CN106146621A - Big rana margaretae skin antioxidation polypeptide AOP OA3 and preparation method thereof - Google Patents
Big rana margaretae skin antioxidation polypeptide AOP OA3 and preparation method thereof Download PDFInfo
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- CN106146621A CN106146621A CN201610714694.4A CN201610714694A CN106146621A CN 106146621 A CN106146621 A CN 106146621A CN 201610714694 A CN201610714694 A CN 201610714694A CN 106146621 A CN106146621 A CN 106146621A
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- rana margaretae
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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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Abstract
The invention discloses big rana margaretae skin antioxidation polypeptide AOP OA3 and preparation method thereof, in the aminoacid sequence of described anti-oxidation peptide AOP OA3 as shown in SEQ ID NO:1 in sequence table.Isolated and purified from big rana margaretae skin secretion obtaining, the antioxidation polypeptide AOP OA3 of institute's isolated has the aminoacid sequence shown in SEQ ID NO:1.The big rana margaretae skin anti-oxidation peptide AOP OA3 that the present invention obtains shows stronger antioxidant activity in free radical ABTS+ removes experiment, and disclosing the present invention big rana margaretae skin anti-oxidation peptide AOP OA3 has potential application prospect.The big beneficial features that rana margaretae skin anti-oxidation peptide AOP OA3 has simple in construction, antioxidant activity is strong.
Description
Technical field
The present invention relates to field of biomedicine technology, the biggest rana margaretae skin antioxidation polypeptide AOP-OA3 and system thereof
Preparation Method.
Background technology
Antioxidant is to stop the dysgenic material of oxygen, and it is that a class can help capture and neutralize free radical, thus
Dispel a class material of radical pair human body infringement.Having had now been found that substantial amounts of antioxidant, these antioxidants are permissible
It being divided into following two big classes: 1) the little molecule of non-genomic coding is helpless, such as vitamin E, vitamin C, glutathion etc.；2) gene
The macromolecular substances of coding, such as superoxide dismutase, catalase, thioredoxin etc..These antioxidants by
It is widely used in various field, including in health, food, cosmetics and various industry, steady for example as fuel and lubricant
Determine the anti-oxidation of agent, it is possible to add and play the purpose preventing polymerization thus avoid engine dirty to be formed in the oil, singly in 2007,
The income just creating about 3,700,000,000 dollars of the antioxidant of industrial use.Importance based on antioxidant, scientists
Find novel antioxidant tireless always.
Amphibian be aquatic animal excessively to the important monoid between terrestrial animal, its skin is exposed, and surface does not has hair
Send out and cover, directly facing various survival pressures, take all more important biological functions, such as resist microorganism invasion, body
Tone joint, water salt balance regulation etc..Having had substantial amounts of research evidence to show, the skin of Amphibian can secrete substantial amounts of activity
Material (mainly albumen and polypeptide) has enabled it to these important biological functions.Recently, (big some Amphibians
Rana margaretae, the Yunnan frog etc.) skin in be found that the antioxidant that a class is novel, its chemical nature is polypeptide, and traditional two big
Class antioxidant is different, and it is by gene code, but molecular weight is the least.Comparing with traditional antioxidant, we are to this class
The 26S Proteasome Structure and Function understanding of novel antioxidant is also known little about it.
Big rana margaretae is the Chinese smelly frog of distinctive one, is distributed widely in the Yunnan-Guizhou Plateau of strong ultra-vioket radiation, raw according to it
Border, enables it to resist the injury that ultra-vioket radiation is caused it is presumed that its skin can secrete efficient antioxidant.Inventor
Having obtained 1 novel antioxidation polypeptide AOP-OA3 in the skin of the most domestic big rana margaretae, its antioxidant activity is very strong, about
For vitamin E 1000 times.By the big rana margaretae skin antioxidation polypeptide AOP-OA3 complete sequence structure of the present invention through albumen prime number
Carry out search according to storehouse to compare, find no any phase homopolypeptide.
Summary of the invention
It is an object of the invention to provide simple in construction, the big rana margaretae skin antioxidation polypeptide AOP-that antioxidant activity is strong
OA3 and preparation method thereof, with the problem solving to propose in above-mentioned background technology.
For achieving the above object, the present invention provides following technical scheme:
Big rana margaretae skin antioxidation polypeptide AOP-OA3, such as sequence in the aminoacid sequence of described anti-oxidation peptide AOP-OA3
In table shown in SEQ ID NO:1.
Another object of the present invention is to provide the preparation method of big rana margaretae skin antioxidation polypeptide AOP-OA3, from big green smelly
Isolated and purified in frog skin secretion obtaining, the antioxidation polypeptide AOP-OA3 of institute's isolated has shown in SEQ ID NO:1
Aminoacid sequence.
As the further scheme of the present invention: the preparation method of described big rana margaretae skin antioxidation polypeptide AOP-OA3, tool
Body comprises the following steps:
1) take big rana margaretae skin secretion by electrostimulation, and be dissolved in normal saline, prepare mixture, vacuum
Lyophilization ,-80 ° save backup；
2) ultrafiltration: mixture uses super filter tube carry out ultrafiltration, molecular cut off is 10KDa；
3) high performance liquid chromatography reversed phase chromatography: by step 2) it is dissolved in deionized water after gained material vacuum lyophilization, system
Obtain sample, sample is splined in the Hypersil ODS2 chromatographic column balanced with the ultra-pure water containing trifluoroacetic acid in advance,
Under conditions of flow velocity is 1ml/min, under linear gradient condition, carries out eluting with the acetonitrile containing trifluoroacetic acid and get final product, monitoring wavelength
For 215nm, linear gradient condition: 0-100%in 100min.
As the further scheme of the present invention: in ultra-pure water, the mass content of trifluoroacetic acid is 0.1%.
As the further scheme of the present invention: in acetonitrile, the mass content of trifluoroacetic acid is 0.1%.
Compared with prior art, the invention has the beneficial effects as follows:
The big rana margaretae skin anti-oxidation peptide AOP-OA3 that the present invention obtains shows in free radical ABTS+ removes experiment
Stronger antioxidant activity, disclosing the present invention big rana margaretae skin anti-oxidation peptide AOP-OA3 has potential application prospect.Big green smelly
The beneficial features that frog skin anti-oxidation peptide AOP-OA3 has simple in construction, antioxidant activity is strong.
Accompanying drawing explanation
Fig. 1 is the anti-phase C of HPLC of the present invention big rana margaretae skin anti-oxidation peptide AOP-OA318Column chromatography figure.
Fig. 2 is the free radical ABTS of the isolated and purified big rana margaretae skin anti-oxidation peptide AOP-OA3 of the present invention+Scavenging capacity
Figure.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all
Belong to the scope of protection of the invention.
The big rana margaretae skin anti-oxidation peptide AOP-OA3 that the present invention provides, its preparation method can pass through bioid credit
From purification process, obtained by big rana margaretae skin secretion.Big rana margaretae skin anti-oxidation peptide AOP-prepared by said method
OA3, can be identified by mass spectrum and antioxidant activity.
The preparation of big rana margaretae skin anti-oxidation peptide AOP-OA3 also can use chemistry to close by the method for this area routine
Becoming or the mode of gene expression obtains, their aminoacid sequence is identical with SEQ ID NO:1 of the present invention.
Embodiment 1
In the embodiment of the present invention, big rana margaretae skin anti-oxidation peptide AOP-OA3 is isolated and purified and identifies
1, isolated and purified
Big rana margaretae live body picks up from the big green Baoshan, takes its skin secretion (being dissolved in normal saline) by electrostimulation, very
Vacuum freecing-dry ,-80 ° save backup.
The first step: ultrafiltration (uses millipore super filter tube, molecular cut off is 10KDa).
Second step: high performance liquid chromatography reversed phase chromatography:
It is splined on after being dissolved in deionized water after first step gained sample vacuum lyophilization and uses ultra-pure water (containing 0.1% in advance
Trifluoroacetic acid) the Hypersil ODS2 5mm pillar (Erie's special product product, a size of 4.6mm × 300mm) that balanced, experiment
Instrument is Waters 1525 high-pressure liquid phase system, under conditions of flow velocity is 1ml/min, with acetonitrile (containing the trifluoro second of 0.1%
Acid) under the conditions of linear gradient (0-100%in 100min), carry out eluting, monitoring wavelength is 215nm.
The anti-phase C of AOP-OA3 correspondence HPLC18Shown in 2 peaks in column chromatography figure (Fig. 1).
2, Molecular Identification
The mensuration of aminoacid sequence:
The big rana margaretae skin anti-oxidation peptide AOP-OA2 that purification obtains is in full-automatic protein sequencing instrument (Shimadzu
PPSQ-31A) through Edman edman degradation Edman on, big rana margaretae skin anti-oxidation peptide AOP-OA2 overall amino acid sequence, result table are determined
Bright big rana margaretae skin anti-oxidation peptide AOP-OA2 has a structure shown in SEQ ID NO:1 of the present invention:
ValThr ProPheGlyLeuLeuCysGly IleLeuGly。
Embodiment 2
The antioxidant activity of big rana margaretae skin anti-oxidation peptide AOP-OA2
The present invention utilizes the experiment of free radical ABTS+ scavenging capacity to detect the anti-of big rana margaretae skin anti-oxidation peptide AOP-OA2
Oxidation activity, result shows that big rana margaretae skin anti-oxidation peptide AOP-OA2 has the ABTS of stronger antioxidant activity 6mM at water
Neutralize the potassium peroxydisulfate mixing of 2.6mM, room temperature dark surrounds reacts 5 hours so that ABTS is oxidized into ABTS+Freely
Base, dilutes 45 times with deionized water afterwards, takes 45 μ L diluents and the mixing of 45 μ L sample, reacts 30 points under room temperature dark surrounds
Clock, afterwards, surveys its absorbance under 415nm wavelength.Free radical ABTS+Clearance rate (%) calculate with following formula:
(Ablank-Asample)×100/Ablank.The antioxidant activity of big Odorrana margaratae antioxidative peptide AOP-OA2 of variable concentrations is as shown in Figure 2.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of the spirit or essential attributes of the present invention, it is possible to realize the present invention in other specific forms.Therefore, no matter
From the point of view of which point, all should regard embodiment as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all by fall in the implication of equivalency and scope of claim
Change is included in the present invention.
Although moreover, it will be appreciated that this specification is been described by according to embodiment, but the most each embodiment only wraps
Containing an independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should
Description can also be formed those skilled in the art through appropriately combined as an entirety, the technical scheme in each embodiment
May be appreciated other embodiments.
Claims (5)
1. big rana margaretae skin antioxidation polypeptide AOP-OA3, it is characterised in that the aminoacid sequence of described anti-oxidation peptide AOP-OA3
In row as shown in SEQ ID NO:1 in sequence table.
2. the preparation method of big rana margaretae skin antioxidation polypeptide AOP-OA3, it is characterised in that from big rana margaretae skin secretion
In isolated and purified obtain, the antioxidation polypeptide AOP-OA3 of institute's isolated has the ammonia as shown in SEQ ID NO:1 in sequence table
Base acid sequence.
The preparation method of big rana margaretae skin antioxidation polypeptide AOP-OA3 the most according to claim 2, it is characterised in that
Specifically include following steps:
1) take big rana margaretae skin secretion by electrostimulation, and be dissolved in normal saline, prepare mixture, vacuum freezing
Being dried ,-80 ° save backup；
2) ultrafiltration: mixture uses super filter tube carry out ultrafiltration, molecular cut off is 10KDa；
3) high performance liquid chromatography reversed phase chromatography: by step 2) it is dissolved in deionized water after gained material vacuum lyophilization, prepare sample
Product, are splined on sample in the Hypersil ODS2 chromatographic column balanced with the ultra-pure water containing trifluoroacetic acid in advance, at flow velocity
Under conditions of 1ml/min, carrying out eluting with the acetonitrile containing trifluoroacetic acid and get final product under linear gradient condition, monitoring wavelength is
215nm, linear gradient condition: 0-100%in 100min.
The preparation method of big rana margaretae skin antioxidation polypeptide AOP-OA3 the most according to claim 3, it is characterised in that
In ultra-pure water, the mass content of trifluoroacetic acid is 0.1%.
The preparation method of big rana margaretae skin antioxidation polypeptide AOP-OA3 the most according to claim 3, it is characterised in that
In acetonitrile, the mass content of trifluoroacetic acid is 0.1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610714694.4A CN106146621A (en) | 2016-08-24 | 2016-08-24 | Big rana margaretae skin antioxidation polypeptide AOP OA3 and preparation method thereof |
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| CN201610714694.4A CN106146621A (en) | 2016-08-24 | 2016-08-24 | Big rana margaretae skin antioxidation polypeptide AOP OA3 and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106632644A (en) * | 2016-11-24 | 2017-05-10 | 云南民族大学 | Antioxidant polypeptide AOP-OA2 in skin of Yunnan odorous frog as well as prepration method and application of antioxidant polypeptide AOP-OA2 |
| CN108586576A (en) * | 2018-04-23 | 2018-09-28 | 昆明医科大学 | A kind of polypeptide OA-FF10 and its method of purification and application |
| CN110642924A (en) * | 2019-10-12 | 2020-01-03 | 杨莹 | Skin protection peptide OM-TV16 and preparation method and application thereof |
| CN111454332A (en) * | 2020-04-14 | 2020-07-28 | 温州大学 | Odorrana livida frog host defense peptide livisin, gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104530192A (en) * | 2015-01-19 | 2015-04-22 | 云南民族大学 | Antioxidant peptide AOP-OM1 as well as preparation method and application thereof |
| CN105713072A (en) * | 2016-02-26 | 2016-06-29 | 昆明昂名科技有限公司 | Antioxidant polypeptide (AOP-OL1) and preparation method and application thereof |
-
2016
- 2016-08-24 CN CN201610714694.4A patent/CN106146621A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104530192A (en) * | 2015-01-19 | 2015-04-22 | 云南民族大学 | Antioxidant peptide AOP-OM1 as well as preparation method and application thereof |
| CN105713072A (en) * | 2016-02-26 | 2016-06-29 | 昆明昂名科技有限公司 | Antioxidant polypeptide (AOP-OL1) and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 凌桂英: "贵州产绿臭蛙（Odorrana margaretae）皮肤活性肽基因克隆和功能研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106632644A (en) * | 2016-11-24 | 2017-05-10 | 云南民族大学 | Antioxidant polypeptide AOP-OA2 in skin of Yunnan odorous frog as well as prepration method and application of antioxidant polypeptide AOP-OA2 |
| CN108586576A (en) * | 2018-04-23 | 2018-09-28 | 昆明医科大学 | A kind of polypeptide OA-FF10 and its method of purification and application |
| CN110642924A (en) * | 2019-10-12 | 2020-01-03 | 杨莹 | Skin protection peptide OM-TV16 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 |
| CN111454332A (en) * | 2020-04-14 | 2020-07-28 | 温州大学 | Odorrana livida frog host defense peptide livisin, gene and application thereof |
| CN111454332B (en) * | 2020-04-14 | 2022-05-27 | 温州大学 | Odorrana livida frog host defense peptide livisin, gene and application thereof |
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Application publication date: 20161123 |