KR20180099047A - Melittin-like recombinant protein, nucleotide encoding the same and composition for skin containing them - Google Patents

Abstract

The present invention relates to a melittin-like recombinant protein, a nucleotide encoding the melittin-like recombinant protein, and a composition for improving a skin function, including the melittin-like recombinant protein and the nucleotide, and more particularly, to a melittin-like recombinant protein derived from a Collinsella sp. Microorganism, a nucleotide with genetic information encoding the recombinant protein, and a composition for skin, which has skin barrier activation and anti-wrinkle effects by including at least one of the recombinant protein and the nucleotide as an active ingredient.

Description

The present invention relates to a melittin-like recombinant protein, a nucleotide encoding the melittin-like recombinant protein, and a composition for skin comprising the same.

The present invention relates to a melittin-like recombinant protein, a nucleotide coding for the melittin-like recombinant protein and a composition for improving skin function comprising the melittin-like recombinant protein. More specifically, the present invention relates to a melittin-like recombinant protein comprising melittin derived from a collinsella sp . Microorganism melittin-like recombinant protein, a nucleotide having genetic information encoding the recombinant protein, and at least one of the recombinant protein or the nucleotide as an active ingredient, thereby to provide a skin composition having skin barrier activation and wrinkle- .

In general, bee venom is a venom from the oviduct of the bee (Apis mellifera), and clinically has a remarkable effect on neuralgia, rheumatism, back pain and the like. Bee venom consists of various constituents, of which one of the pharmacologically active ingredients is melittin.

The melittin is a peptide composed of 26 amino acids and mainly acts as a hemolytic enzyme and an enzyme. The enzyme action stimulates the pituitary gland and adrenal cortex to promote the release of catecholamine and cortisone. The catecholamines include norepinephrine, epinephrine and dopamine. The norepinephrine is associated with emotional, cognitive, and arousal states as sympathetic impulse carriers, and the dopamine has been reported to play an important role in depression.

Bee venom contains a large amount of harmful substances such as phospholipase, hyaluronidase, and histamine, which cause allergic symptoms in the human body in addition to melittin. These allergens can cause urticaria, itching, convulsions, and can cause severe dizziness or dyspnea. Therefore, in order to use bee venom clinically, allergens should be removed for safety. Thus, Korean Patent No. 10-1364506 (Feb. 21, 2014) discloses a method for separating and purifying allergen components from bee venom.

Korean Patent Registration No. 10-1394817 (Apr. 31, 2014) discloses a cosmetic composition containing bee venom or bee venom extract. The cosmetic composition is reported to be effective for antioxidation, anti-aging and wrinkle improvement of skin. However, in order to obtain the bee venom, it is troublesome to perform several steps of separation and purification in order to remove the harmful component from the needle by applying electric shock to the honeybee to recover the needle.

On the other hand, as is well known, the skin of the human body is composed of the epidermal layer, the dermal layer and the subcutaneous fat layer. The skin layer is the outermost layer and mainly functions as a cell barrier that keeps oil and moisture in the skin. In order to improve the cell barrier function of the epidermal layer, the cell permeability of the dermal keratinocytes should be increased by increasing the intercellular binding force.

Also, Free radicals generated during cell metabolism damage DNA through various mechanisms of reaction, causing mutations, and oxidizing membrane lipids to destroy intracellular organelles. This series of processes interferes with the normal division and growth of the cells, promotes skin aging, and causes wrinkles.

Abnormal synthesis or abnormal degradation of collagen, a major component of the extracellular matrix (ECM), is also very closely related to skin aging and the formation of skin wrinkles. In particular, procollagen type I has been reported to have a function of self-regulating collagen synthesis in collagen synthesis cells (Wiestner, M. et al. (1979) J. Biol. Chem. 254, 10482-10484; Paglia, LM Et al. (1979) Biochemistry 18, 5030-5034). Therefore, it is necessary to induce collagen synthesis in dermal fibroblasts to improve skin wrinkles due to collagen reduction.

[Patent Document 1] Korean Patent No. 10-1364506 (Feb. [Patent Document 2] Korean Patent Registration No. 10-1394817 (Apr.

[Non-Patent Document 1] Wiestner, M. et al. (1979) J. Biol. Chem. 254, 10482-10484 [Non-Patent Document 2] Paglia, L. M. et al. (1979) Biochemistry 18, 5030-5034

Accordingly, an object of the present invention is to provide a method for producing a melilin-like recombinant protein which is effective for increasing intercellular binding force of dermal keratinocytes, inhibiting cell permeability, and further promoting collagen synthesis in dermal fibroblasts .

Another object of the present invention is to provide a skin composition comprising the melittin-like recombinant protein or a nucleotide encoding the melittin-like recombinant protein as an active ingredient and having an excellent effect on skin barrier activation and wrinkle improvement of the human body.

Another object of the present invention is to provide a method for producing melittin-like recombinant proteins purified through genetic recombination and microbial cultivation without the need to take a bee venom by applying an electric shock to a bee or to remove harmful components from the bee venom And to provide an efficient method for mass production.

In order to achieve the above object, the present invention provides a melittin-like recombinant protein, which is derived from a microorganism of the genus Collinsella sp., And is composed of the amino acid sequence of SEQ ID NO: 4.

The present invention also provides a nucleotide derived from a Collinsella sp. Microorganism, encoding the melittin-like recombinant protein of claim 1, and comprising the nucleotide sequence of SEQ ID NO: 3.

The present invention also relates to a method for the treatment and / aerofaciens) obtaining from KCTC 15038 strain, primers consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and; Obtaining a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 3 from the primer; Preparing a recombinant vector using the DNA fragment and the pET28a vector; Introducing the recombinant expression vector into E. coli to transform the E. coli; Culturing the Escherichia coli, and isolating and purifying a protein consisting of the amino acid sequence of SEQ ID NO: 4; A recombinant mutantin-like recombinant protein, and a recombinant mutantin-like recombinant protein.

Finally, the present invention provides a composition for skin comprising the melittin-like recombinant protein or at least one of the nucleotides as an active ingredient, and having the skin barrier activation and wrinkle-reducing effect of the human body.

The melittin-like recombinant protein according to the present invention and the nucleotide coating the melittin-like recombinant protein according to the present invention have an effect of enhancing the intercellular binding force of epidermal cells, suppressing cell permeability, and further promoting collagen synthesis in dermal cells.

Accordingly, the composition for skin comprising the melittin-like recombinant protein or nucleotide may be useful for medical or cosmetic purposes because it has an effect of activating skin barrier of the human body and improving skin wrinkles.

Also, according to the present invention, it is possible to efficiently mass-produce purified melittin-like recombinant protein without taking a bee poison by applying an electric shock to a bee, or a purification process for removing harmful components from the bee poison.

Figure 1 shows a DNA map of the pET28a expression vector and the recombinant expression vector pET28a-Meebong,
FIG. 2 is an SDS-PAGE photograph of a melittin-like recombinant protein according to the present invention,
FIG. 3 is a graph showing the cytotoxicity of human epidermal cells according to the concentration of the recombinant protein,
FIG. 4 is a graph showing the cytotoxicity of the recombinant protein to human-derived skin fibroblasts,
FIG. 5 is a graph showing changes in intercellular binding force of human epidermal cells according to the concentration of the recombinant protein,
FIG. 6 is a graph showing changes in cell permeability according to the concentration of the recombinant protein on human-derived epidermal cells,
FIG. 7 is a graph showing changes in cell survival rate of human fibroblasts treated with an oxidative stimulant by concentration of the recombinant protein,
8 is an immunoblot analysis chart showing whether or not the recombinant protein is produced in the form of procollagen against human-derived skin fibroblasts.

Melittin similar according to the invention of recombinant proteins in Choline Cellar (Collinsella sp.) Microorganisms, preferably choline Cellar Aero Pacifico Enschede (Collinsella Aerofaciens KCTC 15038, and consists of the amino acid sequence of SEQ ID NO: 4.

The present inventors confirmed that the recombinant protein has an effect of enhancing the intercellular binding force of epidermal cells, suppressing cell permeability, and further promoting collagen synthesis in dermal cells, and named 'Meebong 81'.

The name 'Meebong 81' means 'bee venom-like protein consisting of 81 amino acid sequences derived from microorganisms of the genus Cholinesella'. Meebong 81 has amino acid sequence and function similar to melittin, which is the main active ingredient of bee venom.

In order to prepare the melitin- like recombinant protein according to the present invention, two primers are isolated from the above-mentioned Collinsella sp. Microorganism, preferably the strain Cholinescela aeropathiens KCTC 15038 as a template. The primers consist of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, respectively.

Subsequently, the primer is subjected to PCR (polymerase chain reaction) to obtain a DNA fragment having the genetic information for Meebong 81, that is, a nucleotide having a nucleotide sequence of SEQ ID NO: 3. Then, the nucleotide is cleaved with a restriction enzyme, inserted into an expression vector to prepare a recombinant expression vector, and the host cell is transformed with the recombinant expression vector.

The expression vector may be a plasmid vector or a viral vector. As the plasmid vector, mammalian expression plasmids known in the art can be used. For example, pET, pcDNA, pRK5 (EP 307,247), pSV16B (WO 91/08291), PVL1392 (PharMingen), pHid / TAT plasmid (Kwon JH et al., 2007, Biochem. Biophys. Res. Commun. 399-404), and the like, but are not limited thereto.

The plasmid vector into which the nucleotide encoding Meebong 81 is introduced may be transient transfection, microinjection, transfection, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran Is introduced into host cells by methods such as DEAE Dextran-mediated transfection, polybrene-mediated transfection, electrophoresis, gene gun, etc. And preferably transient transfection methods can be used.

The viral vector may be a retrovirus vector, an adenovirus vector, a herpes virus vector, or an avipoxvirus vector. For example, the retroviral vector is constructed such that all of the viral genes have been removed or altered so that the non-viral proteins are made in cells infected with the viral vector. Preferably, PA317 amphotropic retrovirus packaging cells, which are FDA-approved retroviral vectors, can be used (Miller, A. D. and Buttimore, C. Mol. Cell Biol. 6: 2985-2902, 1986).

As the host cell, E. coli is preferable.

Meanwhile, the composition for skin according to the present invention contains the melittin-like recombinant protein, that is Meebong 81 or a nucleotide encoding the Meebong 81 as an active ingredient. At this time, the Meebong 81 may further include a polyhistidine region. In one embodiment of the invention, the recombinant protein comprising the polyhistidine region may be a protein in which the polyhistidine region is further fused to Meebong 81 and its N-terminus or C-terminus.

The polyhistidine region may be a His-tag sequence commonly used in the art and includes, for example, a His-tag sequence for protein purification (e.g., affinity chromatography) or Tag, VS-tag, Xpress-tag, T-tag, Ha-tag, FLAG-tag, myc-tag, E-tag, V5-tag, Fc-tag, tag, a streptavidin binding protein (SBP) -tag, a glutathione S-transferase (GST) -tag, a maltose-binding protein (MBP) -tag, Thioredoxin-tag, chitin-binding domain (CBD) -tag, ketosteroid isomerase (KSI) -tag, dihydrofolate reductase (DHFR) -tag, NusA-tag, intein-tag, polu (NANP) -tag, biotin carbohydrate xyl carrier protein, BCCP) -tag, green fluorescent protein (GFP) -tag, and the like.

The composition for skin promotes the intercellular binding force without cytotoxicity in skin epidermal cells, activates skin barrier by inhibiting cell permeability, etc., and promotes antioxidative effect and collagen biosynthesis in human skin fibroblasts without cytotoxicity It has the effect of improving the wrinkles of the skin.

The composition for skin may be prepared in the form of a pharmaceutical composition or a cosmetic composition. The pharmaceutical composition may contain a pharmaceutically acceptable carrier and may be formulated into a parenteral formulation such as a liquid, a suspension, an emulsion, a liposome, an ointment, a lyophilizing agent, a spray or an aerosol according to a conventional method.

Examples of the carrier include aqueous diluents or solvents such as phosphate buffered saline, purified water, sterilized water and mineral water, and non-aqueous diluents or solvents such as propylene glycol, polyethylene glycol and olive oil . Further, if necessary, it may contain a surfactant, an emulsifier, a thickener, an antioxidant, a wetting agent, a fragrance, a preservative and the like.

The content of Meebong 81 or the nucleotide contained in the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition and body weight of the patient, degree of disease, drug form, route of administration and duration. For example, it can be administered or applied in an amount of 0.01 to 1 mg, preferably 0.1 to 1 mg, once a day, or several times a day.

In addition, the cosmetic composition may be manufactured in various forms according to a conventional cosmetic preparation method. The cosmetic composition may be prepared in the form of, for example, shampoo, lotion, cream, gel, essence, or tonic, and may be diluted with a conventional cleansing liquid, a converging liquid, and a humectant. The cosmetic composition may also contain customary adjuvants such as stabilizers, solubilizers, vitamins, pigments, and flavorings commonly used in the cosmetic composition arts.

The content of Meebong 81 or the nucleotide contained in the cosmetic composition may be 0.001 to 50% by weight, preferably 1 to 20% by weight, of the total weight of the composition.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are for illustrating the present invention, and the scope of protection of the present invention is not limited by these examples.

[ Example ] Meebong Of 81 protein  Produce

Collinsella , which was sold by the BRC (KCTC) two kinds of primers by using the genomic DNA of aerofaciens KCTC 15038 strain as a template, i.e., 5'-CACCTA GGATCC ACCGGCGAGCTCACGGCG-3 ' ( SEQ ID NO: 1, BamHⅠ site underlined) and 5'-CAGAGA CTGGAG TTAGGCATCGTTTCGAAT-3' ( SEQ ID NO: 2, Underlined Xho I site) was obtained and PCR was performed to obtain a DNA fragment (SEQ ID NO: 3).

The recombinant expression vector pET28a-Meebong was prepared by digesting the DNA fragment (SEQ ID NO: 3) with restriction enzymes BamHI and XhoI and inserting them into pET28a expression vector (origin: Novagen) . 1 (A) is a DNA map of the pET28a expression vector (source: Novagen), and (B) is a DNA map of the recombinant expression vector pET28a-Meebong in which the Meebong gene is inserted into the expression vector.

The Escherichia coli was cultured for a predetermined time, IPTG (isopropyl- beta -D-thio-galactoside) was added thereto, and further cultured for 12 hours. The cultured Escherichia coli was recovered by centrifugation, disrupted by an ultrasonic grinder, and the recombinant protein Meebong 81 was isolated by Ni-IDA affinity column chromatography.

Purified Meebong 81 was stained with Coomassie brilliant blue, and SDS-PAGE photographs were taken and attached to FIG. 2. In Figure 2, the left lane is the protein size marker. The right lane is the Meebong 81 protein.

[ Test Example  1] Cytotoxicity test for epidermal cells

Human dermal keratinocytes were cultured in DMEM medium supplemented with 10% FBS and antibiotic (penicillin streptomycin), and the cells were transferred to a 96-well plate in a culture dish on the third day of culture. After cells were added, Meebong 81 was added to the culture medium for each concentration and further cultured for 24 hours at 37 ° C in 5% CO ₂ .

Subsequently, MTT solution (Sigma) was treated to quantitate the cell viability, and cell viability was measured with an absorbance meter at 570 nm after completion of the reaction. The results are shown graphically in FIG. As a result of the experiment, the Meebong 81 of the present invention had no effect on the survival of the epidermal cells at all concentrations as compared with the control group.

[ Test Example  2] Cytotoxicity test for dermal fibroblasts

Human dermal fibroblasts were cultured in DMEM medium supplemented with 10% FBS and antibiotic (penicillin streptomycin), and the cells were transferred to a 96-well plate in a culture dish on the third day of culture. After cells were added, Meebong 81 was added to the culture medium for each concentration and further cultured for 24 hours at 37 ° C in 5% CO ₂ .

Subsequently, MTT solution (Sigma) was treated to quantify cell viability. After completion of the reaction, the cell viability was measured using a 570 nm absorptiometer. The results are shown graphically in FIG. As a result of the experiment, Meebong 81 of the present invention had no effect on the survival of dermal fibroblasts at all concentrations as compared with the control group.

[ Test Example  3] Intercellular  Promotion of cell-cell interaction

Human dermal keratinocytes were seeded on 24-well plates at a concentration of 5 × 10 ⁴ , and the same cells were seeded at 1 × 10 ⁵ in a microtube. The cells were incubated with fluorescence with a Vybrant DiO cell labeling dye (ThermoFisher) Lt; / RTI > The Meebong 81 protein was treated at different concentrations for 30 minutes and then washed.

Then, the cells pre-seeded in 24-well plates were treated with fluorescent dyed cells, followed by reaction for 10 minutes, followed by washing. After measuring the binding force between the epidermal cells at 480/501 nm, the results are shown in a graph in FIG. Respectively. As a result of the experiment, it was shown that the intercellular interactions were promoted with increasing the treatment concentration of Meebong 81.

[ Test Example  4] Cell permeability test

Human dermal keratinocytes were cultured and the cells were transferred from the culture dish to a transwell (24-well) insert chamber (Millipore) on the third day of culture. Cell monolayer Lt; / RTI > for 24 hours. Here, Meebong 81 was treated at different concentrations and further cultured for 24 hours.

After 24 hours, the fluorescence emitted from the fluorescence reagent FITC-dextran 4 kDa (Sigma-Aldrich) was measured by excitation 490 nm and emission 520 nm fluorescence measurement to calculate the cell permeability. 6. As a result, the cell permeability was suppressed with increasing Meebong 81 concentration.

[ Test Example  5] Test of cytoprotective effect on oxidative stimulation

Human dermal fibroblasts were cultured and on the third day of culture, the cells were transferred to a 96-well plate in a culture dish. Cells were treated with Meebong 81, treated with 500 μM hydrogen peroxide, and further incubated at 37 ° C in 5% CO _{2 for} 24 hours.

After the reaction, the cell survival rate was measured using a 450 nm absorbance meter, and the results are shown in the attached Table 7. As a result of the test, Meebong 81 was used for the oxidative stimulation It was confirmed that the cell protective effect was obtained.

[ Test Example  6] Promoting effect test of collagen biosynthesis

In order to observe the effect of human dermal fibroblasts on collagen biosynthesis, immunoblot analysis confirmed the promoting effect of procollagen type I on biosynthesis. The cells were cultured in 6-well plates using DMEM medium containing 10% FBS, and Meebong 81 (500 nM) was treated with DMEM without FBS.

The cells were collected and immunoblot analyzed. As shown in FIG. 8, the Meebong 81-treated group was found to significantly increase the amount of procollagen type I biosynthesis in fibroblasts compared to the control group.

<110> UNIMEAT <120> Melittin-like recombinant protein, nucleotide encoding the same          and composition for skin containing them <130> IS17P011 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 cacctaggat ccaccggcga gctcacggcg 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 cagagactgg agttaggcat cgtttcgaat 30 <210> 3 <211> 267 <212> DNA <213> Artificial Sequence <220> <223> Collinsella aerofaciens KCTC 15038 <400> 3 cacctaggat ccaccggcga gctcacggcg gcaagcggag gcgtgctcgg cctgattccc 60 ttttcctggc ctgcacggct cgggtcgctc tccatcgaag cgggcattgc gcgggcaacg 120 gatgccgccc aggccgccac aatcagttcg cttgcctggg cgctcgccat cgcagcggtc 180 gtgctcacca taacggggct cgctgcgctc atagcatggt tcaaccgatt cgaaggagga 240 cgaaacgatg cctaactcca gtctctg 267 <210> 4 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Collinsella aerofaciens KCTC 15038 <400> 4 Met Thr Gly Glu Leu Thr Ala Ala Ser Gly Gly Val Leu Gly Leu Ile   1 5 10 15 Pro Phe Ser Trp Pro Ala Arg Leu Gly Ser Leu Ser Ile Glu Ala Gly              20 25 30 Ile Ala Arg Ala Thr Asp Ala Ala Gln Ala Ala Thr Ile Ser Ser Leu          35 40 45 Ala Trp Ala Leu Ala Ile Ala Ala Val Val Leu Thr Ile Thr Gly Leu      50 55 60 Ala Ala Leu Ile Ala Trp Phe Asn Arg Phe Glu Gly Gly Arg Asn Asp  65 70 75 80 Ala    

Claims (6)

A recombinant melittin-like recombinant protein, which is derived from a microorganism of the genus Collinsella sp., Comprising the amino acid sequence of SEQ ID NO: 4.
The melittin- like recombinant protein according to claim 1, wherein the Collinsella sp. Microorganism is a strain of Collinsella aerofaciens KCTC 15038.
A nucleotide derived from a Collinsella sp. Microorganism, encoding the melittin-like recombinant protein of claim 1, and comprising the nucleotide sequence of SEQ ID NO: 3.
Collinsella aerofaciens) obtaining from KCTC 15038 strain, primers consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and;
Obtaining a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 3 from the primer;
Preparing a recombinant vector using the DNA fragment and the pET28a vector;
Introducing the recombinant expression vector into E. coli to transform the E. coli;
Culturing the Escherichia coli, and isolating and purifying a protein consisting of the amino acid sequence of SEQ ID NO: 4;
Wherein the mutitin-like recombinant protein is a recombinant protein.
Wherein the melitin-like recombinant protein of claim 1 or at least one of the nucleotides of claim 3 is contained as an active ingredient and has the skin barrier activation and wrinkle-reducing effect of the human body.
5. The composition for dermatoses according to claim 4, wherein the melittin-like recombinant protein is a fusion of a polyhistidine domain at the N-terminal or C-terminal.

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