CN104861040B - Tuna dark meat protein anti-cervical cancer polypeptide - Google Patents
Tuna dark meat protein anti-cervical cancer polypeptide Download PDFInfo
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- CN104861040B CN104861040B CN201510198758.5A CN201510198758A CN104861040B CN 104861040 B CN104861040 B CN 104861040B CN 201510198758 A CN201510198758 A CN 201510198758A CN 104861040 B CN104861040 B CN 104861040B
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Abstract
The invention discloses a tuna dark meat protein anti-cervical cancer polypeptide, the amino acid sequence of which is Gln-Tyr-Asp-Glu-Tyr-Trp, ESI-MS detection molecular weightIs 902.88 Da. The polypeptide has significant inhibition effect on proliferation of HeLa cells of human cervical cancer cell strains, IC50Is 0.16mg/mL, and can be used for preparing medicine for preventing and treating cervical cancer.
Description
Technical Field
The invention relates to a fish functional polypeptide, in particular to a tuna dark meat protein anti-cervical cancer polypeptide.
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 technical problem to be solved by the invention is to provide a tuna dark meat protein anti-cervical cancer polypeptide aiming at the technical current situation, and the polypeptide has a remarkable inhibiting effect on the proliferation of human cervical cancer cell strain HeLa cells.
The technical scheme adopted by the invention for solving the technical problems is as follows: an amino acid sequence of the cervical cancer resisting polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW), and ESI-MS detection molecular weight is 902.88 Da.
The tuna dark meat protein cervical cancer resisting polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) has obvious inhibition effect on proliferation of human cervical cancer cell strain HeLa cells, and IC500.16 mg/mL; Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) is safe and nontoxicHas the advantages of strong side effect and activity, and the like, and can be used for preparing the medicine for preventing and treating cervical cancer.
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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.a3) prepared from sephadex G-25 of the present invention.
Fig. 3 is an RP-HPLC chromatogram of the cell membrane chromatographically purified zymolyte (fr. a3-1) 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) pretreatment of tuna dark meat: homogenizing tuna dark meat, heating to 95 ℃, preserving heat for 10min, cooling to room temperature, adding isopropanol according to the feed-liquid ratio of 1g:5mL, degreasing for 24h at room temperature, centrifuging at 4 ℃ and 10000rpm for 20min to remove the isopropanol, and collecting degreased tuna dark meat solid, namely degreased tuna dark meat protein; the tuna is preferably bonito.
2) Enzymolysis of the defatted tuna dark meat protein: taking defatted tuna dark meat protein as a raw material, and adding Gly-NaOH buffer solution (0.05mol/L, pH 9.5) according to the solid-to-liquid ratio of 1g:25mL to obtain a mixed solution; heating the mixed solution to 50 ℃ for preheating for 10min, adding protease according to 2% of the mass of the dark meat of the degreased tuna, carrying out enzymolysis at 50 ℃ for 4h, heating the solution to 95 ℃, keeping the temperature for 10min, centrifuging the solution at 10000g for 25min, and taking supernatant, namely an enzymolysis product;
3) preparing cervical cancer resisting polypeptide from tuna dark meat protein: and (3) performing ultrafiltration treatment on the prepared enzymolysis product by using a 3kDa ultrafiltration membrane, collecting the part with the molecular weight less than 3kDa to obtain ultrafiltration enzymolysis liquid, and purifying the enzymolysis liquid by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the cervical cancer resistant polypeptide.
Gel filtration chromatography: preparing the ultrafiltration enzymolysis solution into a 20mg/mL solution by using a phosphate buffer solution with pH of 7.0, separating by using sephadex G-25 column chromatography (2.6 multiplied by 80cm), eluting by using the phosphate buffer solution with pH of 7.0, and collecting an elution component according to an absorbance curve under 220nm, wherein the component which has a remarkable inhibition effect on the proliferation of human cervical cancer cell strain HeLa cells is gel chromatography zymolyte Fr.A3 (shown in figure 1).
② purifying cell membrane chromatography: preparing the gel chromatography zymolyte Fr.A3 into a solution of 50 mug/mL by using triple distilled water, adding the solution into a cell membrane chromatographic column for purification (the sample introduction amount is 5 muL; Chinese white rabbit red cell membrane chromatographic column (150mm multiplied by 4.6mm, 5μm); the column temperature is 37 ℃, the mobile phase comprises phosphate buffer solution (25mmol/L, pH 7.4), the ultraviolet detection wavelength is 220nm, the flow rate is 0.2mL/min), and collecting the eluted components according to an absorbance curve under 220nm, wherein the component having a significant inhibition effect on the proliferation of human cervical cancer cell strain HeLa cells is the cell membrane chromatography purification zymolyte Fr.A3-1 (see figure 2).
③ RP-HPLC purification: the gel chromatography zymolyte Fr.A3-1 is prepared into a solution of 100 mu g/mL by ultrapure water, and is purified by RP-HPLC (the sample volume is 15 mu L; a chromatographic column is Zorbax C18(250mm multiplied by 4.6mm, 5 mu m), a mobile phase is 35% acetonitrile, and the ultraviolet detection wavelength is 220nm), so that 1 high-activity anti-cervical cancer polypeptide (shown in figure 3) is obtained according to the proliferation inhibition effect on human cervical cancer cell strain HeLa cells.
Fourthly, structure detection: the polypeptide with the strongest proliferation inhibition effect on human cervical cancer cell strain HeLa cells is collected and detected as a single peak, the amino acid sequence determined by a protein/polypeptide sequence analyzer is Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW), and the molecular weight detected by ESI-MS is 902.88 Da.
The prepared tuna dark meat protein cervical cancer-resistant polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) is subjected to cell proliferation inhibition experiment. The experimental results show that: Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) has obvious inhibition effect on proliferation of HeLa cells of human cervical cancer cell strain, and IC50It was 0.16 mg/mL.
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
<120> cervical cancer resisting polypeptide of tuna dark meat protein
<130>zjou-wb-20140411
<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 (1)
1. The tuna dark meat protein anti-cervical cancer polypeptide is characterized in that the amino acid sequence of the anti-cervical cancer polypeptide is Gln-Tyr-Asp-Glu-Tyr-Trp, and the molecular weight of ESI-MS detection is 902.88 Da.
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Address after: 316022 No. 1, Haida South Road, Changzhi Island, Lincheng street, Zhoushan, Zhejiang. Patentee after: Zhejiang Ocean University Address before: 316022 No. 1, Haida South Road, Changzhi Island, Lincheng street, Zhoushan, Zhejiang. Patentee before: ZHEJIANG OCEAN University |
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Effective date of registration: 20230912 Address after: No. 532, middle section of Huaxing Road, Hengyuan street, Linyi County, Dezhou City, Shandong Province, 251500 Patentee after: DEZHOU LANLI BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: Room 834, Unit 2, Building 1, No. 106, Nancheng Section, Guantai Road, Nancheng Street, Dongguan City, Guangdong Province, 523000 Patentee before: Guangdong Kaihong Intellectual Property Agency Co.,Ltd. Effective date of registration: 20230912 Address after: Room 834, Unit 2, Building 1, No. 106, Nancheng Section, Guantai Road, Nancheng Street, Dongguan City, Guangdong Province, 523000 Patentee after: Guangdong Kaihong Intellectual Property Agency Co.,Ltd. Address before: 316022 No. 1, Haida South Road, Changzhi Island, Lincheng street, Zhoushan, Zhejiang. Patentee before: Zhejiang Ocean University |