser 35 40 45 Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr 50 55 60 Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly 65 70 75 80 Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu 85 90 95 Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro 100 105 110 Thr Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala 115 120 125 Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys 130 135 140 Glu Lys Glu Lys Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu 145 150 155 160 Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala 165 170 175 Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val 180 185 190 Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly 195 200 205 Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser 210 215 220 Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu 225 230 235 240 Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu 245 250 255 Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro 260 265 270 Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala 275 280 285 Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile 290 295 300 Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe 305 310 315 320 Ala Pro Ala Arg Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala 325 330 335 Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr 340 345 350 Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala 355 360 365 Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met 370 375 380 <210> 9 <211> 327 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) (327) Partial human IgG4 constant region (Genbank accession No. 2). AAH25985) <400> 9 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 10 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hFc-2 <400> 10 Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 115 120 125 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 11 <211> 225 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hFc-3 <400> 11 Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu 1 5 10 15 Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 20 25 30 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 35 40 45 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 50 55 60 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 65 70 75 80 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 85 90 95 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 100 105 110 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 115 120 125 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 130 135 140 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 145 150 155 160 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 165 170 175 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 180 185 190 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 195 200 205 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 210 215 220 Lys 225 <210> 12 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hFc-4 <400> 12 Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr 1 5 10 15 Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro 20 25 30 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 35 40 45 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 50 55 60 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 65 70 75 80 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 85 90 95 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 100 105 110 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 115 120 125 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 130 135 140 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 145 150 155 160 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 165 170 175 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 180 185 190 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 195 200 205 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 210 215 220 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 225 230 235 <210> 13 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hFc-6 <400> 13 Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu Ser Pro Lys 1 5 10 15 Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala Glu Gly Ser 20 25 30 Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg 35 40 45 Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu 50 55 60 Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly 65 70 75 80 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 85 90 95 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 100 105 110 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 115 120 125 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 130 135 140 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 145 150 155 160 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 165 170 175 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 180 185 190 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 195 200 205 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 210 215 220 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 225 230 235 240 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 245 250 255 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 275 280 285 <210> 14 <211> 660 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for encoding hFc-2 <400> 14 acccccgagt gccccagcca cacccagccc ctgggcgtgt tcctgttccc ccccaagccc 60 aaggacaccc tgatgatcag ccgcaccccc gaggtgacct gcgtggtcgt ggatgtgagc 120 caggaagatc ccgaagtgca gttcaactgg tacgtggatg gcgtggaagt gcacaacgcc 180 aagaccaagc ccagagaaga gcagttcaac tccacctaca gagtggtgag cgtgctgacc 240 gtgctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtgtc caacaaaggc 300 ctgcccagct ccatcgagaa gaccatcagc aaagccaaag gccagcccag agaaccccag 360 gtgtacaccc tgcctcccag ccaggaagag atgaccaaga accaggtgtc cctgacctgc 420 ctggtgaaag gcttctaccc cagcgacatc gccgtggagt gggaaagcaa cggccagccc 480 gagaacaatt acaagacaac ccctcccgtg ctggatagcg atggcagctt ctttctgtac 540 agcagactga ccgtggacaa gagcagatgg caggaaggca acgtgttcag ctgcagcgtg 600 atgcacgaag ccctgcacaa ccactacacc cagaagagcc tgtccctgag cctgggcaag 660 660 <210> 15 <211> 675 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for encoding hFc-3 <400> 15 gagcgcgaga ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg 60 ttccccccca agcccaagga caccctgatg atcagccgca cccccgaggt gacctgcgtg 120 gtcgtggatg tgagccagga agatcccgaa gtgcagttca actggtacgt ggatggcgtg 180 gaagtgcaca acgccaagac caagcccaga gaagagcagt tcaactccac ctacagagtg 240 gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaag 300 gtgtccaaca aaggcctgcc cagctccatc gagaagacca tcagcaaagc caaaggccag 360 cccagagaac cccaggtgta caccctgcct cccagccagg aagagatgac caagaaccag 420 gtgtccctga cctgcctggt gaaaggcttc taccccagcg acatcgccgt ggagtgggaa 480 agcaacggcc agcccgagaa caattacaag acaacccctc ccgtgctgga tagcgatggc 540 agcttctttc tgtacagcag actgaccgtg gacaagagca gatggcagga aggcaacgtg 600 ttcagctgca gcgtgatgca cgaagccctg cacaaccact acacccagaa gagcctgtcc 660 ctgagcctgg gcaag 675 <210> 16 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for encoding hFc-4 <400> 16 aagaaggaga aggagaagga ggagcaggag gagcgcgaga ccaagacccc cgagtgcccc 60 agccacaccc agcccctggg cgtgttcctg ttccccccca agcccaagga caccctgatg 120 atcagccgca cccccgaggt gacctgcgtg gtcgtggatg tgagccagga agatcccgaa 180 gtgcagttca actggtacgt ggatggcgtg gaagtgcaca acgccaagac caagcccaga 240 gaagagcagt tcaactccac ctacagagtg gtgagcgtgc tgaccgtgct gcaccaggac 300 tggctgaacg gcaaggagta caagtgcaag gtgtccaaca aaggcctgcc cagctccatc 360 gagaagacca tcagcaaagc caaaggccag cccagagaac cccaggtgta caccctgcct 420 cccagccagg aagagatgac caagaaccag gtgtccctga cctgcctggt gaaaggcttc 480 taccccagcg acatcgccgt ggagtgggaa agcaacggcc agcccgagaa caattacaag 540 acaacccctc ccgtgctgga tagcgatggc agcttctttc tgtacagcag actgaccgtg 600 gacaagagca gatggcagga aggcaacgtg ttcagctgca gcgtgatgca cgaagccctg 660 cacaaccact acacccagaa gagcctgtcc ctgagcctgg gcaag 705 <210> 17 <211> 864 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide for encoding hFc-6 <400> 17 gctagcaaga gcaagaagga gatcttccgc tggcccgaga gccccaaggc ccaggccagc 60 agcgtgccca ccgcccagcc ccaggccgag ggcagcctgg ccaaggccac caccgccccc 120 gccaccaccc gcaacaccgg ccgcggcggc gaggagaaga agaaggagaa ggagaaggag 180 gagcaggagg agcgcgagac caagaccccc gagtgcccca gccacaccca gcccctgggc 240 gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagccgcac ccccgaggtg 300 acctgcgtgg tcgtggatgt gagccaggaa gatcccgaag tgcagttcaa ctggtacgtg 360 gatggcgtgg aagtgcacaa cgccaagacc aagcccagag aagagcagtt caactccacc 420 tacagagtgg tgagcgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac 480 aagtgcaagg tgtccaacaa aggcctgccc agctccatcg agaagaccat cagcaaagcc 540 aaaggccagc ccagagaacc ccaggtgtac accctgcctc ccagccagga agagatgacc 600 aagaaccagg tgtccctgac ctgcctggtg aaaggcttct accccagcga catcgccgtg 660 gagtgggaaa gcaacggcca gcccgagaac aattacaaga caacccctcc cgtgctggat 720 agcgatggca gcttctttct gtacagcaga ctgaccgtgg acaagagcag atggcaggaa 780 ggcaacgtgt tcagctgcag cgtgatgcac gaagccctgc acaaccacta cacccagaag 840 agcctgtccc tgagcctggg caag 864 <210> 18 <211> 436 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hGH-hyFc without signal peptide <400> 18 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Arg 180 185 190 Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Lys Glu Lys Glu Lys Glu 195 200 205 Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr 210 215 220 Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 225 230 235 240 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 245 250 255 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 260 265 270 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 275 280 285 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 290 295 300 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 305 310 315 320 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 325 330 335 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val 340 345 350 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 355 360 365 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 370 375 380 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 385 390 395 400 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 405 410 415 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 420 425 430 Ser Leu Gly Lys 435

Claims

A composition comprising, as an active ingredient, an extract of mugwort and a fusion protein,
Wherein the fusion protein comprises a growth hormone or a fragment thereof and an Fc region of a modified immunoglobulin.

The composition for preventing or improving skin wrinkles according to claim 1, wherein the fusion protein is represented by the following formula (I):
X- (L) _w1- IgFc (I)
In the above formula (I)
w1 is 0 or 1;
X is a growth hormone or fragment thereof;
L is a linker;
IgFc is the Fc region of the modified immunoglobulin.

The composition for preventing or improving skin wrinkles according to claim 2, wherein the growth hormone has an amino acid sequence of SEQ ID NO: 1.

The composition according to claim 2, wherein the linker is composed of 5 to 20 amino acids.

3. The composition for preventing or improving skin wrinkles according to claim 2, wherein the linker is a polypeptide consisting of a glycine (Gly, G) residue and a serine (Ser, S) residue.

3. The composition according to claim 2, wherein the modified immunoglobulin Fc region is a combination of an Fc region of IgD and an Fc region of IgG4.

The method of claim 6, wherein the Fc region of the modified immunoglobulin comprises a hinge region, a CH2 domain and a CH3 domain in the N-terminal to C-terminal direction,
Wherein the hinge region comprises a human IgD hinge region,
Wherein the CH2 domain comprises a portion of the amino acid residue of the CH2 domain of human IgD and human IgG4,
Wherein the CH3 domain comprises a portion of the amino acid residue of the CH3 domain of human IgG4.

8. The composition according to claim 7, wherein the Fc region of the modified immunoglobulin is represented by the following formula (II).
N '- (Z1) pY-Z2-Z3-Z4-C' (II)
In the above formula (II)
N 'is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide;
p is an integer of 0 or 1;
Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from the 98 position in the amino acid residues 90 to 98 of SEQ ID NO: 8;
Y is an amino acid sequence having 5 to 64 consecutive amino acid residues in the N-terminal direction from the 162 position in the amino acid residues 99 to 162 of SEQ ID NO: 8;
Z2 is an amino acid sequence having 4 to 37 contiguous amino acid residues in the C-terminal direction from the 163 position in the amino acid residue at positions 163 to 199 of SEQ ID NO: 8;
Z3 is an amino acid sequence having 71 to 106 contiguous amino acid residues in the N-terminal direction from the 220 position in the amino acid residues 115 to 220 of SEQ ID NO: 9;
Z4 is an amino acid sequence having 80 to 107 amino acid sequences from the 221 position to the C-terminal direction in the amino acid residues 221 to 327 of SEQ ID NO:

3. The method of claim 2,
Wherein the Fc region of the modified immunoglobulin comprises a polypeptide having an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

The composition for preventing or improving skin wrinkles according to claim 2, wherein the fusion protein is a polypeptide having an amino acid sequence of SEQ ID NO: 18.

11. The composition for preventing or improving skin wrinkles according to any one of claims 1 to 10, wherein the mugwort extract is a ground-based extract of mugwort.

11. The composition for preventing or improving wrinkles of a skin according to any one of claims 1 to 10, wherein the mugwort is Artemisia capillaris .

11. The composition for preventing or improving skin wrinkles according to any one of claims 1 to 10, wherein the extracting solvent of the mugwort extract is water, alcohol or a mixture thereof.

14. The composition for preventing or improving skin wrinkles according to claim 13, wherein the alcohol is a lower alcohol having 1 to 4 carbon atoms.

11. The composition for preventing or improving skin wrinkles according to any one of claims 1 to 10, wherein the weight ratio of the mugwort extract to the fusion protein is 1: 1 to 1:20.