CN110078793A - A kind of polypeptide and application thereof with anti-aging and repair - Google Patents
A kind of polypeptide and application thereof with anti-aging and repair Download PDFInfo
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- CN110078793A CN110078793A CN201910209385.5A CN201910209385A CN110078793A CN 110078793 A CN110078793 A CN 110078793A CN 201910209385 A CN201910209385 A CN 201910209385A CN 110078793 A CN110078793 A CN 110078793A
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- polypeptide
- aging
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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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
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Birds (AREA)
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- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a kind of polypeptide and application thereof with anti-aging and repair.The present invention obtains a kind of novel polypeptide with anti-aging and efficient repair from marine organisms hydra, the polypeptide can promote fibroblastic growth, fibroblast is promoted to generate hyaluronic acid, therefore having, which improves skin quality collagen, is lost, reinforces moistening effect, efficiently repairs skin, highlighting whitening and crease-resistant and other effects, can be used for preparing anti-aging and repairs the drug or cosmetics of skin.
Description
Technical field:
The invention belongs to medical cosmetic product fields, and in particular to a kind of polypeptide with anti-aging and repair and its
Purposes.
Background technique:
The main body of skin is skin corium, is mainly made of fibroblast and matrix components.Fibroblast generates glue
The glycosaminoglycans such as former albumen and hyaluronic acid form connective tissue, play an important role in skin.Fibroblast Function because
The reduction of the other influences such as aging or ultraviolet light may result in the matrix components such as collagen, hyaluronic acid and reduce and be denaturalized.
The oxidative stress such as ultraviolet light cause skin injury, keep its coarse, and cause other adverse reactions.Due to matrix components reduction and
Oxidative stress accelerates skin that aging occurs, and wrinkle, spot, appearance dimness, texture thus occurs and loses smooth, elastic reduction etc.
Aging sign is presented.
Collagen and hyaluronic acid have been main trends instantly as the direction of prevention skin aging.It analyses scientifically transparent
Matter acid is a kind of high molecular polymer, and the way that hyaluronan extraction object is applied to skin is directly detrimental to skin and is absorbed
It utilizes.Hydra body is mainly made of stem cell, while the content of hyaluronic acid and collagen is higher, can maintain hydra
The ability that stem cell is constantly divided.
Summary of the invention:
The object of the present invention is to provide a kind of polypeptide and application thereof with anti-aging and repair.
It is of the invention the study found that extracting a kind of biological polypeptide (its amino acid sequence such as SEQ ID NO.2 institute from hydra
Show) fibroblastic growth can be promoted from cell level, stimulation fibroblast generates hyaluronic acid.
Therefore, the first purpose of the invention is to provide a kind of polypeptides, and amino acid sequence is as shown in SEQ ID NO.2.
A second object of the present invention is to provide the encoding gene of the polypeptide, nucleotide sequence such as SEQ ID
Shown in NO.1.
Third object of the present invention is to provide a kind of prokaryotic expression carriers of encoding gene containing the polypeptide.
The prokaryotic expression carrier is preferably vector pGEX -4T-1.
Fourth object of the present invention is to provide a kind of bacterium containing the prokaryotic expression carrier.
The bacterium is preferably e. coli bl21 (DE3).
Fifth object of the present invention is to provide the polypeptides in the preparation that preparation promotes fibroblastic growth
Using.
Sixth object of the present invention is to provide the polypeptides to promote fibroblast to generate hyaluronic acid in preparation
Application in preparation.
7th purpose of the invention is to provide the polypeptide in the drug or makeup of preparation anti-aging and reparation skin
Application in product.
8th purpose of the invention is to provide the drug or cosmetics of a kind of anti-aging and reparation skin, including described
Polypeptide is as active constituent.
The cosmetics can be prepared into various dosage forms such as freeze-dried powder, Essence, lotion, creme etc..
The present invention obtains the polypeptide of a kind of novel anti-aging and efficient repair from marine organisms hydra, the polypeptide energy
Enough promoting fibroblastic growth, stimulation fibroblast generates hyaluronic acid, therefore there is improvement skin quality collagen to be lost,
Reinforce moistening effect, efficiently repair skin, highlight whitening and crease-resistant and other effects, can be used for preparing anti-aging and repair skin
Drug or cosmetics.
Detailed description of the invention:
Fig. 1 is influence of the polypeptide (its amino acid sequence is as shown in SEQ ID NO.2) to fibroblastic growth.
Fig. 2 is the shadow that polypeptide (its amino acid sequence is as shown in SEQ ID NO.2) generates hyaluronic acid to fibroblast
It rings.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1:
1, RNA is extracted
The extraction of total serum IgE is extracted using Trizol (Invitorgen), the specific steps are as follows:
(1) it takes 50mg clear water Xi Hydra viridis global tissue in the mortar of Liquid nitrogen precooler, tissue is clayed into power
Later, it is transferred in the centrifuge tube equipped with 1mL Trizol, piping and druming mixes；
(2) 200 μ L chloroforms are added, acutely shakes 40s, is placed at room temperature for 5min；
(3) 4 DEG C, 12,000 × g is centrifuged 10min, draws supernatant and is transferred to new pipe；
(4) isopropanol of 0.5mL is added, mixes, -80 DEG C of precipitates overnights；
(5) 4 DEG C, 12,000 × g is centrifuged 10min, abandons supernatant；
(6) 75% ethyl alcohol wash are precipitated twice；
(7) supernatant is abandoned, 100 μ L DEPC water are added and dissolve RNA.
2, cDNA library constructs
CDNA library building uses SMART cDNA library construction Kit (Clontech), tool
Steps are as follows for body:
(1) RNA for utilizing step 1 synthesizes first chain of cDNA using MMLV；
(2) LD PCR amplification cDNA is used；
(3) protease K digesting and cDNA product is purified；
(4) Sfil digests, and carries out primer size separation using Chroma Spin400；
(5) cDNA to λ TripEx2 carrier is connected, after λ bacteriophage is packed, is converted into host cell；
(6) cDNA library titre is detected.
3, Prokaryotic expression vector construction
(1) design a pair covers the primer (being shown in Table 1) of hydra polypeptide sequence, the both ends of primer be separately added into BamH-I and
The site Xho-I and protection base；
1 primer sequence of table
(2) using clear water Xi cDNA as template, PCR amplification is carried out using the primer of step (1)；
(3) PCR product and pGEX-4T-1 carrier use BamH-I and Xho-I to carry out double digestion, and recovery purifying enzyme respectively
Cut product；
(4) PCR product and pGEX-4T-1 carrier after connecting digestion；
(5) connection product being converted into bacillus coli DH 5 alpha, picking positive colony, sequence verification carrier is correct,
Sequencing result shows that the sequence as shown in SEQ ID NO.1 is properly inserted into pGEX-4T-1 carrier, thus obtains propolypeptide
Nuclear expression carrier carries out follow-up test.
4, the prokaryotic expression of recombinant protein and purifying
(1) the polypeptide prokaryotic expression carrier in the bacillus coli DH 5 alpha of extraction step 3, conversion to e. coli bl21
(DE3)；
(2) it selects monoclonal and cultivates to OD=0.6, be added IPTG to final concentration of 0.1mM, 22 DEG C, the item of 180rpm
Inducing expression under part；
(3) bacterium solution is collected after 4h, 4 DEG C, 12,000 × g is centrifuged 10min；
(4) Lysozyme to final concentration 2mg/mL, then ultrasonication is added；
(5) Glutathione Sepharose 4B (GE Healthcare) purification of recombinant proteins is used, in 12%SDS-
PAGE glue analyzes purified product, the polypeptide purified (its amino acid sequence is as shown in SEQ ID NO.2).
5, measurement polypeptide promotes the activity of fibroblastic growth
(1) taking human dermal fibroblasts system BJ cell inoculation, (100 holes μ L/, cell inoculation amount are 1 in 96 orifice plates
×104A/hole), in 37 DEG C, 5%CO2(culture medium is not add the L-15 of growth stimulator containing cow's serum for bed board culture in incubator
Basal medium).
(2) the purified 100 μ L of polypeptide solution of step 4 is added into culture medium after cultivating 6h, and makes polypeptide final concentration point
Wei not be 5,10,20,30 μM (polypeptide group), taking one group of 3 repetition to add isometric PBS to do control is concentration 0 (PBS group), also
Negative control is not added cell list and adds culture medium.
(3) cell cultivates 48h in the incubator, and 10 μ L MTT solution (5mg/mL) are added in every hole, continues to train in incubator
3h is supported, inhales and abandons culture medium, 100 μ L DMSO of every hole addition, which shake to crystallization, is completely dissolved (MTT cell viability detection kit).
(4) absorbance of microplate reader detection 450nm and 625nm, calculates fibroblastic growth rate.
(5) it is presented as the result is shown in 5~30 μM of concentration of range polypeptide and promotes the trend of fibroblastic growth, and polypeptide
The cell growth rate of group (final concentration of 10 μM of polypeptide) is approximately about 2.6 times (see Fig. 1) of PBS group.
6, the effect that measurement polypeptide promotes hyaluronic acid to generate
(1) amount for the hyaluronic acid that ELISA method measurement fibroblast generates.
(2) by human dermal fibroblasts system BJ cell inoculation, (200 holes μ L/, cell inoculation amount are 1 in 48 orifice plates
×105A/hole, culture medium is using the L-15 basal medium containing cow's serum but without growth stimulator), in 37 DEG C, 5%CO2In incubator
It is incubated overnight.
(3) after for 24 hours, toward every group of 20 μ L of addition polypeptide solution, make polypeptide final concentration be respectively 0,100,200,300,400,
500 μM, incubator culture 48h.
(4) supernatant is collected by centrifugation in every group of cell culture.
(5) content of hyaluronic acid in hyaluronic acid assay kit ELISA method measurement supernatant is utilized.
(6) as the result is shown polypeptide within the scope of a certain concentration, hyaluronic acid contents present ascendant trend, after tend towards stability
(see Fig. 2).
Sequence table
<110>Guangdong Tai Nuo stem cell biological Science and Technology Ltd.
<120>a kind of polypeptide and application thereof with anti-aging and repair
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213>clear water Xi (Hydra viridis)
<400> 1
gaatatttct ccgaattctc caaagaattc gtgtccgaag aatcctcctt ccttgaattc 60
<210> 2
<211> 20
<212> PRT
<213>clear water Xi (Hydra viridis)
<400> 2
Leu Ile Lys Arg Leu Lys Arg Phe Leu Lys His Arg Leu Leu Arg Arg
1 5 10 15
Lys Glu Leu Lys
20
Claims (10)
1. a kind of polypeptide, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of encoding gene of polypeptide described in claim 1.
3. encoding gene according to claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.1.
4. a kind of prokaryotic expression carrier of the encoding gene containing polypeptide described in claim 2 or 3.
5. prokaryotic expression carrier according to claim 4, which is characterized in that the prokaryotic expression carrier is carrier
pGEX-4T-1。
6. a kind of bacterium containing prokaryotic expression carrier as claimed in claim 4.
7. bacterium according to claim 6, which is characterized in that the bacterium is e. coli bl21 (DE3).
8. polypeptide described in claim 1 promotes fibroblastic growth in preparation or fibroblast is promoted to generate hyaluronic acid
Preparation in application.
9. application of the polypeptide described in claim 1 in the drug or cosmetics of preparation anti-aging and reparation skin.
10. a kind of anti-aging and the drug or cosmetics for repairing skin, which is characterized in that including polypeptide described in claim 1
As active constituent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910209385.5A CN110078793B (en) | 2019-03-19 | 2019-03-19 | Polypeptide with anti-aging and repairing functions and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910209385.5A CN110078793B (en) | 2019-03-19 | 2019-03-19 | Polypeptide with anti-aging and repairing functions and application thereof |
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| Publication Number | Publication Date |
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| CN110078793A true CN110078793A (en) | 2019-08-02 |
| CN110078793B CN110078793B (en) | 2020-10-16 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201910209385.5A Active CN110078793B (en) | 2019-03-19 | 2019-03-19 | Polypeptide with anti-aging and repairing functions and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113201057A (en) * | 2021-03-16 | 2021-08-03 | 广东佰欧斐丝细胞科研中心有限公司 | Deep-sea top clam antibacterial protein and application thereof |
| CN113876928A (en) * | 2021-10-22 | 2022-01-04 | 北京远胜达生物科技发展有限公司 | Preparation of fibroblast outer vesicle and application of fibroblast outer vesicle in beauty treatment and medicines |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19808258A1 (en) * | 1997-02-28 | 1998-09-03 | Evotec Biosystems Gmbh | Hydra head activator binding protein |
| WO2010063250A1 (en) * | 2008-12-06 | 2010-06-10 | Christian-Albrechts-Universität Zu Kiel | Antimicrobial peptides made of hydra |
| CN108586575A (en) * | 2018-04-11 | 2018-09-28 | 福建省中科生物股份有限公司 | A kind of application of polypeptide and its skin repair function |
| CN108794592A (en) * | 2018-06-29 | 2018-11-13 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide NAGVLQDIRFKQ and its preparation method and application |
-
2019
- 2019-03-19 CN CN201910209385.5A patent/CN110078793B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19808258A1 (en) * | 1997-02-28 | 1998-09-03 | Evotec Biosystems Gmbh | Hydra head activator binding protein |
| WO2010063250A1 (en) * | 2008-12-06 | 2010-06-10 | Christian-Albrechts-Universität Zu Kiel | Antimicrobial peptides made of hydra |
| CN108586575A (en) * | 2018-04-11 | 2018-09-28 | 福建省中科生物股份有限公司 | A kind of application of polypeptide and its skin repair function |
| CN108794592A (en) * | 2018-06-29 | 2018-11-13 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide NAGVLQDIRFKQ and its preparation method and application |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113201057A (en) * | 2021-03-16 | 2021-08-03 | 广东佰欧斐丝细胞科研中心有限公司 | Deep-sea top clam antibacterial protein and application thereof |
| CN113876928A (en) * | 2021-10-22 | 2022-01-04 | 北京远胜达生物科技发展有限公司 | Preparation of fibroblast outer vesicle and application of fibroblast outer vesicle in beauty treatment and medicines |
| CN113876928B (en) * | 2021-10-22 | 2022-08-05 | 北京赛尔再生医学生物科技有限公司 | Preparation of fibroblast outer vesicle and application of fibroblast outer vesicle in beauty treatment and medicines |
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| CN110078793B (en) | 2020-10-16 |
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Effective date of registration: 20220117 Address after: 510700 Room 203, building 3, No. 9, Xiangshan Road, Huangpu District, Guangzhou City, Guangdong Province Patentee after: Guangdong baioufei silk Cell Research Center Co.,Ltd. Address before: 510000 210, No. 110, Zhucun East Ring Road, Tianhe District, Guangzhou City, Guangdong Province Patentee before: Guangdong Peptide Normal Stem Cell Biotechnology Co.,Ltd. |
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