KR20190120905A - Composition for prevention and treatment of muscle strengthening or treating of sarcopenia - Google Patents

Abstract

The present invention relates to a composition for muscle strengthening or preventing and treating sarcopenia. More particularly, the present invention relates to the composition for muscle strengthening preventing and treating sarcopenia, which can have functions of preventing and treating sarcopenia by a recombinant protein having anti-myostatin activity that can inhibit a function of myostatin, and thus muscle strengthening.

Description

Composition for prevention and treatment of muscle strengthening or treating of sarcopenia}

The present invention relates to a composition for preventing and treating muscle strengthening or muscular dystrophy, and more particularly, the prevention and treatment of muscular dystrophy through a recombinant protein having antimyostatin activity that can inhibit the function of myostatin or muscle strengthening function. It relates to a composition for preventing and treating muscle strengthening or muscular dystrophy that may have.

In the 20th century, the number of populations has doubled, but in the 21st century, it is difficult to double the growth of the population due to lower birth rates and longer life expectancy. According to the 2015 United Nations World population prospects: the 2015 revision, the population over 60, from 910 million in 2015, will grow to about 1.4 billion by 2030, to 2.1 billion by 2050. It is estimated. It is also anticipated that the age of 80 and older will be about 443 million by 2050, and this increase is expected to continue for decades to come. In South Korea, the population aged 65 and over reached 7.2% of the total population in 2000, entering the aging society.In 2018, the aged population reached 20.8%. It is expected that the National Statistical Office will enter a post-aged society. In addition, it is pointed out that compared to other countries, Korea is approaching a very early age to reach an aging society or a very old society. This problem is thought to be due to the recent rapid decrease in fertility rate, which has led to a sharp increase in the number of elderly population. The increase in the elderly population is causing economic problems due to the burden of medical expenses due to increased support for the elderly population per household, which is also affecting the potential decrease in national growth rate due to the decrease in the labor force.

Representative physiological changes with aging are in the reduction of muscle mass and strength. It is known that the decrease in muscle mass occurs after about 40 years, and about 8% decrease occurs in 10 years until the 70s. After that, a sharp decrease occurs and it is known to occur up to 15% in 10 years.

Sarcopenia causes a direct decrease in muscle strength, which increases the risk of death due to diminished or impaired physical function, as well as metabolic effects such as high blood pressure, diabetes, arthritis, obesity, and cancer, including decreased metabolism and decreased immunity. It can also increase the prevalence of the disease.

Myostatin (Myostatin, MSTN) is a protein that regulates muscle growth, belongs to transforming growth factor-β (TGF-β) family and is also known as growth and differentiation factor-8 (GDF-8). Begian blue is a MSTN mutation in the natural state. The 11 bases of the third exon are missing and the MSTN protein does not function normally, resulting in two to three times more muscle mass than normal cows.

In addition, the gene knock-out technique controls the function of the myostatin gene in mice, and skeletal muscle is known to show two to three times more phenotypes than normal mice.

Research into various antagonists for inhibiting the function of myostatin is ongoing in various fields. MSTN prodomains, activin type II receptors, follistatin, follistatin related proteins, etc. may be used to inhibit the function of myostatin at the molecular level.

These proteins are known to interfere with signal transduction into cells by directly binding to myostatin and inhibiting myostatin binding to the receptor. According to Lee and Mcpherron, the overexpression of activin type II receptor protein in vivo in mice resulted in a 27% increase in myofiber growth and a 19% increase in myofiber hypertrophy. Recently, an antibody that specifically inhibits the function of MSTN was produced to confirm muscle growth in animal experiments. However, in clinical studies based on these studies, the increase in muscle is less than expected or the study has been discontinued due to various side effects.

Therefore, there is a need for the development of new materials having low side effects and high economic value for various therapeutic compositions that can prevent or treat myopathy.

The present inventors have developed a recombinant protein having antimyostatin activity, thereby completing the present invention by confirming that it exhibits the effect of preventing and treating muscle strengthening and myopathy.

Accordingly, it is an object of the present invention to provide a composition for treating or preventing myopathy comprising a novel recombinant protein.

In addition, another object of the present invention is to provide a composition for strengthening muscle strength, which comprises a novel recombinant protein.

It is still another object of the present invention to provide a composition for treating or preventing myotropin as a recombinant protein capable of inhibiting the function of myostatin.

In addition, another object of the present invention is to provide a composition that can be used as a health functional food for the prevention of muscle loss for health promotion and diet.

According to one embodiment of the present invention to achieve the above object, it provides a pharmaceutical composition for treating or preventing myopathy comprising at least one selected from the amino acid sequence represented by SEQ ID NO: 2 to SEQ ID NO: 9.

In addition, the composition is characterized by having anti myostatin activity.

In addition, the composition is characterized by improving muscle strength and muscle density.

On the other hand, according to another embodiment of the present invention, it provides a health functional food for preventing or improving muscle reduction comprising at least one selected from the amino acid sequence represented by SEQ ID NO: 2 to SEQ ID NO: 9.

On the other hand, according to another embodiment of the present invention, the present invention provides a pharmaceutical composition for treating or preventing myopathy, comprising at least one sungotadinin in the amino acid sequence represented by SEQ ID NO: 10 to SEQ ID NO: 17.

In addition, the composition is characterized by having anti myostatin activity.

In addition, the composition is characterized by improving muscle strength and muscle density.

In addition, SEQ ID NO: 10 to SEQ ID NO: 17 may be derived from SEQ ID NO: 2.

On the other hand, according to another embodiment of the present invention, it provides a health functional food for preventing or improving muscle reduction comprising at least one selected from the amino acid sequence represented by SEQ ID NO: 10 to SEQ ID NO: 17.

On the other hand, there is provided a protein having an anti myostatin activity comprising at least one selected from the amino acid sequence represented by SEQ ID NO: 2 to 17.

On the other hand, there is provided a protein having an anti myostatin activity comprising the amino acid sequence of SEQ ID NO: 12.

In addition, the pharmaceutical composition of the present invention may be administered with a physiologically acceptable carrier, and when orally administered, a binder, a suspending agent, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, a flavoring agent, and the like. In the case of injections, buffers, preservatives, analgesic agents, solubilizers, isotonic agents, stabilizers and the like can be used in combination, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. .

In addition, the formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elesir, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or multiple dosage forms. Other pharmaceutical compositions of the present invention may be prepared according to techniques commonly used in the form of various formulations.

In addition, the pharmaceutical compositions of the present invention are administered to humans and animals orally or parenterally, intravenously, subcutaneously, intranasally or intraperitoneally. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.

In addition, the effective dosage range of the recombinant protein contained in the pharmaceutical composition of the present invention may vary depending on various factors such as sex, severity, age, method of administration, target cell, expression level, and the like and can be easily determined by those skilled in the art. Can be determined. As one example, it may range from about 0.01 ug to about 100 ug / kg.

In addition, the health functional food of the present invention has the effect of preventing, treating or alleviating myotropia or strengthening muscle, and may include food, food additives, beverages or beverage additives. The food composition may further comprise suitable carriers, excipients and diluents commonly used in the preparation thereof.

In addition, the term food in the present invention means a natural product or processed product containing one or more nutrients, and preferably means that it can be directly eaten through a certain processing step, and means It is intended to include all foods, food additives, health functional foods and beverages, preferably gum or candy.

In addition, foods to which the composition of the present invention can be added include various foods, beverages, gums, candy, tea, vitamin complexes, and functional foods. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements. The food, beverage or food additives may be prepared by a conventional manufacturing method.

In addition, the functional food of the present invention is to control the biological defense rhythm, disease prevention of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the food for a specific purpose Means foods that are designed and processed to express fully the gymnastic function related to the recovery and recovery, and preferably means foods capable of sufficiently expressing the effects of preventing and alleviating muscle reduction or strengthening the muscles. . The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The additive may be 0 to 20 parts by weight, preferably 0.00001 to 15 parts by weight, per 100 parts by weight of the antimicrobial composition of the present invention.

According to the composition for preventing and treating muscle strength or muscular dystrophy of the present invention as described above, it has high antimyostatin activity, increases muscle strength, muscular endurance and muscle mass, and consequently according to senile muscular dystrophy, hereditary disease metabolic disease It can be used for the treatment or prevention of myopathy.

In addition, it can be used as a health functional food to prevent muscle loss for health promotion and diet.

Effects according to the present invention in the above is not limited to the above, other effects of the present invention through the matters described in the embodiments and claims to be described later clearly by those skilled in the art to which the present invention belongs. It can be understood.

Figure 1 shows the sequencing analysis and amino acid sequence of the MSTN prodomain vector produced by site-specific mutagenesis of the present invention.
Figure 2 shows the results of electrophoresis of the recombinant protein of the present invention.
Figure 3 shows the anti myostatin activity of the MSTN prodomain of the present invention.
Figure 4 shows the nucleopeptide, amino acid sequence and electrophoresis results of pMALc5x-Pro45-70-Hisx6 of the present invention.
Figure 5 shows the results of measuring the anti myostatin activity of pMALc5x-Pro45-70-Hisx6 of the present invention.
Figure 6 shows the Western blot results for blocking MSTN signal on the Samd path of pMALc5x-Pro45-70-Hisx6 of the present invention.
Figure 7 shows the increase in muscle strength and endurance of the mouse model administered pMALc5x-Pro45-70-Hisx6 of the present invention.
Figure 8 shows the results of the measurement of muscle mass of the mouse model administered with pMALc5x-Pro45-70-Hisx6 of the present invention.
Figure 9 is the result of confirming the production of antibodies with blood collected from the mouse model administered pMALc5x-Pro45-70-Hisx6 of the present invention.
10 shows the results of electrophoresis of myostatin prodomains constructed from pMALc5x-Pro45-70 of the present invention.
Figure 11 shows the results of luciferase assay of pMALc5x-Pro45-67, pMALc5x-Pro45-66 and pMALc5x-Pro45-65 recombinant protein of the present invention.
Figure 12 shows the results of measuring the anti myostatin activity of Pep45-65-NH2 of the present invention.
FIG. 13 shows Western blot results for MSTN signal blocking on the Samd path of Pep45-65-NH2 of the present invention. FIG.
Figure 14 shows the results of muscle strength and endurance measurement of the mouse model administered Pep45-65-NH2 of the present invention.
Figure 15 shows the muscle mass measurement results of the mouse model administered with Pep45-65-NH2 of the present invention.
Figure 16 shows the results of confirming the antibody production by blood collected from the mouse model administered Pep45-65-NH2 of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for preventing and treating muscle strengthening or muscular dystrophy, by selecting a recombinant protein having antimyostatin activity that can inhibit the function of myostatin, inhibiting myostatin activity, muscle mass, muscle density, etc. The effect of was confirmed through the experiment. Through the present invention it can be used as a material that can be used to treat senile muscular dystrophy, myopathies caused by genetic diseases metabolic diseases.

In the present invention, sarcopenia is a disease caused by a decrease in skeletal muscle mass, and is known to directly cause a decrease in muscle strength, thereby reducing various physical functions and causing disorders, thereby increasing the risk of death. It is a disease that is a result of gradual skeletal muscle loss associated with aging (Park Sung-won, 2007, Muscle Reduction of the Elderly, Journal of the Korean Endocrinology Vol. 22, No. 1). In particular, the present invention has a therapeutic and prophylactic effect of senile myotropia, myotropenia caused by genetic diseases metabolic disease.

In the present invention, a therapeutic or prophylactic composition comprising a recombinant protein includes a health functional composition or a food composition. Here, the increase in muscle strength refers to an increase in overall muscle strength due to an increase in skeletal muscle mass or an improvement in muscle function, and is not an effect that is limited to a specific group of patients with specific vagina.

Myostatin is one of the transforming and growth factor-β (TGF-β) and is known as growth and differentiation factor-8 (GDF-8) that regulates growth and differentiation. have.

Similar to other proteins belonging to the transforming growth factor, the activity of myostatin is regulated by myostatin prodomains located at the N-terminus of the precursor. The full-length protein of myostatin prodomains induced an increase in muscle mass in adult mouse models. In addition, MSTNPro1 (Pro1-265) and Pro45-80 derived from flounder myostatin inhibit myostatin signaling.

In the present invention, to determine the amino acid sequence that can suppress myostatin signal without immunogenicity. Eleven truncated myostatin prodomains were constructed from pMALc5x-Pro45-70 and antimyostatin activity was measured by luciferase assay. pMALc5x-Pro45-65 (IC50: 3.1 μM) showed the strongest anti myostatin activity, and Pep-45-65-NH2 (IC50: 3.67 μM) synthesized from this amino acid was similar to pMALc5x-Pro45-65 It exhibited strong anti myostatin activity. In addition, the improvement of muscle strength and endurance by subcutaneous injection in the mouse model was confirmed.

The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more thereof conventionally used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as the composition is suitable for in vivo delivery, and can be used, for example, in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. And other conventional additives such as antioxidants, buffers, bacteriostatics, etc. may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. In addition, the composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions.

The compositions of the present invention can be administered orally or by parenteral administration (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method. Suitable dosages of the pharmaceutical compositions of the present invention may be determined depending on the weight, age, sex, health condition, diet, method of formulation, time of administration, method of administration, rate of excretion and severity of the patient, and the like.

The health functional food of the present invention is to have the effect of preventing, treating or alleviating or reducing muscle strength, and may include food, food additives, beverages or beverage additives. The food composition may further comprise suitable carriers, excipients and diluents commonly used in the preparation thereof.

In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages, preferably gum or candy.

Examples of the food to which the composition of the present invention can be added include various foods, beverages, gums, candy, tea, vitamin complexes, and functional foods. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements. The food, beverage or food additives may be prepared by a conventional manufacturing method.

Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food that is designed and processed to express fully the gymnastics function related to the living body, and preferably refers to a food that can sufficiently express the prevention and alleviation effect of muscle reduction or muscle strengthening effect on the living body. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The additive may be 0 to 20 parts by weight, preferably 0.00001 to 15 parts by weight, per 100 parts by weight of the antimicrobial composition of the present invention, but is not limited thereto.

Myostatin is composed of three exons and two introns, and myostatin mRNA, which has undergone RNA splicing, is about 3.1 kb, mainly expressed in muscle tissue. The human myostatin gene is translated into 375 amino acids and consists of a 28 kDa prodomain region and a 12 kDa mature region (promyostatin) containing a signal peptide. Promyostatin maintains homodimer protein form through disulfide binding. A total of three proteolytic events occur in order for myostatin protein to remain active. First, signal peptides in the prodomain region are cleaved by signal peptidase and divided into prodomain and mature regions by furin (paired basic amino acid enzyme). The cleaved prodomain region regulates the activity of the mature region by maintaining a non-covalent bond with the mature region. Finally, BMP-1 / TLD metalloproteinase cleaves between arginine No. 75 and aspartic acid No. 76 in the prodomain to inhibit prodomain activity. By inhibiting, non-covalent bonds between the prodomain and mature region are broken down to produce an active mature MSTN.

The mature region of the active type binds to and phosphorylates activin type II receptors present in the cell membrane and transmits signals to activin type I receptors to induce intracellular smad2 phosphorylation. Phosphorylated smad2 forms a complex with smad3, binds to smad4 and regulates transcription in the target gene.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are only intended to embody the content of the present invention, thereby not limiting the present invention.

Example 1 Construction of Recombinant Protein Expression Vector

In order to construct a recombinant protein capable of inhibiting myostatin activity from flounder-derived myostatin prodomains, a recombinant protein expression vector of various lengths was constructed using a site directed mutagenesis system.

Site directed mutagenesis system is a method of inducing deletion, substitution and insertion of bases by PCR reaction using specific primers. In this example, eight recombinant protein expression vectors were constructed from pMALc5x-Pro45-80 recombinant protein expression vector. .

Primers specific for each recombinant protein expression vector were prepared using NEB's NEBaseChanger tool (NEB, USA) and subjected to KLD reaction after PCR using site directed mutagenesis kit (Site directed mustagenesis kit, NEB, USA).

The constructed vector was transduced into DH5α competent cell (Solgent, Korea) by thermal shock method, and 100% ug / ml of ampicillin added LB solid medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1% agar). ) And incubated for 16 hours at 37 ° C.

Selected colonies were inoculated in LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with 100 ug / ml ampicillin and incubated for 16 hours using plasmid extract kit (Solgnet, Korea). The plasmid DNA was extracted.

The extracted plasmid DNA was identified by sequencing analysis (Solgent, Korea), and the identified nucleotide sequence is shown in FIG. 1. Constructed recombinant proteins were pMALc5x-Pro45-70, pMALc5x-Pro45-69, pMALc5x-Pro45-68, pMALc5x-Pro45-67, pMALc5x-Pro46-70, pMALc5x-Pro46-69, pMALc5x-Pro47-70, pMALc5x- Named Pro49-70.

Figure 1 shows the nucleotide and amino acid sequence of each recombinant protein expression vector as a result of sequencing analysis of the MSTN prodomain vector produced by site-specific mutagenesis of the present invention.

Example 2 Recombinant Protein Expression and Purification

The vectors identified in the base sequence in Example 1 were transduced into a K12TB1 competent cell by thermal shock method, LB solid medium (1% tryptone, 0.5% added 100 ug / ml ampicillin and 10 ug / ml streptomycin) yeast extract, 1% NaCl, 1% agar) and incubated for 16 hours at 37 ° C.

Selected colonies were inoculated in LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl) to which 100 ug / ml ampicillin and 10 ug / ml streptomycine were incubated for 16 hours.

To express recombinant protein, 5 ml of medium incubated for 16 hours in 500 ml of LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 100 ug / ml ampicillin, 10 ug / ml streptomycine) was added. Shaking was incubated until the OD600 value of 0.3 ~ 0.4 at 150 ° C, ° C, IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added so that the final concentration is 0.3 mM.

After incubation for 3 hours at 30 ° C, 150 rpm, the strain was recovered by centrifugation at 4 ° C, 4000 rpm for 20 minutes, 20 ml of column buffer (20 mM tris-HCl, pH 8.0, 200 mM NaCl , 1 mM EDTA) was resuspended, and PMSF (Phenylmethane sulfonyl fluoride) was added to a final concentration of 1 mM.

E. coli was crushed using a sonicator and centrifuged at 4 ° C and 12,000 rpm for 20 minutes.

The recovered supernatant was loaded on amylose resin (NEB, USA) and eluted with elution buffer (20 mM tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 10 mM maltose).

Purified recombinant protein was confirmed by the expression and purification of recombinant protein by electrophoresis using 12.5% polyacrylamide gel, the electrophoresis results are shown in FIG.

Figure 2 shows the results of electrophoresis of the recombinant protein of the present invention. In Figure 2, M is the standard marker to know the protein size, T is the extract sample of E. coli induced recombinant protein expression, S is centrifuged after the T sample supernatant sample, P is centrifuged T sample pellet sample , FT is the sample passed through the column by loading the S sample into amylose resin in affinity chromatography, W is the washing step sample after loading the sample, E is the sample eluting the recombinant protein using elution buffer. The red arrow indicates the recombinant protein purified using amylose resin.

Example 3 Antimyostatin Activity by Luciferase Assay System

Antimyostatin activity at the cellular level was confirmed by luciferase assay system.

Hek293 cells were seeded with 2.0 × 10 ^ 4 cells per well in 96 well plates in DMEM medium (10% FBS, 1% penicillin / streptomycine, 1% geneticine) and cultured in a 5% CO2 incubator.

After 24 hours, the medium was replaced with DMEM without FBS, and cultured in a CO 2 incubator by treatment with 1 nM of recombinant myostatin (R & D system, USA) and recombinant protein by concentration.

After 24 hours, the medium was removed and treated with 65 ul of DMEM medium and 65 ul of reagent (Bright-Glo luciferase assay system, USA) to measure luminescence on a micro plate luminometer.

The measured values are shown in FIGS. 3A and 3B. Here, antimyostatin activity was expressed in% using the values of positive and negative control.

The measured values were obtained using the following equations for the positive control (positive control, test treated with myostatin only, MSTN only) and negative control (no test treated without myostatin, No MSTN). The antimyostatin activity is expressed in%.

Myostatin Inhibitory Activity (%) = (Luminescence Value of Experimental Water Treated with 1 nM Myostatin Only-Luminous Value of Experimental Water Treated with Myostatin and Recombinant Protein Simultaneously) × 100 / (1 nM Myostatin Only Luminescence values of one experimental group-Luminous values of experimental group not treated with myostatin)

Figure 3 shows the anti myostatin activity of the MSTN prodomain of the present invention.

As a result of confirming the anti myostatin activity at the cellular level using the Luciferase assay system in Figure 3, among the total 8 recombinant proteins pMALc5x-Pro45-70 recombinant protein IC1 value of 1 nM myostatin to 1.10 nM It was confirmed that the highest anti myostatin activity.

In addition, the IC 50 value of each MSTN prodomain is shown in Table 1 below.

As shown in Table 1, the IC50 value of pMALc5x-Pro45-69 is 2.3 nM, the IC50 value of pMALc5x-Pro45-68 is 13.27 nM, the IC50 value of pMALc5x-Pro46-70 is 28.62 nM, and the IC50 value of pMALc5x-Pro46-69 is The IC50 value of 52.22 nM and pMALc5x-Pro47-70 was able to confirm an IC50 value of 13.52 nM, and pMALc5x-Pro45-67 and pMALc5x-Pro49-70 did not show antimyostatin activity below 60 nM.

Example 4 Expression and Purification of pMALc5x-Pro45-70-Hisx6 Recombinant Protein

In order to improve the purity of the recombinant protein, His tag was inserted into the C-terminal region of the pMALc5x-Pro45-70 recombinant protein identified in Example 3 using a site directed mutagenesis system and named pMALc5x-Pro45-70-Hisx6. It was.

The nucleotide sequence of pMALc5x-Pro45-70-Hisx6 recombinant protein in the form of 6 Histidine bound to the C terminus of pMALc5x-Pro45-70 was confirmed by sequencing analysis, and is shown in FIG. 4.

Purification of the pMALc5x-Pro45-70-Hisx6 recombinant protein was carried out by adding PMSF to 20 ml of binding buffer (20 mM tris-HCl, pH8.0, 500 mM NaCl, 5 mM imidazole) to a final concentration of 1 mM pMALc5x-Pro45. Strains expressing the -70-Hisx6 recombinant protein were resuspended and disrupted using a sonicator.

Then, the supernatant recovered by centrifugation at 4 ° C and 12,000 rpm for 20 minutes was filtered through a syringe filter (pore size: 0.2 um) and loaded into Ni-NTA resin, followed by elution buffer (20 mM tris-HCl, pH8. 0, 500 mM NaCl, 40 mM imidazole).

Purified recombinant protein was dialyzed for 24 hours using a dialysis tube, and purified secondly using amylose resin according to the method described above.

Figure 4 shows the nucleopeptide, amino acid sequence and electrophoresis results of pMALc5x-Pro45-70-Hisx6 of the present invention.

In Figure 4, M is the standard marker to know the protein size, T is the extract sample of E. coli induced recombinant protein expression, S is centrifuged T sample supernatant sample, P is centrifuged T sample pellet sample , FT1 is the sample passed through the column by loading the S sample in Ni-NTA resin, W1 is the washing step sample of the column, E1 is the sample eluting the recombinant protein using elution buffer, and FT2 is the primary purification (Ni-NTA After purification, the sample was eluted, loaded into amylose resin, loaded through the column, W2 was the washing step sample, and E2 was the sample eluted with the recombinant protein using elution buffer.

In the part indicated by the two red arrows in E1, other proteins besides the desired recombinant protein were seen after Ni-NTA purification, but in the case of the red arrow shown in E2, only the desired recombinant protein was clearly purified.

Example 5 Anti Myostatin Activity of pMALc5x-Pro45-70-Hisx6

The antimyostatin activity of pMALc5x-Pro45-70-Hisx6 of Example 4 was measured using a Luciferase assay system, and the results are shown in FIG. 5.

Figure 5 shows the results of measuring the anti myostatin activity of pMALc5x-Pro45-70-Hisx6 of the present invention, it can be seen that the IC 50 value of 0.29 nM for 1 nM myostatin.

Compared to the IC50 value of pMALc5x-Pro45-70 of 1.1 nM, the purity of the target protein is increased by purification using affinity chromatography, which is 2 step (purification of Ni-NTA resin and amylose resin). This can be seen as higher.

In addition, the inhibitory activity against activin A and GDF11 belonging to the same family as myostatin and having similar amino acid sequences was confirmed by luciferase assay.

pMALc5x-Pro45-70-Hisx6 had an IC50 value of 32.84 nM for 1 nM of activinA and an IC50 value of 1 nM for GDF11 of 22.65 nM.

The antimyostatin IC50 value of 0.29 nM has no inhibitory effect on activinA and GDF11, but at 20 nM or higher concentrations, the side effects of anti-activinA or anti-GDF11 effects should be considered.

Here, ActivinA and GDF11 belong to the TGF-b superfamily, such as myostatin, and form a nucleotide sequence similar to that of myostatin. ActivinA shares receptors, such as myostatin, and acts on a wide variety of cells, all of which are difficult to mention, including early morphogenesis and repair, neurodevelopment, inflammation, and cancer.

GDF11 is recently known as an aging inhibitor, but its function is not yet clear. Myostatin and amino acid sequence is very similar, it is involved in fore-and-aft formation in the early stage of development, and when skeletal formation is not possible in the early stage of development. It is also involved in cerebral vessels and neurogenesis.

Both ActivinA and GDF11 are closely related to cell survival and differentiation, and use signaling pathways similar to myositan. Therefore, the concentration of recombinant proteins and peptides that specifically inhibit the activity of myostatin without affecting ActivinA and GDF11 is very important.

Example 6 Effect of pMALc5x-Pro45-70-Hisx6 on Myostatin Signaling Pathway

In order to confirm the effect of pMALc5x-Pro45-70-Hisx6 of Example 4 on the myostatin signaling pathway, the degree of phosphorylation of smad 2 and smad 3 transcription factors was confirmed through a wester blot.

Western blot was cultured in 5% CO2 incubator for seeding HepG2 cells in DMEM medium (10% FBS, 1% penicillin / streptomycine) in 6 well plater at 2.0 × 10 ^ 5 cells per well.

After 24 hours, the medium was replaced with DMEM without FBS, and after 4 hours, 10 nM of recombinant myostatin and 600 nM of pMALc5x-Pro45-70-Hisx6 were treated for 30 minutes.

Cells were washed twice with PBS followed by RIPA buffer (20 mM tris-HCl, pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxychloate, 2.5 mM sodium pyrophosphate, 1 Treatment was performed with mM β-glycerophosphate, 1 mM NA ₃ NO _4, 1 ug / ml leupeptin (cell signaling, USA), protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche, USA).

The cells were then disrupted using a sonicator and centrifuged at 4 ° C and 12,000 rpm for 20 minutes.

Supernatants were quantified by BCA assay and electrophoresed on 10% polyacryamide gel.

After electrophoresis, transfer was made to PVDF membrane and blocked for 2 hours at room temperature using 5% BSA or 5% skim milk.

PVDF Membrane was washed three times at room temperature for 10 minutes using TBS-T buffer and the primary antibodies (Samd2, P-Smad2, Smad3, P-Smad3, β-actin, Cell signaling, USA) for 2 hours at room temperature. Reacted.

After washing three times for 10 minutes at room temperature using TBS-T buffer and the secondary antibody (anti-Mouse-IgG, anti-Rabbit IgG, Cell signaling, USA) was reacted for 2 hours at room temperature.

PVDF membranes were washed three times for 10 minutes at room temperature using TBS-T buffer, and then exposed to x-ray films using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, USA).

Figure 6 shows the Western blot results for MSTN signal block on the Samd pathway of pMALc5x-Pro45-70-Hisx6 of the present invention.

In FIG. 6, it was confirmed that pMALc5x-Pro45-70-Hisx6 inhibits myostatin signal transduction and inhibits the signal propagating inside the cell (phosphorylation of Smad transcription factor).

Figure 6 (A) shows the Western blot results for the role of pMALc5x-Pro45-70-Hisx6 as MSTN antagonist, (B) shows the results of the measurement of the concentration of total Smad2 and phospho-Smad2 in HepG2 cells. (C) measured the concentrations of total Smad3 and phospho-Smad3 in HepG2 cells with or without pMALc5x-Pro45-70-Hisx6.

Lane (A) in (A) is a lane without any treatment to the cell as a control. Lane 2 can be confirmed that the expression level of smad2, smad3 phosphorylated by lanes treated only with myostatin. Lane 3 is a positive control lane that is simultaneously treated with myostatin and SB431542 (small molecule that inhibits phosphorylation of the receptor that myostatin binds), and does not increase the amount of phosphorylated Smad2, 3 that should be elevated by myostatin. I could confirm that. Lane 4 is a lane treated with both myostatin and pMALc5x-Pro45-70-Hisx6 recombinant protein. Similar to lane treated with SB431542, lane 4 did not increase the expression level of pMALc5x-Pro45-70-Hisx6. Has been shown to interfere with myostatin binding to receptors.

Example 7 Effect of pMALc5x-Pro45-70-Hisx6 on Muscle

To determine the effect of pMALc5x-Pro45-70-Hisx6 on muscle at the in vivo level, we examined the change in muscle strength and endurance by subcutaneous injection into ICR mice.

The mice used in the experiment were 5 week old male ICR mice, which were injected subcutaneously in total for 5 weeks. The injection amount was subcutaneously injected at a concentration of 20 mg / kg relative to the mouse body weight, and the weight, strength test, and muscular endurance test were performed at 0, 7, and 14 days. After the experiment, muscle density was calculated by measuring the weight and volume of the forelimb and hind limb muscles, and the production of antibodies to pMALc5x-Pro45-70-Hisx6 recombinant protein was determined from blood samples.

Figure 7 shows the increase in muscle strength and endurance of the mouse model administered pMALc5x-Pro45-70-Hisx6 of the present invention.

In FIG. 7, the mouse body weight was not significantly different through the subcutaneous injection of pMALc5x-Pro45-70-Hisx6. However, muscle strength increased significantly by 6.3% (p <0.001) at 7 days and decreased significantly compared with 7 days at 14 days (p <0.01).

In the swimming endurance test, which is an indicator of muscular endurance, it increased by 106% (p <0.005) compared to the control group on the 7th day and decreased significantly compared to the 7th day on the 14th day, but increased significantly compared to the 0th day (p <0.05). Indicated.

Figure 8 shows the results of the measurement of muscle mass of the mouse model administered with pMALc5x-Pro45-70-Hisx6 of the present invention.

In FIG. 8, the muscle mass of both the forelimbs and hind limbs was significantly increased (p <0.1, hind limbs: p <0.001) compared to the control group by subcutaneous injection of pMALc5x-Pro45-70-Hisx6. It also increased significantly (p <0.05).

Muscle density, which was calculated using muscle mass and volume, was increased 4.9% (p <0.05) in the hind legs compared to the control.

Figure 9 is the result of confirming whether the antibody produced by blood collected from the mouse model administered pMALc5x-Pro45-70-Hisx6 of the present invention.

The production of antibodies to pMALc5x-Pro45-70-Hisx6 from blood collected on days 7 and 14 was confirmed by ELISA method. The collected blood was centrifuged at 3000 rpm for 20 minutes at room temperature to separate only serum. pMALc5x-Pro45-70-Hisx6 was dispensed at 100 ng per well in a 96 well plate and coated at 4 ° C for 24 hours. After washing three times with PBS using 1% BSA was blocked for 3 hours at room temperature. The serum collected on days 7 and 14 was added to the wells and reacted at room temperature for 3 hours. After washing four times with PBS, anti-mouse IgG alkaline phosphate was treated for 3 hours at room temperature. After washing four times with PBS, treated with 100 ul of p-nitrophenyl phosphate (pNPP, Sigma, USA) and reacted for 30 minutes while blocking the light at 37 ° C and the absorbance was measured at 405 nm.

In FIG. 9, one group of subcutaneous injections showed 309% high absorbance at 7 days (p <0.001) in the control group and 509% high absorbance at 14 days (p <0.001), indicating that pMALc5x-Pro45-70 It is thought that antibodies were produced from day 7 through -Hisx6 subcutaneous injection. These results affected muscle strength and endurance on day 14, and increased muscle strength and endurance on day 7, whereas on day 14, the antibody canceled the function of pMALc5x-Pro45-70-Hisx6, resulting in longer muscle strength and endurance. It did not increase and decreased similarly to the control.

pMALc5x-Pro45-70-Hisx6 was subjected to five subcutaneous injections for 14 days, and then serum was isolated from blood samples. Total cholesterol, triglyceride, and glucose were measured using Lipidocare (SD biosense, Korea) equipment, and LDH activity, GPx activity, HDL, LDL, and FFA were measured using Biovision kit (Biovision, USA). The measured values are shown in Table 2 below.

Total cholesterol was significantly reduced by 16.4% compared to the control group in Table 2 (p <0.01). However, no significant difference was found in the contents of HDL and LDL.

In addition, the triglyceride content was significantly decreased (p <0.001) by 41% compared to the control group, and the glucose content was decreased by 9.7% compared to the control group, but there was no significant difference.

FFA was significantly decreased (p <0.005) by 31% compared to the control group due to the decrease in triglycerides.

Example 8 Construction of pMALc5x-Pro45-70-Hisx6 Based Recombinant Protein

In vivo experiments using the pMALc5x-Pro45-70-Hisx6 recombinant protein of Example 7 showed that after a certain time as the antibody is formed, muscle strength and muscle endurance no longer increase. Therefore, a recombinant protein without antigenicity is required, and thus, a shorter recombinant protein was constructed.

Eight recombinant proteins were constructed based on the pMALc5x-Pro45-70 sequence identified in Example 3, pMAL-c5x-Pro45-67, pMAL-c5x-Pro45-66, pMAL-c5x-Pro45-65, pMAL-c5x-Pro45-64, pMAL-c5x-Pro45-63, pMAL-c5x-Pro45-62, pMAL-c5x-Pro45-61, pMAL-c5x-Pro45-60.

Among them, pMAL-c5x-Pro45-66, pMAL-c5x-Pro45-65, pMAL-c5x-Pro45-64, pMAL-c5x-Pro45-63, pMAL-c5x-Pro45-62, pMAL-c5x-Pro45-61, pMAL-c5x-Pro45-60 recombinant protein was constructed using the site directed mutagenesis system to determine the expression, and the results were shown in FIG. 10.

10 shows the results of electrophoresis of myostatin prodomains constructed from pMALc5x-Pro45-70 of the present invention. In Figure 10, M is the standard marker to know the protein size, T is the extract sample of E. coli induced recombinant protein expression, S is centrifuged T sample supernatant sample, P is centrifuged T sample pellet sample , FT is the sample passed through the column by loading the S sample into amylose resin in affinity chromatography, W is the washing step sample after loading the sample, E is the sample eluting the recombinant protein using elution buffer. The red arrow indicates the recombinant protein purified using amylose resin.

Figure 11 shows the results of luciferase assay of pMALc5x-Pro45-67, pMALc5x-Pro45-66 and pMALc5x-Pro45-65 recombinant proteins of the present invention, pMALc5x-Pro45-67, pMALc5x-Pro45-66, pMALc5x-Pro45-65 The recombinant protein was able to confirm antimyostatin activity by luciferase assay system. Antimyostatin activity could not be confirmed in the remaining recombinant proteins.

Luciferase assay and in silico analysis of pMALc5x-Pro45-70 based recombinant proteins in the present invention are shown in Table 3 below.

Luciferase assay showed that pMALc5x-Pro45-67 and pMALc5x-Pro45-66 showed IC6 values of 0.16 uM and 8.0 uM for myostatin of 1 nM, but were immunogenic through in silico analysis.

pMALc5x-Pro45-65 was analyzed to be immunogenic without in silico analysis, and luciferase assay showed that IC50 value was 3.1 uM.

The remaining pMALc5x-Pro45-64, pMALc5x-Pro45-63, pMALc5x-Pro45-62, pMALc5x-Pro45-61, pMALc5x-Pro45-60 were immunogenic but did not show antimyostatin activity below 5 uM.

Recombinant proteins contain fusion proteins for the convenience of purification, and thus, peptides having the amino acid sequence pMALc5x-Pro45-65 were synthesized because the protein was large in size and highly likely to have antigenicity.

The synthesized peptide was named Pep45-65-NH2, and anti-myostatin activity was confirmed by luciferase assay system. In addition, the inhibitory activity against activinA and GDF11 belonging to the same family as myostatin and having similar amino acid sequences was confirmed by luciferase assay system.

Figure 12 shows the results of measuring the anti myostatin activity of Pep45-65-NH2 of the present invention. In FIG. 12, Pep45-65-NH2 was able to confirm an IC50 value of 3.6 uM against 1 nM myostatin, and inhibitory activity was less than 10 uM in Pep45-65-NH2 of 1 nM of activinA and 1 nM of GDF11. It can be seen that Pep45-65-NH2 specifically inhibits only myostatin.

Western blot confirmed the effect of Pep45-65-NH2 on myostatin signaling. Western blot method of Pep45-65-NH2 was performed in the same manner as the western blot method of pMALc5x-Pro45-70-Hisx6 of Example 6. Western blot results of Pep45-65-NH2 is shown in Figure 13, it was confirmed that the concentration-dependent inhibition of phosphorylation of the smad2 transcription factor.

FIG. 13 shows Western blot results for MSTN signal blocking on the Samd path of Pep45-65-NH2 of the present invention. FIG.

In FIG. 13A, lane 1 is a lane in which nothing is treated in the cell as a control.

Lane 2 can be confirmed that the expression level of smad2 phosphorylated by lanes treated with myostatin only increased.

Lane 3 is a positive control lane that is treated with both myostatin and SB431542 (small molecule that inhibits phosphorylation of the receptor that myostatin binds to), and confirms that phosphorylated Smad2 expression level that should be elevated by myostatin does not increase. Could.

Lanes 4 and 5 were lanes treated with myostatin and Pep45-65-NH2 peptides at the same time and could be found to interfere with myostatin binding.

Figure 14 shows the results of muscle strength and endurance measurement of the mouse model administered Pep45-65-NH2 of the present invention, in the in vivo experiment using ICR mice Pep45-65-NH2 was injected subcutaneously five times over two weeks and the injection amount was Injection was at a concentration of 20 mg / kg of mouse body weight.

Weight, strength test, and endurance test on day 0, 7, and 14 showed significant weight gain (p <0.05) compared to the control on both 7 and 14 days. Significantly increased (muscle strength: p <0.01, muscle endurance: p <0.05).

Figure 15 shows the results of the measurement of muscle mass of the mouse model administered Pep45-65-NH2 of the present invention, the muscle mass of both the front and hind legs through the subcutaneous injection of Pep-45-65-NH2 significantly compared to the control group In addition, the muscle volume of the hind limb increased significantly (p <0.05).

Muscle density, calculated from muscle mass and volume, was significantly increased (p <0.1) in the hind legs compared to the control.

Figure 16 shows the results of the antibody produced by blood collected from the mouse model administered Pep45-65-NH2 of the present invention, for the Pep-45-65-NH2 from the blood collected on the 7th and 14th day Whether the antibody was produced was confirmed by the ELISA method. One group of subcutaneous injections showed no antibody formation against the control and absorbance Pep-45-65-NH2 at both 7 and 14 days.

Triglycerides, total cholesterol, glucose, LDH activity, GPx activity, HDL, LDL, and FFA were measured using serum isolated from blood collected after subcutaneous injection of Pep-45-65-NH2. Total cholesterol, triglyceride, and glucose were measured using Lipidocare (SD biosense, Korea) equipment, and LDH activity, GPx activity, HDL, LDL, and FFA were measured using Biovision kit (Biovision, USA). The measured values are shown in Table 4 below.

As shown in Table 4, due to the subcutaneous injection of Pep45-65-NH2, the serum triglyceride content was significantly reduced (p <0.05) by 26.5% compared to the control. In addition, the glucose content was also significantly reduced (p <0.001) by 19.1% compared to the control.

FFA was significantly decreased (p <0.01) by 73% compared to the control group due to the decrease in triglycerides.

Therefore, the muscle density and blood volume of Pep45-65-NH2 increased significantly compared to the control group due to the increase of muscle mass and volume, and the content of triglyceride, glucose and FFA in blood were significantly increased according to the increased muscle mass. It was confirmed that the decrease. In addition, unlike the results of pMALc5x-Pro45-70-Hisx6, it was confirmed that muscle strength and endurance continued to increase until 14 days.

As described above, the composition for preventing or treating muscle strengthening or myopathy according to the present invention has been described in detail with reference to specific embodiments of the present invention, but those skilled in the art who understand the spirit of the present invention are within the scope of the same idea. Through the addition, modification, deletion, etc., it will be able to easily suggest other embodiments that fall within the scope of the invention or other regression. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the invention is indicated by the following claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention. Should be interpreted as

<110> GANGNEUNG-WONJU NATIONAL UNIVERSITY INDUSTRY ACADEMY COOPERATION GROUP <120> Composition for prevention and treatment of muscle strengthening          or treating of sarcopenia <130> PN1712-474 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro45-80 <400> 1 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser Lys Leu Arg Met Lys Glu Ala Pro Asn Ile Ser              20 25 30 Arg Asp Ile Val          35 <210> 2 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro45-70 <400> 2 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser Lys Leu Arg Met Lys              20 25 <210> 3 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro45-69 <400> 3 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser Lys Leu Arg Met              20 25 <210> 4 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro45-68 <400> 4 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser Lys Leu Arg              20 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro45-67 <400> 5 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser Lys Leu              20 <210> 6 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro46-70 <400> 6 Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile Ser   1 5 10 15 Gln Ile Leu Ser Lys Leu Arg Met Lys              20 25 <210> 7 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro46-69 <400> 7 Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile Ser   1 5 10 15 Gln Ile Leu Ser Lys Leu Arg Met              20 <210> 8 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro47-70 <400> 8 Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile Ser Gln   1 5 10 15 Ile Leu Ser Lys Leu Arg Met Lys              20 <210> 9 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> pMALc5x-Pro49-70 <400> 9 Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile Ser Gln Ile Leu   1 5 10 15 Ser Lys Leu Arg Met Lys              20 <210> 10 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-67 <400> 10 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser Lys Leu              20 <210> 11 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-66 <400> 11 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser Lys              20 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-65 <400> 12 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu Ser              20 <210> 13 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-64 <400> 13 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile Leu              20 <210> 14 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-63 <400> 14 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser Gln Ile             <210> 15 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-62 <400> 15 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser gln         <210> 16 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-61 <400> 16 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15 Ser     <210> 17 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> pMAL-c5x-Pro45-60 <400> 17 Cys Asp Val Arg Gln Gln Ile Lys Thr Met Arg Leu Asn Ala Ile Ile   1 5 10 15

Claims (11)

A pharmaceutical composition for treating or preventing muscle erosion comprising at least one selected from amino acid sequences represented by SEQ ID NOs: 2 to 9.
According to claim 1,
The composition is a pharmaceutical composition for treating or preventing myotropin, characterized in that it has an anti myostatin activity.
The method according to claim 1 or 2,
The composition is a pharmaceutical composition for treating or preventing myopathy, characterized in that to improve muscle strength and muscle density.
A health functional food for preventing or improving muscular dystrophy comprising at least one selected from amino acid sequences represented by SEQ ID NOs: 2 to 9.
A pharmaceutical composition for treating or preventing muscle erosion comprising at least one selected from amino acid sequences represented by SEQ ID NOs: 10 to 17.
The method of claim 5,
The composition is a pharmaceutical composition for treating or preventing myopathy, characterized in that it has an anti myostatin activity.
The method of claim 5 or 6,
The composition is a pharmaceutical composition for treating or preventing myopathy, characterized in that to improve muscle strength and muscle density.
The method of claim 5,
The SEQ ID NO: 10 to SEQ ID NO: 17 is derived from SEQ ID NO: 2, pharmaceutical composition for the treatment or prevention of muscle reduction.
Health functional food for preventing or improving myopathy comprising at least one selected from amino acid sequences represented by SEQ ID NO: 10 to SEQ ID NO: 17.
A protein having anti myostatin activity comprising at least one selected from the amino acid sequences represented by SEQ ID NOs: 2 to 17.
A protein having anti myostatin activity comprising the amino acid sequence of SEQ ID NO.

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