CN104774898B - Application of tuna dark meat protein anti-cervical cancer polypeptide - Google Patents
Application of tuna dark meat protein anti-cervical cancer polypeptide Download PDFInfo
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- CN104774898B CN104774898B CN201510197968.2A CN201510197968A CN104774898B CN 104774898 B CN104774898 B CN 104774898B CN 201510197968 A CN201510197968 A CN 201510197968A CN 104774898 B CN104774898 B CN 104774898B
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Abstract
The invention discloses an application of a tuna dark meat protein anti-cervical cancer polypeptide, wherein the amino acid sequence of the anti-cervical cancer polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW), the ESI-MS detection molecular weight is 902.88 Da, the tuna dark meat is taken as a raw material during preparation, and the anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp is obtained by degreasing, alkaline protease enzymolysis, ultrafiltration, gel filtration chromatography, cell membrane chromatography purification and reversed phase high performance liquid purification, so that the polypeptide has a significant inhibition effect on the proliferation of cells of human cervical cancer cell strains He L a, and IC is IC50Is 0.16 mg/m L, and can be used for preparing medicine, health food or related additives for preventing and treating cervical cancer.
Description
Technical Field
The invention relates to application of a functional polypeptide of aquatic organisms, in particular to application of a cervical cancer resisting polypeptide of tuna dark meat protein.
Background
Tuna is an oceanic fish, is a key target fish species for the development of oceanic fishery in the world, and is also one of three kinds of nutritional fishes recommended by the international society for nutrition in the world. According to statistics of grain and agriculture organizations in the united nations, the total output of oceanic fishery in the world is 850 ten thousand tons at present, wherein the output of tuna exceeds 600 ten thousand tons, and accounts for more than 70% of the total output of fishery in the open sea. During tuna processing, a large amount of dark meat is produced, accounting for approximately 11% of the raw material. The existing research shows that the tuna dark meat has rich and comprehensive essential amino acid content, is easy to digest and absorb, and is a high-quality raw material for preparing active peptide. But are not currently being utilized effectively.
The research of the applicant finds that the process research for preparing the anti-cervical cancer polypeptide by using the tuna dark meat as the raw material and utilizing the enzymolysis technology is in a blank stage, and the preparation of the high-activity anti-cervical cancer polypeptide by using the enzymolysis product as the material is not reported.
Disclosure of Invention
The first technical problem to be solved by the invention is to provide the application of the tuna dark meat protein anti-cervical cancer polypeptide with a remarkable inhibition effect on the proliferation of the cells of the human cervical cancer cell line He L a aiming at the technical current situation.
The amino acid sequence of the tuna dark meat protein anti-cervical cancer polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW), and the ESI-MS detected molecular weight is 902.88 Da.
A preparation method of cervical cancer resisting polypeptide of tuna dark meat protein is characterized by comprising the following steps:
1) pretreating tuna dark meat, namely homogenizing the tuna dark meat, heating to 95-100 ℃, preserving heat for 10-15 min, cooling to room temperature, adding isopropanol according to the feed-liquid ratio of 1g: 4-5 m L, degreasing for 22-24 h at room temperature, centrifuging at 4 ℃ and 10000 rpm for 15-20 min to remove the isopropanol, and collecting a solid matter of the degreased tuna dark meat, namely degreased tuna dark meat protein;
2) carrying out enzymolysis on the defatted tuna dark meat protein, namely taking the defatted tuna dark meat protein as a raw material, adding Gly-NaOH buffer solution (0.05 mol/L, pH 9-10) into the defatted tuna dark meat protein according to the solid-to-liquid ratio of 1g to 20-25 m L to obtain mixed solution, heating the mixed solution to 45-55 ℃, preheating for 5-10 min, adding protease according to 1.5-2.5% of the mass of the defatted tuna dark meat, carrying out enzymolysis at 45-55 ℃, heating the solution to 90-95 ℃ after carrying out enzymolysis for 4-5 h, keeping the temperature for 10-15 min, centrifuging for 20-25 min at 10000g, and taking supernatant fluid to obtain an enzymolysis product;
3) the preparation method of the tuna dark meat protein anti-cervical cancer polypeptide comprises the steps of carrying out ultrafiltration treatment on a prepared enzymolysis product by using a3 kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3 kDa to obtain an ultrafiltration enzymolysis liquid, and purifying the enzymolysis liquid by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HP L C) in sequence to obtain the anti-cervical cancer polypeptide.
Preferably, the tuna in step 1) is usedIs prepared from bonitoKatsuwonus pelamis）。
Preferably, the protease in the step 2) is alkaline protease, and the enzyme activity is more than or equal to 2.0 × 105U/g。
As an improvement, the specific processes of gel filtration chromatography, cell membrane chromatography and RP-HP L C purification in the step 3) are as follows:
and (3) performing gel filtration chromatography, namely preparing the ultrafiltration enzymolysis liquid into a solution of 15-25 mg/m L by using a phosphate buffer solution with the pH of 6.5-7.5, separating by using sephadex G-25 column chromatography (2.6 × 80 cm), eluting by using a phosphate buffer solution with the pH of 6.5-7.5, and collecting an elution component according to an absorbance curve at 220nm, wherein the component which has a remarkable inhibition effect on the proliferation of a human cervical cancer cell strain He L a is a gel chromatography enzymolysis product.
And (3) cell membrane chromatographic purification, namely preparing the gel chromatographic zymolyte into a solution of 40-50 mu g/m L by using triple distilled water, adding the solution into a cell membrane chromatographic column for purification, and collecting eluted components according to an absorbance curve at 220nm, wherein the component which has a remarkable inhibition effect on proliferation of human cervical cancer cell strain He L a cells is the cell membrane chromatographic purification zymolyte.
And (3) RP-HP L C purification, namely preparing the purified zymolyte of the cell membrane chromatography into a solution of 80-100 mu g/m L by using triple distilled water, purifying by using RP-HP L C, and obtaining 1 high-activity anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) according to the proliferation inhibition effect on cells of a human cervical cancer cell strain He L a, wherein the molecular weight is 902.88 Da detected by ESI-MS.
Preferably, the cell membrane chromatographic conditions comprise a sample injection amount of 5-10 mu L, a cell membrane chromatographic column (150 mm × 4.6.6 mm, 5 mu m), a column temperature of 37 ℃, a mobile phase of phosphate buffer solution (25 mmol/L, pH 7.4), an ultraviolet detection wavelength of 220nm and a flow rate of 0.2 m L/min.
Preferably, the RP-HP L C conditions include a sample injection amount of 15-20 mu L, a chromatographic column of Zorbax C18 (250 mm × 4.6.6 mm, 5 mu m), a mobile phase of 35% acetonitrile, and an ultraviolet detection wavelength of 220 nm.
More preferably, the cell membrane chromatographic column adopts Chinese white rabbit red cell membrane as cell membrane, and the carrier for fixing the cell membrane is silica gel (5 μm, 200 Å).
The technical scheme adopted for solving the technical problem is that the prepared tuna dark meat protein anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) has a remarkable inhibition effect on proliferation of human cervical cancer cell strain He L a, and IC (integrated circuit) has the advantages of500.16 mg/m L, Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW), has the advantages of safety, no toxic or side effect, strong activity and the like, and can be used for preparing the medicine for preventing and treating the cervical cancer.
Drawings
FIG. 1 is a chromatogram of Sephadex G-25 column chromatography of the ultrafiltrated enzymatic hydrolysate (3 kDa component or less) of the present invention.
Fig. 2 is a cell membrane chromatogram of a substrate (fr.a 3) prepared from sephadex G-25 of the present invention.
Fig. 3 is a RP-HP L C chromatogram of a cell membrane chromatography purified zymolyte (fr. a 3-1) of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
A preparation method of tuna dark meat protein anti-cervical cancer polypeptide comprises the following preparation process flow: tuna dark meat → degreasing → enzymolysis → ultrafiltration → gel filtration chromatography → cell membrane chromatography purification → high performance liquid chromatography purification → anti-cervical cancer polypeptide.
Example (b):
1) pretreating tuna dark meat, namely homogenizing the tuna dark meat, heating to 95 ℃, preserving heat for 10 min, cooling to room temperature, adding isopropanol into the mixture according to the feed-liquid ratio of 1g to 5m L, degreasing for 24 h at the room temperature, centrifuging for 20min at 4 ℃ and 10000 rpm to remove the isopropanol, and collecting degreased tuna dark meat solid, namely degreased tuna dark meat protein;
2) carrying out enzymolysis on the defatted tuna dark meat protein, namely taking the defatted tuna dark meat protein as a raw material, adding Gly-NaOH buffer solution (0.05 mol/L, pH 9.5) into the defatted tuna dark meat protein according to the solid-to-liquid ratio of 1g to 25 m L to obtain mixed solution, heating the mixed solution to 50 ℃, preheating for 10 min, adding protease according to 2 percent of the mass of the defatted tuna dark meat, carrying out enzymolysis at 50 ℃ for 4 h, heating the solution to 95 ℃, keeping the temperature for 10 min, centrifuging for 25 min at 10000g, and taking supernatant fluid, namely an enzymolysis product;
3) the preparation method of the tuna dark meat protein anti-cervical cancer polypeptide comprises the steps of carrying out ultrafiltration treatment on a prepared enzymolysis product by using a3 kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3 kDa to obtain an ultrafiltration enzymolysis liquid, and purifying the enzymolysis liquid by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HP L C) in sequence to obtain the anti-cervical cancer polypeptide.
① gel filtration chromatography, which comprises preparing the above ultrafiltrate hydrolysate into 20 mg/m L solution with pH 7.0 phosphate buffer solution, separating by Sephadex G-25 column chromatography (2.6 × 80 cm), eluting with pH 7.0 phosphate buffer solution, and collecting eluate according to absorbance curve at 220nm, wherein the component with significant inhibition effect on proliferation of human cervical cancer cell strain He L a is gel chromatography zymolyte Fr.A3 (see figure 1).
② cell membrane chromatographic purification, preparing the gel chromatography zymolyte Fr.A3 into 50 μ g/m L solution with triple distilled water, adding the solution into a cell membrane chromatographic column for purification (the sample introduction amount is 5 μ L; Chinese white rabbit red cell membrane chromatographic column (150 mm × 4.6.6 mm, 5 μm), the column temperature is 37 ℃, the mobile phase comprises phosphate buffer solution (25 mmol/L, pH 7.4), the ultraviolet detection wavelength is 220nm, the flow rate is 0.2 m L/min), and collecting elution components according to an absorbance curve under 220nm, wherein the component which has a significant inhibition effect on the proliferation of human cervical cancer cell line He L a is the cell membrane chromatographic purification zymolyte Fr.A3-1 (see figure 2).
③ RP-HP L C, preparing the gel chromatography zymolyte Fr.A3-1 into a solution of 100 mu g/m L with ultrapure water, purifying by RP-HP L C (the sample volume is 15 mu L; the chromatographic column is Zorbax C18 (250 mm × 4.6.6 mm, 5 mu m), the mobile phase is 35% acetonitrile, the ultraviolet detection wavelength is 220 nm), and obtaining 1 high-activity anti-cervical cancer polypeptide according to the proliferation inhibition effect on human cervical cancer cell strain He L a cells (see figure 3).
④ structure detection, collecting polypeptide with strongest proliferation inhibition effect on human cervical cancer cell strain He L a cell, detecting the polypeptide as single peak, determining the amino acid sequence as Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) by using a protein/polypeptide sequence analyzer, and detecting the molecular weight as 902.88 Da by ESI-MS.
Cell proliferation inhibition experiments are carried out on the prepared tuna dark meat protein cervical cancer resistant polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW). Experimental results show that the Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) has obvious inhibition effect on proliferation of human cervical cancer cell strain He L a cells, and IC (integrated Circuit) shows that50It was 0.16 mg/m L.
Finally, it should be noted that the above-mentioned list is only one specific embodiment of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
SEQUENCE LISTING
<110> Zhejiang ocean academy
Application of <120> cervical cancer resisting polypeptide of tuna dark meat protein
<130>zjou-wb-20150411
<160>1
<170>PatentIn version 3.5
<210>1
<211>6
<212>PRT
<213> Artificial Synthesis
<400>1
Gln Tyr Asp Glu Tyr Trp
1 5
Claims (2)
1. The application of the tuna dark meat protein anti-cervical cancer polypeptide in preparing the medicine for treating the cervical cancer is that the amino acid sequence of the tuna dark meat protein anti-cervical cancer polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp, and the ESI-MS detection molecular weight is 902.88 Da.
2. The tuna dark flesh protein cervical cancer resisting polypeptide has an amino acid sequence of Gln-Tyr-Asp-Glu-Tyr-Trp, and has a molecular weight of 902.88 Da in ESI-MS detection.
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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 |
| 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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| US20150274791A1 (en) * | 2012-10-29 | 2015-10-01 | Matis Ohf. | Use of natural antioxidants during enzymatic hydrolysis of aquatic protein to obtain high quality aquatic protein hydrolysates |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| 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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