CN104961805A - Fibroin polypeptide, and preparation and application thereof - Google Patents
Fibroin polypeptide, and preparation and application thereof Download PDFInfo
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- CN104961805A CN104961805A CN201510451393.2A CN201510451393A CN104961805A CN 104961805 A CN104961805 A CN 104961805A CN 201510451393 A CN201510451393 A CN 201510451393A CN 104961805 A CN104961805 A CN 104961805A
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Abstract
The invention discloses a fibroin polypeptide, relating to the field of polypeptide preparation in biotechnology. The sequence is DDTDKSIAILNVQEILKDM. The preparation method comprises the following steps: (1) shredding 500g of cleaned and dried cocoon, and adding into 500-1000ml of deionized water; (2) boiling, carrying out ultrasonic treatment for 1-2 hours, and slowly stirring; (3) after the solution is cooled, adding a hydrolase, and reacting at 20-40 DEG C for 30-60 minutes; (4) centrifuging at 2000-3000 rpm for 5-10 minutes, and taking the supernatant; (5) adding the supernate into a slightly acidic buffer solution with the pH value of 6.0-6.5, stirring, and centrifuging at 10000-15000 rpm for 30-60 minutes to obtain a precipitate; and (6) redissolving the precipitate in a buffer solution with the pH value of 5.0-5.5, and separating out the polypeptide with the molecular weight of 2-3KDa by a sepharose chromatography technique. The fibroin polypeptide can be used as an additive for preparing cosmetics. The purity of the fibroin polypeptide is greater than 70%. The experiment indicates that the polypeptide can resist oxidation and has equivalent actions to the original fibroin. The antigen experiment result indicates that the polypeptide has no antigenicity.
Description
Technical field
The present invention relates to biotechnology field of polypeptide preparation, be specifically related to fibroin field of polypeptide preparation.
Technical background
Fibroin (Fibroin) has another name called: silk fibroin.Silk fibroin, it is the natural polymer scleroproein extracted from silk, content accounts for 70% ~ 80% of silk, and containing 18 seed amino acids, wherein glycine (gly), L-Ala (ala) and Serine (ser) account for more than 80% of total composition.
Silk protein is exploited at cosmetic field.Due to silk protein and human body skin keratin matter fairly similar, both have fabulous affinity; Due to the chemical structure that silk protein molecule is special, so silk protein has good water absorbability and loose wet performance, the change of surrounding environment can be adapted to, play natural damping, improve human body skin nutrition, prevent skin wrinkling, strengthen skin cells vigor and elasticity, promotion cell metabolism and maintain skin normal physiological condition; Silk protein also has absorption ultraviolet, prevents the effect that solar radiation damages human body skin, so silk protein has been widely used in the excellent additive of cosmetics.
But fibroin has 66 amino acid whose protein, molecular weight is comparatively large, often causes allergic reaction, causes skin pruritus, thus cannot play due skin care effect.Small protein or polypeptide are just not easy to bring out allergic immune reaction.But small protein often loses effect of original shape protein.Therefore, how could allow the effect of the existing crude protein of fibroin small molecules after decomposing, and not cause allergy, be a problem being difficult to coordinate always.
Summary of the invention
Goal of the invention
For the present situation of existing fibroin makeup, invent a kind of fibroin polypeptide and Synthesis and applications, the fibroin polypeptide of preparation, this polypeptide is applied to makeup, effect of its existing former fibroin, and does not produce anaphylaxis.
Technical scheme
The present invention is a kind of fibroin polypeptide, and its sequence is DDTDKSIAILNVQEILKDM.Preparation process comprises (1) shreds the silk cocoon 500g after cleaning, oven dry, adds in 500-1000ml deionized water; (2) boil, ultrasonic 1-2 hour, slowly stir simultaneously; (3) after solution cooling, lytic enzyme is added, at 20-40 DEG C of reaction 30-60 minute; (4) centrifugal 2000-3000rpm, 5-10 minute, get supernatant; (5) added by supernatant liquor in the slant acidity damping fluid of pH6.0-6.5, stir, centrifugal 10000-15000rpm, 30-60 minute, be precipitated thing; (6) throw out is redissolved in pH5.0-5.5 damping fluid, adopt sephadex chromatography technology, isolate the polypeptide that molecular weight is 2-3KDa, to obtain final product.Described lytic enzyme can be papoid, ficin, bromeline, Ginger Protease, silk tree proteolytic enzyme, choke proteolytic enzyme etc.Described fibroin polypeptide, is preparing the application in makeup.
Polypeptide of the present invention also can entrust Shanghai gill synthesis, and purity is more than 95%.
Beneficial effect
The fibroin polypeptide prepared of the present invention, through protein sequencing, sequence is DDTDKSIAILNVQEILKDM, and purity is greater than 70%.Experiment shows, the oxidation resistant effect of this polypeptide energy, acts on suitable with former fibroin.Antigen assay result shows, this polypeptide does not have antigenicity, and the experiment of vitro inhibition tyrosine oxidase confirms that it has whitening function.
Embodiment
Embodiment 1
Silk cocoon 500g after cleaning, oven dry is shredded, adds in 500-1000ml deionized water; Boil, ultrasonic 1-2 hour, slowly stir simultaneously; After solution cooling, add bromeline, room temperature reaction 60 minutes; Centrifugal 2000rpm, 5-10 minute, get supernatant; Added by supernatant liquor in the slant acidity damping fluid of pH6.0-6.5, stir, centrifugal 10000-15000rpm, 30-60 minute, be precipitated thing; Throw out is redissolved in pH5.0-5.5 damping fluid, adopt sephadex chromatography technology, isolate the polypeptide that molecular weight is 2-3KDa, to obtain final product.The sequence adopting solid phase protein automatic sequencer to measure this polypeptide is DDTDKSIAILNVQEILKDM, and purity is 87.8%.
Embodiment 2
Silk cocoon 500g after cleaning, oven dry is shredded, adds in 500-1000ml deionized water; Boil, ultrasonic 1-2 hour, slowly stir simultaneously; After solution cooling, add ficin, 37 DEG C of reactions 30 minutes; Centrifugal 3000rpm, 5-10 minute, get supernatant; Added by supernatant liquor in the slant acidity damping fluid of pH6.0-6.5, stir, centrifugal 10000-15000rpm, 30-60 minute, be precipitated thing; Throw out is redissolved in pH5.0-5.5 damping fluid, adopt sephadex chromatography technology, isolate the polypeptide that molecular weight is 2-3KDa, to obtain final product.The sequence adopting solid phase protein automatic sequencer to measure this polypeptide is DDTDKSIAILNVQEILKDM, and purity is 76.5%.
Embodiment 3
The polypeptide that Shanghai gill synthesizes is added in emulsifiable paste matrix, carries out immunogenicity detection.BALB/c white mouse 6, male and female half and half, lose hair or feathers mouse back, baring skin.Get the fibroin polypeptide solution (0.5 of different concns respectively, 1.0,2.0mg/ml) apply 1 coating of density short-term with 1.25 μ L/cm2, apply 1 every day, continuous 8 weeks, get blood in 1-12 week orbital venous plexus fixed point weekly, the centrifugal 2min of 12000rpm, supernatant liquor got by separated plasma, and-20 DEG C of cryopreservation are for subsequent use.After room-temperature dissolution, get 0.1ml supernatant liquor indirect ELISA and detect Serum Antibody titre.Namely obtain RA value with the actual A value (OD450) of each sample than negative control sera A value, RA >=2.1 are the positive, detect after Blood bio-samples sample is done a series of dilution, and the most high dilution be positive is the titre of this sample.Measurement result shows.The blood sample that 1-12 week collects, detects polypeptid induction antibody through ELISA indirect method and is feminine gender.As can be seen here, the fibroin polypeptide non-immunogenicity that obtains of the present invention.
Embodiment 4
The polypeptide that Shanghai gill synthesizes is added in emulsifiable paste matrix, adopts bitter diazanyl (1,1-diphenyl-2-picryhydrazyl, the DPPH) method of 1,1-phenylbenzene-2-to measure fibroin polypeptide Free-radical scavenging activity.Get the fibroin 2.0mg/ml of different concns, fibroin polypeptide solution (0.5 respectively, 1.0,2.0mg/ml) 1.0ml, put in 10ml centrifuge tube, add the DPPH solution of 3.0ml, room temperature lucifuge reaction 30min, is simultaneously blank with dehydrated alcohol, measures light absorption value in 517nm wavelength place.By following formulae discovery DPPH free radical scavenging activity.DPPH free radical scavenging activity (%)=A
0-(A
s-A
c)/A
0× 100% (in formula, A
0the absorbance of-1.0ml distilled water+3.0mlDPPH solution; A
sthe absorbance of-1.0ml sample solution+3.0mlDPPH solution; A
cthe absorbance of-1.0ml sample solution+3.0ml dehydrated alcohol), will test in triplicate, the clearance rate of 2.0mg/ml fibroin, 0.5,1.0,2.0, mg/ml fibroin polypeptide solution is respectively 34.89,24.43,32.65,54.98%.Visible, fibroin polypeptide solution has oxidation resistant effect, and compared with prototype fibroin, its antioxygenation is constant.
Embodiment 5
The experiment of restraint of tyrosinase
The polypeptide 1mL that the polypeptide administration group reaction mixture 2mL:0.1M pH6.8PBS 0.9mL that Shanghai gill synthesizes adds different concns adds 0.03% tyrosine 0.1mL.The thermostat container of 37 DEG C hatches 10min, adds tyrosine oxidase (350u/mL) aqueous solution of 0.1mL, is mixed by solution, after the thermostat container being placed in 37 DEG C reacts 25min, measures absorbance with spectrophotometer in 475mm; Negative control group replaces polypeptide spectrophotometer to measure absorbance in 475mm with isopyknic distilled water; Positive controls arbutin replaces polypeptide spectrophotometer to measure absorbance in 475mm; 0.1M pH 6.8PBS prepares according to 2005 editions " Chinese Pharmacopoeias ".Polypeptide is divided into 1,5,10mg/mL.Meanwhile, using arbutin (10mg/mL) as positive controls.Inhibiting rate %=(1-administration group/negative group) * 100%; Each concentration polypeptide experiment all repeats statistical procedures testing data and represents with mean ± standard deviation (x ± s), adopts t inspection, uses Excel software to do Data Analysis Services.
The black pigment of skin formation is a very complicated process, owing to being subject to the impact of ultraviolet, heredity, internal secretion, inflammatory mediator, the factor such as improper diet in the forming process of the black pigment of skin, metabolism of pigment is abnormal, the black pigment of skin excessively rapid growth and skewness, local skin will be caused to cross black and pigmentation, show as pigmentation etc. after chloasma, freckle and inflammation.Tyrosine oxidase is the oxydase containing Cu ion, it is prevalent in Mammals, plant and microorganism, the forming process of melanochrome and other polyphenols in main participation organism, it is the key enzyme of organism synthesis of melanin, tyrosinase inhibitor is exactly active by restraint of tyrosinase thus check melanin generates, and thus can be used to prevention and therapy pigmentation etc.The present invention observes the restraining effect of polypeptide to tyrosinase activity by experiment in vitro, and result shows that the polypeptide liquid of different concns all has restraining effect to tyrosinase activity, and has significant dose-effect relationship, confirms that it has definite whitening effect.
SEQUENCE LISTING
Pu Luoda bio tech ltd, <110> Suzhou
<120> fibroin polypeptide and Synthesis and applications thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> PRT
<213> artificial sequence
<400> 1
Asp Asp Thr Asp Lys Ser Ile Ala Ile Leu Asn Val Gln Glu Ile Leu
1 5 10 15
Lys Asp Met
Claims (4)
1. a fibroin polypeptide, is characterized in that, sequence is DDTDKSIAILNVQEILKDM.
2. the preparation method of fibroin polypeptide according to claim 1, is characterized in that, step comprises (1) by cleaning, dry after silk cocoon 500g shreds, add in 500-1000ml deionized water; (2) boil, ultrasonic 1-2 hour, slowly stir simultaneously; (3) after solution cooling, lytic enzyme is added, at 20-40 DEG C of reaction 30-60 minute; (4) centrifugal 2000-3000rpm, 5-10 minute, get supernatant; (5) added by supernatant liquor in the slant acidity damping fluid of pH6.0-6.5, stir, centrifugal 10000-15000rpm, 30-60 minute, be precipitated thing; (6) throw out is redissolved in pH5.0-5.5 damping fluid, adopt agarose gel chromatography technology, isolate the polypeptide that molecular weight is 2-3KDa, to obtain final product.
3. the preparation method of fibroin polypeptide according to claims 2, it is characterized in that, described lytic enzyme is papoid, ficin, bromeline, Ginger Protease, silk tree proteolytic enzyme or choke proteolytic enzyme.
4. the fibroin polypeptide according to claims 1 is preparing the application in makeup.
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| CN201510451393.2A CN104961805A (en) | 2015-07-28 | 2015-07-28 | Fibroin polypeptide, and preparation and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107469138A (en) * | 2017-10-09 | 2017-12-15 | 常州金艺广告传媒有限公司 | A kind of preparation method of high imbibition promoting healing type compound hemostatic material |
| CN107686856A (en) * | 2017-09-27 | 2018-02-13 | 广西壮族自治区农业科学院农产品加工研究所 | A kind of preparation method of Pitaya Flower polypeptide |
| CN110937595A (en) * | 2020-02-10 | 2020-03-31 | 江苏科技大学 | Method for preparing polypeptide-graphene composite material by utilizing silk fibroin oligopeptide water-phase stripping graphite and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948524A (en) * | 2010-09-02 | 2011-01-19 | 天津大学 | Multi-phosphopeptide-enriched casein hydrolyzate and preparation method thereof |
| CN102212107A (en) * | 2011-04-12 | 2011-10-12 | 江南大学 | Rice protein polypeptide and preparation method thereof |
| CN103923965A (en) * | 2014-05-11 | 2014-07-16 | 徐华 | Fibroin micro-molecule polypeptide preparing method |
| CN104098702A (en) * | 2014-07-23 | 2014-10-15 | 湖北工业大学 | Method for preparation of GLP-1 polypeptide or analogue thereof through MFH fusion protein and application of GLP-1 polypeptide or analogue thereof |
-
2015
- 2015-07-28 CN CN201510451393.2A patent/CN104961805A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948524A (en) * | 2010-09-02 | 2011-01-19 | 天津大学 | Multi-phosphopeptide-enriched casein hydrolyzate and preparation method thereof |
| CN102212107A (en) * | 2011-04-12 | 2011-10-12 | 江南大学 | Rice protein polypeptide and preparation method thereof |
| CN103923965A (en) * | 2014-05-11 | 2014-07-16 | 徐华 | Fibroin micro-molecule polypeptide preparing method |
| CN104098702A (en) * | 2014-07-23 | 2014-10-15 | 湖北工业大学 | Method for preparation of GLP-1 polypeptide or analogue thereof through MFH fusion protein and application of GLP-1 polypeptide or analogue thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107686856A (en) * | 2017-09-27 | 2018-02-13 | 广西壮族自治区农业科学院农产品加工研究所 | A kind of preparation method of Pitaya Flower polypeptide |
| CN107686856B (en) * | 2017-09-27 | 2021-04-27 | 广西壮族自治区农业科学院农产品加工研究所 | Preparation method of pitaya flower protein polypeptide |
| CN107469138A (en) * | 2017-10-09 | 2017-12-15 | 常州金艺广告传媒有限公司 | A kind of preparation method of high imbibition promoting healing type compound hemostatic material |
| CN110937595A (en) * | 2020-02-10 | 2020-03-31 | 江苏科技大学 | Method for preparing polypeptide-graphene composite material by utilizing silk fibroin oligopeptide water-phase stripping graphite and application thereof |
| CN110937595B (en) * | 2020-02-10 | 2023-04-18 | 江苏科技大学 | Method for preparing polypeptide-graphene composite material by utilizing silk fibroin oligopeptide water phase to strip graphite and application of polypeptide-graphene composite material |
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