CN107619437B - A kind of skin ultrastructure peptide WHPP-OA1 and its method of purification and application - Google Patents
A kind of skin ultrastructure peptide WHPP-OA1 and its method of purification and application Download PDFInfo
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Abstract
The invention discloses a kind of skin injuries to repair peptide WHPP-OA1WHPP-OA1, and the amino acid sequence that the reparation peptide WHPP-OA1 includes is IGKPPDTWKHLASKLKCI, and the invention also discloses the methods of purification and application for repairing peptide WHPP-OA1.Skin ultrastructure peptide WHPP-OA1 of the present invention can accelerate the Wound healing of skin surface, reduce scar and generate, have broad application prospects.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of skin ultrastructure peptide WHPP-OA1 and its method of purification
With application.
Background technique
Skin ultrastructure is always to compare stubborn problem in clinical medicine domain.Skin is the maximum organ of human body,
It is the physical barriers between human body and external environment, and carries all more important physiological functions, including perspires, thermal sensation and pain
Feel etc..Once skin is destroyed, body internal and external environment will be caused to lack of proper care, so that secondary malnutritive, multiple organ failure is even
Lead to death.Traditional rush wound repair drug has certain limitation, for example, it is at high cost, activity is relatively low and is easy
Hyperplastic scar is caused to heal.Therefore, finding novel rush wound repair drug is extremely important and is highly desirable.With tradition
Promote wound repair drug to compare, polypeptide has the characteristics such as high activity, high specific and high stability.At present, it has been found that
Many active peptides, such as antibacterial peptide, analgesia peptide, anticancer polypeptide, but there is the polypeptide for promoting wound repair effect also seldom to report
Road.
The living environment of amphibian is complicated, their skin is exposed, smooth, moist, is easy by the micro- life of various cause of diseases
Object invasion and ultraviolet radiation damage.During long-term natural selection, amphibian has gradually evolved unique, high
The skin polypeptide system of defense of effect.It is extensive research shows that: amphibian skin can be repaired quickly by after wound, therefore amphibious
Animal skin is considered as the rush wound repair drug resource treasure-house with huge potentiality to be exploited.Amphibian skin includes abundant
Micromolecule polypeptide, these small-molecular peptides have various bioactivity, such as antibiotic property, inoxidizability and immunocompetence.However,
Up to the present, have the cognition of skin rush wound repair active peptides also very deficient amphibian skin.
For this purpose, the present invention provides one kind from the smelly frog in Yunnan (Odorrana andersonii) Tool promote wound
The polypeptide and its method of purification of repairing activity and application.
Summary of the invention
The first object of the present invention is to provide a kind of skin ultrastructure peptide WHPP-OA1, the reparation peptide WHPP-OA1
The amino acid sequence for including is IGKPPDTWKHLASKLKCI.
The second object of the present invention is to provide the method for purification of skin ultrastructure peptide WHPP-OA1 a kind of, special
Sign is, comprising the following steps:
(1) the smelly frog skin of living body secretion in Yunnan is taken with electrostimulation, is dissolved in PBS, vacuum freeze drying obtains skin
- 80 DEG C of secretion freeze-dried powder saves backup；
(2) freeze-dried powder is separated with Sephadex G75 exclusion chromatography；
(3) separation product of step (2) is subjected to first time high performance liquid chromatography reversed phase chromatography, and collects tool rush wound and repairs
The active component of multiple function；
(4) active component is subjected to second of high performance liquid chromatography reversed phase chromatography, the reparation peptide WHPP- purified
OA1。
The third object of the present invention is to provide the application of the skin ultrastructure peptide WHPP-OA1, which is characterized in that
The wound repair peptide WHPP-OA1 is used to accelerate the Wound healing of skin surface, scar is reduced and generates.
The fourth object of the present invention is to provide a kind of topical drug containing the skin ultrastructure peptide WHPP-OA1.
The fifth object of the present invention is to provide a kind of external-use skin care containing the skin ultrastructure peptide WHPP-OA1
Product.
Detailed description of the invention
Fig. 1 is the Sephadex G75 sieve chromatography that the smelly frog skin in Yunnan of the present invention promotees wound repair peptide WHPP-OA1
Figure.
Fig. 2 is the first time HPLC reverse phase C18 column chromatography that the smelly frog skin in Yunnan of the present invention promotees wound repair peptide WHPP-OA1
Figure.
Fig. 3 is second of HPLC reverse phase C18 column chromatography that the smelly frog skin in Yunnan of the present invention promotees wound repair peptide WHPP-OA1
Figure.
Fig. 4 is that the rush wound for the HaCat that the present invention isolates and purifies the smelly frog skin rush wound repair peptide WHPP-OA1 in Yunnan is repaired
Active figure；
In figure:
It is living that A-HaCat cell shows stronger rush wound repair in the case where 10 μm of ol/L repair peptide WHPP-OA1 effect
Property；
After mitomycin C inhibition cell Proliferation is added in B-, 10 μm of ol/L repair peptide WHPP-OA1 and cell are not promoted to create
Wound is repaired；
C- under the action of repairing peptide WHPP-OA1, the wound repair activities present of HaCaT cell go out time and dosage according to
Lai Xing；
After mitomycin C inhibition cell Proliferation is added in D, under the action of repairing peptide WHPP-OA1, HaCaT cell does not have
Show wound repair activity and time and dose dependent；
E- repairs the proliferation that peptide WHPP-OA1 promotes HaCaT cell.
Fig. 5 is the HSF rush wound repair activity figure of reparation peptide WHPP-OA1 after present invention purification；
In figure:
A-HSF cell shows stronger rush wound repair activity in the case where 10 nmol/L repair peptide WHPP-OA1 effect；
After mitomycin C inhibition cell Proliferation is added in B-, 10 nmol/L repair peptide WHPP-OA1 and cell trauma are promoted to repair
It is active；
For C- under the action of repairing peptide WHPP-OA1, the wound repair activities present of HSF cell goes out time and dose-dependant
Property；
After mitomycin C inhibition cell Proliferation is added in D-, under the action of repairing peptide WHPP-OA1, the wound of HSF cell
Repairing activity shows time and dose dependent；
E- repairs the proliferation function that peptide WHPP-OA1 does not promote HSF cell.
Fig. 6 is that the animal model rush wound of the invention for isolating and purifying the smelly frog skin rush wound repair peptide WHPP-OA1 in Yunnan is repaired
Active figure.
Specific embodiment
The present invention will be further described below with reference to the drawings, but the present invention is limited in any way, base
In present invention teach that done it is any transform or replace, all belong to the scope of protection of the present invention.
Of the present invention kind of skin ultrastructure peptide WHPP-OA1, from the smelly frog in Yunnan (Odorrana andersonii), the amino acid sequence that the reparation peptide WHPP-OA1 includes is IGKPPDTWKHLASKLKCI.
Since the skin ultrastructure peptide WHPP-OA1 derives from the smelly frog in Yunnan, the present invention provides a kind of from the smelly frog in Yunnan
Living body purifies the method for purification of the skin ultrastructure peptide WHPP-OA1, comprising the following steps:
(1) the smelly frog skin of living body secretion in Yunnan is taken with electrostimulation, is dissolved in PBS, vacuum freeze drying obtains skin
- 80 DEG C of secretion freeze-dried powder saves backup；
(2) freeze-dried powder is crossed into Sephadex G75 molecular sieve: by skin secretion freeze-dried powder, is dissolved in deionized water
In, it is configured to the solution of 100 mg/mL, is taken on 1 mL in advance with 20 mmol/L Tris-HCl buffers (pH=7.8, containing 0.1
Mol/L NaCl) balance 24 hours Sephadex G75 (GE Healthcare, ultra-fine) pillar (40 cm of length, internal diameter
1.5 cm of width), eluted with same buffer, flow velocity 1.5mL/10min, every 10 min is collected 1 time, measure its
Absorbance under 280 nm；
(3) product of over-molecular sieve is subjected to first time high performance liquid chromatography reversed phase chromatography, and collects tool and promotees wound repair
Active section product: the product that will be sieved is splined on the Hypersil balanced in advance with ultrapure water (trifluoroacetic acid containing 0.1%)
ODS2 5mm pillar (Erie's specialty product, having a size of the mm of 4.6 mm × 300), laboratory apparatus are 1525 high pressure liquid phase of Waters
System, under conditions of flow velocity is 1 ml/min, with acetonitrile (trifluoroacetic acid containing 0.1%) in linear gradient (0-100% in
100 min) under the conditions of eluted, monitoring wavelength be 220 nm.
(4) tool is promoted into wound repair activity section product and carries out second of high performance liquid chromatography reversed phase chromatography, obtained
The reparation peptide WHPP-OA1 of purifying: the first time of collection is had and promotees wound repair Peak Activity, is dissolved in after vacuum freeze drying
Ionized water, then repeatedly first time HPLC process, obtains purified product.
Other than directly extracting from the smelly frog living body in Yunnan, reparation peptide WHPP-OA1 of the present invention can also be by artificial
Synthetic method obtains.At present there are many kinds of the preparation methods of biologically active peptide, including protection chemical synthesis, Hydrolyze method, again
Group DNA technique etc., these methods are suitable for preparing reparation peptide WHPP-OA1 of the present invention.
It, can since reparation peptide WHPP-OA1 of the present invention has the characteristics that source is natural, activity is high, it is strong to promote repair ability
For accelerating the Wound healing of skin surface, reduces scar and generate.Production contains the skin ultrastructure peptide WHPP-OA1's
The products such as topical drug, external use skin care, have broad application prospects.
Below in conjunction with specific embodiment, the present invention is further detailed.
Embodiment 1: the purification and Molecular Identification of peptide WHPP-OA1 are repaired
1, it purifies
The smelly frog living body in Yunnan picks up from Baoshan, Yunnan, takes its skin secretion (being dissolved in PBS), vacuum refrigeration with electrostimulation
Dry, -80 ° save backup.
It will obtain skin secretion freeze-dried powder, be dissolved in deionized water, be configured to the solution of 100 mg/mL, take 1
ML(protein content is (pH=7.8 are balanced containing 0.1 M NaCl) with 20 mmol/L Tris-HCl buffers in advance on 100 mg)
24 hours Sephadex G75 (GE Healthcare, ultra-fine) pillar (40 cm of length, 1.5 cm of internal diameter width), with same
The buffer of sample is eluted, and flow velocity 1.5ml/10min every 10 min collection 1 time, measures its extinction at 280 nm
Degree, it is resulting to isolate and purify map as shown in Figure 1, wherein OA1 is present in peak shown in figure.
Sample obtained by the first step is splined on to the Hypersil balanced in advance with ultrapure water (trifluoroacetic acid containing 0.1%)
ODS2 5mm pillar (Erie's specialty product, having a size of the mm of 4.6 mm × 300), laboratory apparatus are 1525 high pressure liquid phase of Waters
System, under conditions of flow velocity is 1 mL/min, with acetonitrile (trifluoroacetic acid containing 0.1%) in linear gradient (0-100% in
100 min) under the conditions of eluted, monitoring wavelength be 220 nm, it is resulting to isolate and purify map as shown in Fig. 2, arrow is signified
For with wound repair Peak Activity is promoted, there are shown peaks by WHPP-OA1.
It collects to have for the first time and promotees wound repair Peak Activity, be dissolved in deionized water after vacuum freeze drying, then repeatedly the
HPLC process, it is resulting to isolate and purify map as shown in figure 3, arrow meaning is peak where the WHPP-OA1 of purifying.
2, Molecular Identification
Obtained reparation peptide WHPP-OA1 is purified to pass through on full-automatic protein sequencing instrument (Shimadzu PPSQ-31A)
Edman edman degradation Edman measures overall amino acid sequence, as the result is shown: the amino acid sequence for repairing peptide WHPP-OA1 is
IGKPPDTWKHLASKLKC。
Embodiment 2: the repairing activity of peptide WHPP-OA1 is repaired in measurement in cellular level and animal model
1, activity is measured on cellular level
Containing 5%CO237 DEG C of insulating box in, with the DMEM/F12 containing 10% fetal calf serum and 1% Pen .- Strep
The epithelial cell (HaCaT) of (BI, Israel) culture medium culture people and fibroblast (HSF), to cell in culture bottle
In cover with after, cell is digested in culture bottle with pancreatin, by cell kind in 24 orifice plates, 2.5 × (10- is about planted in every hole
5) a cell, then cultivate 12-14 hours, every hole cell covers with to form cell monolayer, with 200 μ L pipette tips of yellow (Axygen,
USA) in every hole scratch, every hole is cleaned twice with PBS, primarily to dead cell is cleaned up.Finally, adding in every hole
Enter containing various concentration WHPP-OA1 (10 nmol/L, 100 nmol/L, 1 μm of ol/L, 10 μm of ol/L for HaCaT
The and nmol/L for of 100 pmol/L, 1 nmol/L, 10 HSF) the DMEM/F12 culture medium without fetal calf serum
500 μ L continue to cultivate cell, then take pictures every 6h, calculate wound repair rate.
Cell migration and proliferation is two main causes for causing wound repair, therefore we also use mitomycin C (10
μ g/ml, Sigma-Aldrich, St Louis, MO, USA) cell inhibitory effect, whether further probe into is by thin
Born of the same parents' migration causes wound repair；The effect for detecting WHPP-OA1 cell proliferation by 96 hole cell proliferation detecting kits is living
Property: the cell suspension (5000 HaCaT/hole, 10000 HSF/hole, 90 μ L) without fetal calf serum is moved into 96 holes
Plate culture 2-4 h, after cell is adherent, every hole be added various concentration WHPP-OA1 (10 nmol/L, 100 nmol/L,
1 μm of ol/L, and 10 μm of ol/L HaCaT cells, 10 pmol/L, 100 pmol/L, 1 nmol/L, and 10 nmol/
L HSF cell) 10 μ L, behind insulating box culture 24 hours of 37 DEG C, finally with cell proliferation detecting kit detection cell
Proliferation activity.
2, activity is measured in animal model
It selects the SPF grade Kun Ming mice of weight 22-25 g to be tested, mouse is randomly divided into two groups, every group 10.
First to yellow Jackets (0.1 mL/20g) anesthetized mice of mouse peritoneal injection 1%, then back of mice hair is shaved dry
Only, and with 75% alcohol disinfecting, two symmetrical holes of size finally are dug out with punch in back of mice two sides, about 8mm ×
8mm size.Postoperative mouse is placed on by heater is put back to receptacle and is normally raised after it is waken up.Loading twice daily, every hole are every
About 20 μ L of secondary loading, first group of back of mice left side apply physiological saline (NaCl), right side WHPP-OA1；Second group of back of mice
Left side applies epidermal growth factor (EGF), right side OA1.It took pictures every 3 days to back of mice wound, it is soft finally by Image J
Part calculates wound repair rate.The smelly frog skin in Yunnan promotees the rush wound repair activity of wound repair peptide WHPP-OA1 as shown in Figure 4.
Cellular level (see Fig. 4,5) and animal model test (see figure 6) result show: skin trauma of the present invention
Repairing peptide WHPP-OA1 has stronger rush wound repair activity.
SEQUENCE LISTING
<110>Kunming Medical University
<120>a kind of skin ultrastructure peptide WHPP-OA1 and its method of purification and application
<130> 2017
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> PRT
<213> Odorrana andersonii
<400> 1
Ile Gly Lys Pro Pro Asp Thr Trp Lys His Leu Ala Ser Lys Leu Lys
1 5 10 15
Cys Ile
Claims (4)
1. a kind of skin ultrastructure peptide WHPP-OA1, it is characterised in that it is described repair peptide WHPP-OA1 amino acid sequence be
IGKPPDTWKHLASKLKCI。
2. skin ultrastructure peptide WHPP-OA1 described in a kind of claim 1 accelerates the Wound healing of skin surface preparing, subtracts
Application in the topical drug, external use skin care of scar generation less.
3. a kind of topical drug containing skin ultrastructure peptide WHPP-OA1 described in claim 1.
4. a kind of external use skin care containing skin ultrastructure peptide WHPP-OA1 described in claim 1.
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| CN108484747B (en) * | 2018-03-22 | 2019-07-12 | 昆明医科大学 | Japan frog skin repair peptide cathelicidin-NV and its gene and application |
| CN108586575B (en) * | 2018-04-11 | 2020-08-14 | 福建省中科生物股份有限公司 | Polypeptide and application of polypeptide in skin repair function |
| CN110386961B (en) * | 2019-08-29 | 2022-05-10 | 昆明医科大学 | Skin repair polypeptide RL-RL10 and application thereof |
| CN110563811B (en) * | 2019-09-25 | 2022-05-10 | 昆明医科大学 | Skin protection peptide HW-P1 and preparation method and application thereof |
| CN110642924B (en) * | 2019-10-12 | 2022-10-28 | 杨莹 | Skin protection peptide OM-TV16 and preparation method and application thereof |
| CN111187344A (en) * | 2019-12-19 | 2020-05-22 | 北京安锐鑫生物科技有限公司 | Polypeptide, composition containing polypeptide and application of polypeptide in preventing or treating skin injury or resisting oxidation |
| CN114957394B (en) * | 2022-04-22 | 2023-06-09 | 成都佩德生物医药有限公司 | Polypeptide PM-7 for promoting skin repair and application thereof |
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| CN104840483A (en) * | 2015-06-08 | 2015-08-19 | 济南博创医药科技有限公司 | Medicine composition for treating skin wounds and medicinal preparation thereof |
| CN106518998A (en) * | 2016-11-24 | 2017-03-22 | 昆明医科大学 | Antioxidant polypeptide AOP-OA3 from skin of rana andersonli as well as preparation method and application of antioxidant polypeptide AOP-OA3 |
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Effective date of registration: 20220106 Address after: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Patentee after: Liu Jiaojiao Address before: Kunming Medical University, 1168 Chunrong West Road, Chenggong District, Kunming, Yunnan 650500 Patentee before: KUNMING MEDICAL University Effective date of registration: 20220106 Address after: 510000 room 4106, No. 888, Yuncheng West Road, Baiyun District, Guangzhou City, Guangdong Province Patentee after: Guangzhou Qianxiang Biotechnology Co.,Ltd. Address before: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Patentee before: Liu Jiaojiao |
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