CN104774898A - Application of anti-cervical-carcinoma polypeptide prepared by using tuna dark-meat proteins - Google Patents
Application of anti-cervical-carcinoma polypeptide prepared by using tuna dark-meat proteins Download PDFInfo
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- CN104774898A CN104774898A CN201510197968.2A CN201510197968A CN104774898A CN 104774898 A CN104774898 A CN 104774898A CN 201510197968 A CN201510197968 A CN 201510197968A CN 104774898 A CN104774898 A CN 104774898A
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Abstract
The invention discloses an application of an anti-cervical-carcinoma polypeptide prepared by using tuna dark-meat proteins. The amino acid sequence of the anti-cervical-carcinoma polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW), and the molecular weight detected through ESI-MS (electrospray ionization-mass spectrometry) is 902.88 Da. In the process of preparation, by taking tuna dark-meat as a raw material, the tuna dark-meat is sequentially subjected to degreasing, alkaline protease enzymolysis, ultrafiltration, gel filtration chromatography, cell membrane chromatographic purification, and reversed-phase high-performance liquid purification, so that an anti-cervical-carcinoma polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp is obtained. The polypeptide has a significant inhibitory effect on the proliferation of human cervical carcinoma cell lines HeLa cells, and the IC50 is 0.16 mg/mL, so that the polypeptide can be used for preparing drugs, health foods and related additives for preventing and treating cervical carcinoma.
Description
Technical field
The present invention relates to a kind of purposes of hydrobiont functional polypeptide, particularly relate to the purposes of tuna dark-coloured meat albumen anti-cervical cancer polypeptide.
Background technology
Tuna is oceanic fishes, is the highest priority fingerling of world's Development for Distant Water Fishery, and the international NI of Ye Shi is recommended as one of large nutrition fish in the world three.According to Food and Argriculture OrganizationFAO's statistics, current world ocean property fishery ultimate production is 8,500,000 tons, and wherein tuna output is more than 6,000,000 tons, accounts for high sea fishery ultimate production more than 70%.In the tuna course of processing, produce a large amount of dark-coloured meat, account for 11% of raw material.Now there are some researches show, the dark-coloured meat essential amino acids content of tuna is abundant, comprehensively, cutting absorption easy to digest, is the high quality raw material preparing bioactive peptide.But be not utilized effectively at present.
Applicant studies discovery, and with the dark-coloured meat of tuna for raw material, the technical study utilizing zymolysis technique to prepare anti-cervical cancer polypeptide is in the blank stage, and is that material is prepared high reactivity anti-cervical cancer polypeptide and had no report especially with enzymolysis product.
Summary of the invention
First technical problem to be solved by this invention provides a kind of increment to Human cervical carcinoma cell line HeLa cell to have the purposes of significant restraining effect tuna dark-coloured meat albumen anti-cervical cancer polypeptide for the above-mentioned state of the art.
The aminoacid sequence of tuna of the present invention dark-coloured meat albumen anti-cervical cancer polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp(QYDEYW), ESI-MS detection molecules amount is 902.88 Da.
A preparation method for tuna dark-coloured meat albumen anti-cervical cancer polypeptide, is characterized in that comprising the following steps:
1) pre-treatment of the dark-coloured meat of tuna:the dark-coloured meat homogenate of tuna, 10 ~ 15min is incubated after being heated to 95 ~ 100 DEG C, then room temperature is down to, Virahol is added according to solid-liquid ratio 1g:4 ~ 5mL, degreasing 22 ~ 24 h under room temperature, then in 4 DEG C, centrifugal 15 ~ 20 min removing Virahols of 10000 rpm, collect the dark-coloured meat solid substance of degreasing tuna, be the dark-coloured meat albumen of degreasing tuna;
2) enzymolysis of the dark-coloured meat albumen of degreasing tuna:using the dark-coloured meat albumen of degreasing tuna as raw material, add Gly-NaOH damping fluid (0.05 mol/L, pH 9 ~ 10) by solid-to-liquid ratio 1 g: 20 ~ 25 mL, obtain mixed solution; Mixeding liquid temperature is risen to 45 ~ 55 DEG C of preheating 5 ~ 10 min, proteolytic enzyme is added according to 1.5 ~ 2.5% of the dark-coloured meat quality of degreasing tuna, hydrolysis temperature is 45 ~ 55 DEG C, after enzymolysis 4 ~ 5 h, by solution warms to 90 ~ 95 DEG C, and after this temperature keeps 10 ~ 15 min, centrifugal 20 ~ 25 min of 10000g, get supernatant liquor, be enzymolysis product;
3) preparation of tuna dark-coloured meat albumen anti-cervical cancer polypeptide:3 kDa ultra-filtration membranes are adopted by the enzymolysis product of preparation to carry out uf processing, collect molecular weight and be less than 3 kDa parts, obtain ultrafiltration enzymolysis solution, then by enzymolysis solution successively through gel permeation chromatography, membrane flexibility and RPLC (RP-HPLC) purifying, obtain anti-cervical cancer polypeptide.
As preferably, the tuna in described step 1) be stripped tuna (
katsuwonus pelamis).
As preferably, described step 2) in proteolytic enzyme be Sumizyme MP, enzyme activity>=2.0 × 10
5u/g.
As improvement, the detailed process of the gel permeation chromatography in described step 3), membrane flexibility and RP-HPLC purifying is:
gel permeation chromatography:above-mentioned ultrafiltration enzymolysis solution pH 6.5 ~ 7.5 phosphate buffered saline buffer is made into the solution of 15 ~ 25 mg/mL, be separated through sephadex G-25 column chromatography (2.6 × 80 cm), wash-out is carried out with pH 6.5 ~ 7.5 phosphate buffered saline buffer, elution fraction is collected according to the absorbance curve under 220 nm, wherein, having remarkable inhibiting component to the increment of Human cervical carcinoma cell line HeLa cell is gel chromatography zymolyte.
membrane flexibility purifying:above-mentioned gel chromatography zymolyte tri-distilled water is made into the solution of 40 ~ 50 μ g/mL, join membrane flexibility post and carry out purifying, elution fraction is collected according to the absorbance curve under 220 nm, wherein, having remarkable inhibiting component to the increment of Human cervical carcinoma cell line HeLa cell is membrane flexibility purifying zymolyte.
rP-HPLC purifying:above-mentioned membrane flexibility purifying zymolyte tri-distilled water is made into the solution of 80 ~ 100 μ g/mL, RP-HPLC is utilized to carry out purifying, according to the increment restraining effect of Human cervical carcinoma cell line HeLa cell being obtained to 1 high reactivity anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp(QYDEYW), ESI-MS detection molecules amount is 902.88 Da.
Preferably, described membrane flexibility condition is: sample size 5 ~ 10 μ L; Membrane flexibility post (150 mm × 4.6mm, 5 μm); Column temperature is 37 DEG C; Moving phase: phosphate buffered saline buffer (25 mmol/L, pH 7.4); Ultraviolet detection wavelength 220 nm; Flow velocity: 0.2 mL/min.
Preferably, described RP-HPLC condition is: sample size 15 ~ 20 μ L; Chromatographic column is Zorbax C18(250 mm × 4.6 mm, 5 μm); Moving phase is 35 % acetonitriles; Ultraviolet detection wavelength is 220 nm.
Preferred again, adopt cytolemma to be Chinese White Rabbit erythrocyte membrane in described membrane flexibility post, the carrier of fixed cell film used is silica gel (5 μm, 200).
The technical scheme that the present invention takes for technical solution problem for: the dark-coloured meat albumen of the tuna prepared by the present invention anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp(QYDEYW) to the increment of Human cervical carcinoma cell line HeLa cell, there is remarkable restraining effect, IC
50be 0.16 mg/mL; Gln-Tyr-Asp-Glu-Tyr-Trp(QYDEYW) there is safe without toxic side effect and the advantage such as activity is strong, can be used for preparing the medicine preventing and treating cervical cancer.
Accompanying drawing explanation
Fig. 1 is sephadex G-25 column chromatography chromatogram figure of ultrafiltration enzymolysis solution of the present invention (≤3 kDa component).
Fig. 2 is the membrane flexibility figure that sephadex G-25 of the present invention prepares zymolyte (Fr.A3).
Fig. 3 is the RP-HPLC color atlas of membrane flexibility purifying zymolyte (Fr.A3-1) of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
A preparation method for tuna dark-coloured meat albumen anti-cervical cancer polypeptide, preparation technology's flow process is as follows: the dark-coloured meat → degreasing → enzymolysis → ultrafiltration → gel permeation chromatography → membrane flexibility purifying → high-efficient liquid phase chromatogram purification → anti-cervical cancer polypeptide of tuna.
Embodiment:
1) pre-treatment of the dark-coloured meat of tuna:the dark-coloured meat homogenate of tuna, 10 min are incubated after being heated to 95 DEG C, then room temperature is down to, Virahol is added according to solid-liquid ratio 1g:5mL, degreasing 24 h under room temperature, then in 4 DEG C, the centrifugal 20 min removing Virahols of 10000 rpm, collect the dark-coloured meat solid substance of degreasing tuna, be the dark-coloured meat albumen of degreasing tuna;
2) enzymolysis of the dark-coloured meat albumen of degreasing tuna:using the dark-coloured meat albumen of degreasing tuna as raw material, add Gly-NaOH damping fluid (0.05 mol/L, pH 9.5) by solid-to-liquid ratio 1 g: 25 mL, obtain mixed solution; Mixeding liquid temperature is risen to 50 DEG C of preheating 10 min, add proteolytic enzyme according to 2% of the dark-coloured meat quality of degreasing tuna, hydrolysis temperature is 50 DEG C, after enzymolysis 4 h, by solution warms to 95 DEG C, and after this temperature keeps 10 min, centrifugal 25 min of 10000g, get supernatant liquor, are enzymolysis product;
3) preparation of tuna dark-coloured meat albumen anti-cervical cancer polypeptide:3 kDa ultra-filtration membranes are adopted by the enzymolysis product of preparation to carry out uf processing, collect molecular weight and be less than 3 kDa parts, obtain ultrafiltration enzymolysis solution, then by enzymolysis solution successively through gel permeation chromatography, membrane flexibility and RPLC (RP-HPLC) purifying, obtain anti-cervical cancer polypeptide.
1. gel permeation chromatography: the solution above-mentioned ultrafiltration enzymolysis solution pH 7.0 phosphate buffered saline buffer being made into 20 mg/mL, be separated through sephadex G-25 column chromatography (2.6 × 80 cm), wash-out is carried out with pH 7.0 phosphate buffered saline buffer, elution fraction is collected according to the absorbance curve under 220 nm, wherein, having remarkable inhibiting component to the increment of Human cervical carcinoma cell line HeLa cell is that gel chromatography zymolyte Fr.A3(is shown in Fig. 1).
2. membrane flexibility purifying: the solution above-mentioned gel chromatography zymolyte Fr.A3 tri-distilled water being made into 50 μ g/mL, joins membrane flexibility post and carries out purifying (sample size 5 μ L; Chinese White Rabbit erythrocyte membrane chromatographic column (150 mm × 4.6 mm, 5 μm); Column temperature is 37 DEG C; Moving phase: phosphate buffered saline buffer (25 mmol/L, pH 7.4); Ultraviolet detection wavelength 220 nm; Flow velocity: 0.2 mL/min), collect elution fraction according to the absorbance curve under 220 nm, wherein, having remarkable inhibiting component to the increment of Human cervical carcinoma cell line HeLa cell is that membrane flexibility purifying zymolyte Fr.A3-1(is shown in Fig. 2).
3. RP-HPLC purifying: the solution above-mentioned gel chromatography zymolyte Fr.A3-1 ultrapure water being made into 100 μ g/mL, utilizes RP-HPLC to carry out purifying (sample size 15 μ L; Chromatographic column is Zorbax C18(250 mm × 4.6 mm, 5 μm); Moving phase is 35 % acetonitriles; Ultraviolet detection wavelength is 220 nm), according to the increment restraining effect of Human cervical carcinoma cell line HeLa cell being obtained to 1 high reactivity anti-cervical cancer polypeptide (see figure 3).
4. structure detection: collecting the polypeptide the strongest to the increment restraining effect of Human cervical carcinoma cell line HeLa cell is simple spike after testing, utilizing protein/polypeptide sequenator to measure aminoacid sequence is Gln-Tyr-Asp-Glu-Tyr-Trp(QYDEYW), ESI-MS detection molecules amount is 902.88 Da.
By above-mentioned obtained tuna dark-coloured meat albumen anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp(QYDEYW) carry out cell growth inhibition assay.Experimental result shows: Gln-Tyr-Asp-Glu-Tyr-Trp(QYDEYW) to the increment of Human cervical carcinoma cell line HeLa cell, there is remarkable restraining effect, IC
50be 0.16 mg/mL.
Finally, still need it is noted that what enumerate above is only a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
SEQUENCE LISTING
<110> Oceanography Institute Of Zhejiang
The purposes of <120> tuna dark-coloured meat albumen anti-cervical cancer polypeptide
<130> zjou-wb-20150411
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213> synthetic
<400> 1
Gln Tyr Asp Glu Tyr Trp
1 5
Claims (1)
1. the purposes of tuna dark-coloured meat albumen anti-cervical cancer polypeptide, is characterized in that described tuna dark-coloured meat albumen anti-cervical cancer polypeptide is for the preparation of the protective foods or the foodstuff additive that prevent cervical cancer; The aminoacid sequence of described tuna dark-coloured meat albumen anti-cervical cancer polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp, ESI-MS detection molecules amount is 902.88 Da.
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Citations (4)
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|---|---|---|---|---|
| CN103275181A (en) * | 2013-05-23 | 2013-09-04 | 浙江海洋学院 | Tuna ground meat polypeptide angiogenesis inhibiting factor as well as preparation method and application thereof |
| WO2014068601A1 (en) * | 2012-10-29 | 2014-05-08 | Matis Ohf. | Use of natural antioxidants during enzymatic hydrolysis of aquatic protein to obtain high quality aquatic protein hydrolysates |
| CN104031136A (en) * | 2014-06-06 | 2014-09-10 | 杭州普英生物科技有限公司 | Functional region variants of sea purse angiogenesis inhibitor 1 and application of variants |
| CN104530191A (en) * | 2014-12-29 | 2015-04-22 | 浙江海洋学院 | Anti-prostate-cancer tegillarca granosa protein polypeptide as well as preparation method and application thereof |
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- 2015-04-24 CN CN201510197968.2A patent/CN104774898B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014068601A1 (en) * | 2012-10-29 | 2014-05-08 | Matis Ohf. | Use of natural antioxidants during enzymatic hydrolysis of aquatic protein to obtain high quality aquatic protein hydrolysates |
| CN103275181A (en) * | 2013-05-23 | 2013-09-04 | 浙江海洋学院 | Tuna ground meat polypeptide angiogenesis inhibiting factor as well as preparation method and application thereof |
| CN104031136A (en) * | 2014-06-06 | 2014-09-10 | 杭州普英生物科技有限公司 | Functional region variants of sea purse angiogenesis inhibitor 1 and application of variants |
| CN104530191A (en) * | 2014-12-29 | 2015-04-22 | 浙江海洋学院 | Anti-prostate-cancer tegillarca granosa protein polypeptide as well as preparation method and application thereof |
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Effective date of registration: 20230111 Address after: 238014 Room 904, Unit 2, Building 20, Phoenix City, Dongfeng Neighborhood Committee, Chaohu City, Anhui Province Patentee after: Liu Hongmei Address before: 710000 No. B49, Xinda Zhongchuang space, 26th Street, block C, No. 2 Trading Plaza, South China City, international port district, Xi'an, Shaanxi Province Patentee before: Xi'an Huaqi Zhongxin Technology Development Co.,Ltd. Effective date of registration: 20230111 Address after: 710000 No. B49, Xinda Zhongchuang space, 26th Street, block C, No. 2 Trading Plaza, South China City, international port district, Xi'an, Shaanxi Province Patentee after: Xi'an Huaqi Zhongxin Technology Development Co.,Ltd. Address before: 316022 No. 1, Haida South Road, Changzhi Island, Lincheng street, Zhoushan, Zhejiang. Patentee before: ZHEJIANG OCEAN University |
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