KR101657297B1 - Triple fusion protein comprising human growth hormone, human epidermal growth factor and human insulin-like growth factor-1 with increased cell proliferation effect and cosmetic composition for anti-wrinkle and anti-aging comprising the same as effective component - Google Patents
Triple fusion protein comprising human growth hormone, human epidermal growth factor and human insulin-like growth factor-1 with increased cell proliferation effect and cosmetic composition for anti-wrinkle and anti-aging comprising the same as effective component Download PDFInfo
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- KR101657297B1 KR101657297B1 KR1020160053831A KR20160053831A KR101657297B1 KR 101657297 B1 KR101657297 B1 KR 101657297B1 KR 1020160053831 A KR1020160053831 A KR 1020160053831A KR 20160053831 A KR20160053831 A KR 20160053831A KR 101657297 B1 KR101657297 B1 KR 101657297B1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF] (urogastrone)
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/62—DNA sequences coding for fusion proteins
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Abstract
Description
The present invention relates to a triple fusion protein comprising human growth hormone, a human epithelial cell regenerating factor, human insulin like growth factor-1, and a skin wrinkle improving and anti-aging cosmetic composition containing the same as an effective ingredient.
Human growth hormone (hGH) affects many organs in the body, including skeletal growth, muscle growth, fat breakdown, organs growth, and sexual maturation in the growth phase. In general, human growth hormone has been used as an injectable drug in strengthening the cardiovascular system, improving exercise capacity, and strengthening muscles. It is known that growth hormone has an excellent effect in preventing aging by proving the effect of improving metabolism and elastic skin such as fat decomposition effect, arthritis improvement, skin thickness increase, cardiopulmonary function improvement, muscle mass increase.
Growth hormone secretion is the best in puberty, gradually decline after the 20s, but the growth hormone secretion is still 40 years old. However, if it is over 60, it will fall below 50% of the 20s. The decrease in growth hormone affects skin elasticity, especially the increase of eye wrinkles around the eyes and mouth, as the skin thickness and skin collagen decrease. Growth hormone has been used as a typical drug administration method to prevent this aging process.
Growth hormone plays an important role in maintaining elastic skin such as cardiovascular disease treatment and wrinkle improvement. It can be used not only for skin beauty but also for skin care to prevent aging.
Human Epidermal Growth Factor (hEGF) binds to epithelial cell regenerative factor receptors on the cell surface and induces dimerization of epithelial regenerative factor receptors. The dimeric epithelial regenerative factor receptor activates the tyrosine kinase present in the receptor and induces an intracellular signaling system. Through such a series of processes, glycolysis and protein synthesis in the cells are promoted, and the cell growth finally proceeds.
The human epithelial cell regenerating factor, which plays a pivotal role in skin regeneration, decreases as aging progresses. As a result, skin cell proliferation and migration are reduced, which may cause skin aging, wrinkles increase, decrease in elasticity, and the like. Currently, it is used as a pharmaceutical or functional cosmetic for promoting skin cell growth.
Human Insulin-like Growth Factor-1 (hIGF-1) is a hormone analogous to insulin and molecular structure, plays an important role in the growth of children and has an anabolic effect in adults . Human insulin-like growth factor-1 is a polypeptide molecular structure in which 70 amino acids are linked in a single chain, with three disulfide bonds in the molecule and a molecular weight of 7,649 daltons.
The main function of human insulin-like growth factor-1 is to stimulate the Akt / Pkb signaling system by stimulating the mammalian Target of Rapamycin (mTOR), which activates the intracellular signaling system by binding to specific receptors and stimulates muscle protein synthesis, Induce proliferation.
Human insulin-like growth factor-1 mediates primarily the effects of growth hormone, and human insulin-like growth factor-1 is also involved in DNA synthesis to regulate cell growth and differentiation. Human insulin-like growth factor-1 induces collagen synthesis, and as a result, human insulin-like growth factor-1 plays a role in regenerating damaged cells and delaying aging by increasing skin elasticity.
Under these circumstances, the inventors of the present invention have found that the effect of the present invention on wrinkle improvement and anti-aging function of human growth hormone protein, maintenance of skin regeneration function of human epithelial cell growth factor protein, delayed aging function of human insulin like growth factor- As a result, we succeeded in developing a triple fusion protein containing human growth hormone, human epithelial cell growth factor, and human insulin-like growth factor-1.
Korean Patent No. 1551728 discloses a functional cosmetic composition comprising a growth factor and an amino acid. Korean Patent Laid-Open Publication No. 2015-0056021 discloses a cosmetic composition comprising a human growth hormone Discloses a cosmetic composition for skin improvement comprising a fusion protein of a human growth hormone, a human epithelial cell regenerative factor and a human insulin-like growth factor-1, There is no description of a composition for improving skin wrinkles and an anti-aging cosmetic composition contained as an active ingredient.
The present invention has been made in view of the above-mentioned needs, and the present inventors have produced triple fusion proteins including human growth hormone, human epithelial cell growth factor and human insulin like growth factor-1, The skin cell proliferation and the antioxidative effect were superior to each other, thereby completing the present invention.
In order to solve the above problems, the present invention provides a triple fusion protein comprising human growth hormone, human epithelial cell growth factor, and human insulin-like growth factor-1 having increased cell proliferation effect comprising the amino acid sequence of SEQ ID NO: 2 .
The present invention also provides a gene encoding a triple fusion protein comprising human growth hormone, human epithelial cell growth factor, and human insulin-like growth factor-1 with increased cell proliferation effect.
The present invention also provides a recombinant vector comprising the gene.
The present invention also provides a host cell transformed with the recombinant vector.
The present invention also provides a method for producing a triple fusion protein comprising the step of transfecting a host cell with the recombinant vector to overexpress a gene encoding a triple fusion protein comprising human growth hormone, human epithelial cell growth factor and human insulin like growth factor- A method for producing a triple fusion protein comprising human growth hormone, a human epithelial cell growth factor and human insulin-like growth factor-1 in a host cell.
The present invention also provides a triple fusion protein comprising human growth hormone, human epithelial cell growth factor, and human insulin-like growth factor-1 produced by the above method.
The present invention also relates to a pharmaceutical composition comprising a human growth hormone, a human epithelial cell growth factor, and a triple fusion protein comprising human insulin-like growth factor-1, which has an increased cell proliferation effect comprising an amino acid sequence of SEQ ID NO: And an anti-aging cosmetic composition.
The triple fusion protein comprising human growth hormone, human epithelial cell growth factor, and human insulin like growth factor-1, which have increased cell proliferation and antioxidant effects of the present invention, is excellent in skin regeneration function, skin elasticity enhancement and anti- It may be useful as a functional cosmetic raw material.
Figure 1 shows a gene encoding a triple fusion protein (GH-EGF-IGF-1) comprising human growth hormone (GH), human epithelial cell regenerative factor (EGF) and human insulin like growth factor- (PET22b :: GH-EGF-IGF-1) containing the recombinant plasmid (pET22b :: IGF-1) and the transformation into E. coli.
FIG. 2 shows the result of electrophoresis on SDS-polyacrylamide gel to confirm the expression of triple fusion protein (GEI-1) in the transformed E. coli. A is the electrophoresis result of the separated and purified fusion protein, and B is the result of confirming the triple fusion protein using the EGF confirmation kit. M, a protein size marker; 1, before induction of fusion protein expression; 2, after induction of fusion protein expression; C, control group; T, test sample.
FIG. 3 is a graph showing the effect of human fibroblast (HDFa) on human growth hormone (GH), human epithelial cell regenerating factor (EGF), human insulin like growth factor-1 (IGF-1) and triple fusion protein (GEI- The results of this study are as follows.
FIG. 4 shows the wound healing effects of triple fusion proteins at different concentrations (0 to 20 ppm) in HaCaT cells for 24 hours.
FIG. 5 shows the cell proliferation effect of triple fusion protein (GEI-1) at different concentrations (0 to 20 ppm) in HaCaT cells.
FIG. 6 shows the cell adhesion effect of triple fusion protein (GEI-1) at different concentrations (0 to 20 ppm) in HaCaT cells.
FIG. 7 is a graph comparing the DPPH radical scavenging activity of human growth hormone (GH), human epithelial cell regenerating factor (EGF), human insulin like growth factor-1 (IGF-1) and triple fusion protein (GEI-1) This is a result. L-ascorbic acid, positive control.
In order to accomplish the object of the present invention, the present invention provides a method for producing a fusion protein comprising human growth hormone, human epithelial cell growth factor, and human insulin-like growth factor-1 comprising the amino acid sequence of SEQ ID NO: to provide.
The range of the triple fusion protein comprising human growth hormone, human epithelial cell regenerating factor and human insulin like growth factor-1, which has increased cell proliferation effect according to the present invention, is a protein having the amino acid sequence of SEQ ID NO: 2, Includes equivalents. As used herein means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, more preferably 95% or more, Or more homologous to the amino acid sequence of SEQ ID NO: 2 and exhibiting substantially the same activity as the protein of SEQ ID NO: 2. "Substantially homogeneous activity" refers to the activity of promoting skin elasticity and preventing skin aging while having cell proliferation effect.
The present invention also provides a gene encoding a triple fusion protein comprising human growth hormone, human epithelial cell regenerating factor and human insulin-like growth factor-1 with increased cell proliferation effect. The gene may comprise the nucleotide sequence of SEQ ID NO: 1, which is optimized for E. coli codon, but is not limited thereto.
The gene coding for the triple fusion protein comprising human growth hormone, human epithelial cell regenerating factor and human insulin like growth factor-1 having increased cell proliferation effect according to the present invention may include the nucleotide sequence of SEQ ID NO: 1 . In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% sequence identity with the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: Or more of the sequence homology. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >
"Codon optimization" means altering the codon of a polynucleotide that encodes a protein that is preferentially used in a particular organism so that the encoded protein is more efficiently expressed in the organism. The codon usage by a particular organism is not arbitrary but is biased towards a particular codon triplet, although the genetic code is axial accumulation, in that most amino acids are represented by some codons, referred to as "synonymous" or "consent" codons. Such codon usage bias may be higher in relation to a given gene, a gene of common function or ancestral origin, a highly expressed protein versus a low copy number protein, and a collective protein coding region of the organism genome. The nucleotide sequence of SEQ ID NO: 1 of the present invention is a sequence optimized for E. coli codons so that the human epithelial cell regenerating factor and the human growth hormone protein coding gene can be expressed in E. coli.
The present invention also provides a recombinant vector comprising the gene and a host cell transformed with the recombinant vector.
The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.
In the present invention, the gene can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.
An expression vector comprising a gene sequence coding for a triple fusion protein comprising human growth hormone, human epithelial cell growth factor and human insulin like growth factor-1 and an appropriate transcription / translation regulatory signal with increased cell proliferation effect can be obtained by those skilled in the art Can be constructed by well-known methods. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.
The recombinant vector according to one embodiment of the present invention comprises a gene (SEQ ID NO: 1) encoding a triple fusion protein comprising human growth hormone, human epithelial cell growth factor and human insulin like growth factor-1 synthesized on a pET22b vector 5 'end (Nde I restriction enzyme site) and 3' ends (Xho I restriction enzyme site) in the mainframe (in frame) as was fusion produced, lac promoter (lac promoter), the gene and the lac I repressor (lac I repressor) to produce a triple fusion protein.
Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention while being stable in prokaryotic cells. Examples of the host cell include E. coli Rosetta, E. coli JM109, E. coli BL21, Enterobacteria and strains such as Escherichia coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus subtilis, Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species .
Saccharomyce cerevisiae , insect cells, human cells (for example, CHO (Chinese hamster ovary), W138, BHK, COS-7, 293, etc.) are used as host cells when the vector of the present invention is transformed into eukaryotic cells. HepG2, 3T3, RIN and MDCK cell lines) and plant cells.
The host cell transformed with the recombinant vector according to an embodiment of the present invention may be preferably E. coli, more preferably E. coli Rosetta2 (DE3) pLysS, but is not limited thereto.
The method of delivering the vector of the present invention into a host cell can be carried out by a CaCl 2 method, a single method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580) Method or the like. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .
The present invention also provides a method for producing a triple fusion protein comprising the step of transfecting a host cell with the recombinant vector to overexpress a gene encoding a triple fusion protein comprising human growth hormone, human epithelial cell growth factor and human insulin- A method for producing a triple fusion protein comprising human growth hormone, human epithelial cell growth factor, and human insulin-like growth factor-1 and human growth hormone, human epithelial cell growth factor and human insulin like growth factor-1 Lt; RTI ID = 0.0 > fusion protein. ≪ / RTI >
In the method according to one embodiment of the present invention, the host cell can be preferably E. coli, more preferably, but not limited to, Escherichia coli Rosetta2 (DE3) pLysS.
The present invention also relates to a skin wrinkle-improving and anti-aging cosmetic composition comprising, as an active ingredient, a triple fusion protein comprising human growth hormone, human epithelial cell growth factor and human insulin-like growth factor- Lt; / RTI >
The triple fusion protein comprising the human growth hormone, human epithelial cell growth factor and human insulin like growth factor-1 of the present invention having increased cell proliferation effect is a fusion protein consisting of the amino acid sequence of SEQ ID NO: 2, Wrinkle improvement effect and anti-aging function maintenance effect; Skin regeneration function of human epithelial cell growth factor protein; And the human insulin-like growth factor-1 protein can be expected to have a delayed aging function and a skin elasticity-enhancing effect.
In the cosmetic composition according to an embodiment of the present invention, the content of the triplet fusion protein comprising the human growth hormone, the human epithelial cell growth factor and the human insulin like growth factor-1, which have increased cell proliferation effect, 0.000001 to 0.0001% by weight.
If the content of the protein is less than 0.000001% by weight, the effect is insufficient in the skin. If the content of the protein is more than 0.0001% by weight, the effect of increasing the content of the protein is insignificant.
The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the above-mentioned effective components, and examples thereof include lipids, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents , Conventional adjuvants such as lipid vesicles, and carriers.
The composition of the present invention may be prepared in any form conventionally produced in the art and may be prepared in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation , A wax foundation, and a spray, but the present invention is not limited thereto. More specifically, the present invention relates to a cosmetic composition for skin, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, eye cream, moisturizer cream, hand cream, essence, Foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap and 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 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 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.
Hereinafter, the present invention will be described in detail with reference to examples. The following examples are illustrative of the present invention, but the present invention is not limited to the following examples.
Example 1 Preparation of Recombinant Expression Vectors and Transforming Microorganisms for Production of Triple Fusion Proteins Containing Human Growth Hormone, Human Epithelial Cell Regenerator and Human Insulin-Like Growth Factor-1
The optimized gene, recombinant expression vector and transforming recombinant microorganism encoding a triple fusion protein comprising human growth hormone, human epithelial cell regenerating factor and human insulin like growth factor-1 were prepared by the following method.
A triple fusion protein consisting of 314 amino acids optimized to be expressed in a host microorganism using as a template a gene encoding a triple fusion protein comprising human growth hormone, human epithelial cell regenerative factor and human insulin like growth factor-1 (SEQ ID NO: 1) were synthesized and synthesized. The primers used for the synthesis of triplet fusion proteins including human growth hormone, human epithelial cell regenerating factor and human insulin like growth factor-1 were as follows: forward primer: 5'-aaggagatatacatatTTCCCCACTATTCCGCTG-3 '(SEQ ID NO: 3) and reverse primer : 5'-ggtggtggtgctcgagGGCTGATTTTGCCGGTTTC-3 '(SEQ ID NO: 4).
The gene fragment and the recombinant plasmid were digested with the same restriction enzymes (5'-terminal NdeI restriction enzyme and 3'-terminal XhoI restriction enzyme) and inserted into the recombinant plasmid (pET22b :: GH-EGF-IGF -1) was produced. The recombinant plasmid reactant prepared above was transformed into E. coli TOP10, and a large amount of the gene construct was obtained from the host microorganism. In addition, the recombinant plasmid was transformed into E. coli Rosetta2 (DE3) pLysS to prepare a recombinant microorganism for producing a triple fusion protein in which a gene construct was inserted into the host microorganism (FIG. 1).
Example 2 Induction and Isolation of Human Growth Hormone, Human Epithelial Cell Regenerator and Triple Fusion Protein Containing Human Insulin-Like Growth Factor-1
E. coli Rosetta2 (DE3) pLysS prepared in Example 1 was inoculated in a 1 L LB or BSB medium until OD 600 = 0.6-0.8 in a batch culture or in a 20 L fermentor And cultured until OD 600 = 15-20. Then, final 1 ~ 5mM IPTG or 2% lactose was added to the cell culture medium to induce the gene expression of the recombinant E. coli. After induction of gene expression, cells were further cultured for 3 to 4 hours and recovered by centrifugation. The cells were completely suspended in a buffer solution (phosphate buffered saline, NaCl 8 g, KCl 0.2 g, Na 2 HPO 4 1.44 g, KH 2 PO 4 0.24 g / l, pH 7.4), and then the cells were disrupted using an ultrasonic disrupter And the protein-containing solution was separated.
Protein expression was confirmed by 15% SDS-polyacrylamide gel electrophoresis using the separated solution as a sample. As a result, expression of a triple fusion protein was confirmed in a cell lysate inducing protein expression through IPTG or lactose (Fig. 2A). FIG. 2B shows the results of confirming the presence of EGF in the triple fusion protein isolated and purified using the EGF detection kit.
Example 3 HDFa cell proliferation effect of triple fusion protein including human growth hormone, human epithelial cell regenerating factor and human insulin like growth factor-1
The presence of the triple fusion protein isolated and purified in Example 2 was confirmed, and the cell proliferation effect was confirmed in human dermal fibroblasts adult (HDFa).
0.02 ppm and 0.2 ppm of each protein was treated with each of human growth hormone, human epithelial cell regenerating factor, and skin fibroblast cultured for measuring the activity of human insulin-like growth factor-1 and triple fusion protein, respectively Lt; RTI ID = 0.0 > 37 C < / RTI > Thereafter, cell viability was confirmed by crystal violet staining.
As a result, it was confirmed that the effect of proliferation of dermal fibroblasts was better in the cells treated with the triple fusion protein than the untreated control (Fig. 3). In addition, the cell proliferation effect of the triple fusion protein was higher than that of each single protein treatment group. This corresponds to the respective active domains used for the production of the triple fusion protein, but not to the respective full-length proteins, of the treated GH, EGF and IGF-1, and to the same concentration (for example, 0.02 ppm ), The moles of triple-fused protein are about 1/3 of those of GH, EGF and IGF-1, respectively. Therefore, if the similar concentration of the dermal fibroblast proliferation effect is exhibited at the same concentration, the triple fusion protein has a dermal fibroblast proliferation effect about three times as much as that of the respective GH, EGF and IGF-1. As can be seen from FIG. 3, it was found that the treatment of the triple fusion protein had at least three times more dermal fibroblast proliferation effect than that of each of GH, EGF and IGF-1 It is. From the above results, it could be deduced that the triplet fusion protein increased cell proliferation effect.
Example 4: Cell proliferation, wound healing and cell fusion effect in HaCaT cells of human growth hormone, human epithelial cell regenerating factor and triple fusion protein including human insulin like growth factor-1
The activity of the fusion protein in HaCaT (normal keratinocyte cell) cells was measured by selecting a sample in which the presence of the triple fusion protein isolated and purified in Example 2 was confirmed. Briefly, the method for measuring the activity of a fusion protein comprises culturing HaCaT cells and treating the cells with a concentration of 0, 0.02 ppm, 0.2 ppm, 2 ppm, and 20 ppm of the triple fusion protein to inhibit cell proliferation, wound healing, Were analyzed and evaluated.
When cell proliferation was analyzed using PrestoBlue TM Cell Viability reagent (Invitrogen, USA), the cell proliferation effect of HaCaT cells by triple fusion protein treatment was confirmed (FIG. 4), and in particular, 2ppm triple fusion protein treatment Cell proliferation was observed. From the above results, it was confirmed that the triple fusion protein penetrated into the skin cells and showed excellent cell proliferation effect.
Secondly, after culturing HaCaT cells, the wells were treated with 0, 0.02ppm, 0.2ppm, 2ppm and 20ppm concentrations of the triple fusion proteins and then observed with a microscope (Olympus CK40, Olympus, Japan) for 24 hours The wound healing effect of HaCaT cells was observed. HaCaT cells treated with the triple fusion protein exhibited superior wound healing effects compared with the untreated control. Especially, the effect was apparent in the 2 ppm concentration range (FIG. 5).
Finally, the triple fusion proteins at concentrations of 0, 0.02 ppm, 0.2 ppm, 2 ppm and 20 ppm were coated on 96 well plates and treated with HaCaT cells. Unincorporated cells were removed after the reaction at 37 ° C for 20 min in the incubator and cultured in a 37 ° C incubator for 12 hrs to recover the spliced cells. Thereafter, the binding effect between the cell and the triple fusion protein was analyzed using the PrestoBlue TM Cell Viability reagent. As a result, it was confirmed that HaCaT cells treated with the fusion protein at a concentration of 0.02 to 20 ppm showed a superior bonding effect to that of the negative control treated with BSA (bovine serum albumin) (FIG. 6).
Example 5 Antioxidant Efficacy of Human Growth Hormone, Human Epithelial Cell Regeneration Factor and Triple Fusion Protein Containing Human Insulin Like Growth Factor-1
DPPH (1,1-diphenyl-2-picryl hydrazyl) analysis was performed to measure the antioxidant activity of triple fusion proteins. The above reagents exist as relatively stable free radicals and thus are one of the in vitro methods used primarily for confirming free radical scavenging action.
L-ascorbic acid was used as a positive control in DPPH analysis. Experimental methods were as follows: human growth hormone (GH), human epithelial cell regeneration factor (EGF), human insulin-like growth factor-1 (IGF-1), triple fusion protein and L- ascorbic acid 0.2 mM concentration. The mixture was mixed at a ratio of 1: 1 and left at 37 ° C for 30 minutes. The absorbance at 520 nm was measured with an ELISA reader. The free radical scavenging activity (%) was calculated by the following
Free radical scavenging ability (%) = 100 - ((B / A) * 100)
[A: Absorbance of the control group not treated with the sample
B: absorbance of the control group or experimental group treated with the sample]
As a result, the triple fusion protein including human growth hormone, human epithelial cell regenerating factor and human insulin like growth factor-1 showed 1.5-fold more free radical scavenging ability than the control group, and the free radical scavenging ability (Fig. 7). Thus, the triple fusion protein has a high antioxidative effect, which means that it can have an antioxidant effect according to the antioxidant effect of the protein.
<110> Nexgen Biotechnologies, Inc. <120> Triple fusion protein comprising human growth hormone, human epidermal growth factor and human insulin-like growth factor-1 with increased cell proliferation effect and cosmetic composition for anti-wrinkle and anti-aging component <130> PN16153 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> GEI-1 <400> 1 atgttcccca ctattccgct gtcgcgactt tttgacaatg caatgctccg tgcccacagg 60 ctacatcaac tcgcgttcga tacctatcag gagttcgaag aggcttatat tccaaaggag 120 caaaaataca gcttccttca gaaccctcaa acgtcactct gtttctccga atccatcccc 180 actccatcca atcgcgagga gactcagcaa aagtctaacc ttgagctgct gcggataagt 240 ttgctgttga tccagtcctg gttggaacct gtgcagttcc ttaggagcgt tttcgctaac 300 agcctcgtat acggagccag cgacagcaat gtttatgatc tccttaagga tttggaagaa 360 ggtatccaaa cactgatggg ccggctggag gatggatcac caagaaccgg acaaattttt 420 aagcagacct actctaaatt cgacaccaat tcgcataacg atgacgcttt gctcaagaac 480 tacgggcttc tctactgttt tcgtaaagat atggacaagg tggaaacatt tctcaggatc 540 gtccaatgcc gctctgtcga gggttcctgc ggctttaact cagactctga gtgcccactg 600 tctcacgacg gctactgcct tcacgacgga gtctgcatgt acatcgaggc tttggataag 660 tacgcttgta attgcgtcgt tggttacatt ggagagcgct gccaataccg tgacttaaaa 720 tggtgggagt tgcgcggtcc agaaacttta tgcggcgccg aactggtgga cgccttacaa 780 tttgtctgtg gtgaccgcgg attttatttt aataagccaa cgggttatgg tagtagcagt 840 cgccgtgcgc cgcagaccgg cattgttgac gagtgttgct ttcgttcctg cgatctgcgc 900 cgcctggaaa tgtattgcgc accgctgaaa ccggcaaaat cagcc 945 <210> 2 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> GEI-1 <400> 2 Met Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu 1 5 10 15 Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe 20 25 30 Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn 35 40 45 Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn 50 55 60 Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser 65 70 75 80 Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser 85 90 95 Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr 100 105 110 Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg 115 120 125 Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr 130 135 140 Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn 145 150 155 160 Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr 165 170 175 Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 195 200 205 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 210 215 220 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 225 230 235 240 Trp Trp Glu Leu Arg Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val 245 250 255 Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys 260 265 270 Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile 275 280 285 Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met 290 295 300 Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala 305 310 315 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aaggagatat acatatgttc cccactattc cgctg 35 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggtggtggtg ctcgagggct gattttgccg gtttc 35
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| KR101551728B1 (en) * | 2013-07-09 | 2015-09-09 | 휴먼바이오텍 주식회사 | A functional cosmetic composition comprising growth factors and amino acids |
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| KR20150056021A (en) * | 2013-11-14 | 2015-05-22 | 주식회사 엘지생활건강 | Cosmetic composition for skin care comprising fusion protein with hGH |
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