CN104774899B - Preparation method of tuna dark meat protein anti-cervical cancer polypeptide - Google Patents

Abstract

The invention discloses a preparation method of a tuna dark meat protein cervical cancer resistant polypeptide, wherein the tuna dark meat is used as a raw material, and the cervical cancer resistant 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 chromatography purification, wherein the polypeptide has a significant inhibition effect on proliferation of human cervical cancer cell strain He L a cells, and IC (integrated circuit) is used for inhibiting proliferation of human cervical cancer cell strain He L a cells₅₀Is 0.16 mg/m L, and can be used for preparing medicine for preventing and treating cervical cancer.

Description

Preparation method of tuna dark meat protein anti-cervical cancer polypeptide

technical field

The invention relates to a preparation method of a fish functional polypeptide, in particular to a preparation method of a tuna dark meat protein anti-cervical cancer polypeptide.

Background technique

Tuna is an oceanic fish and is the key target species for the development of the world's distant-water fisheries. It is also recommended by the International Society of Nutrition as one of the world's three major nutritious fish. According to the statistics of the United Nations Food and Agriculture Organization, the current total output of oceanic fisheries in the world is 8.5 million tons, of which the output of tuna exceeds 6 million tons, accounting for more than 70% of the total output of high seas fisheries. A large amount of dark flesh is produced during the processing of tuna, accounting for about 11% of the raw material. Existing research shows that the dark meat of tuna is rich in essential amino acids, comprehensive, easy to digest and absorb, and is a high-quality raw material for preparing active peptides. But it has not been used effectively.

The applicant's research found that the research on the preparation of anti-cervical cancer polypeptides by enzymatic hydrolysis technology using tuna dark meat as raw materials is in the blank stage, and the preparation of highly active anti-cervical cancer polypeptides using enzymatic hydrolysis products as materials has not been reported.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a preparation method of tuna dark meat protein anti-cervical cancer polypeptide which has a significant inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells, and the technology is scientific and advanced.

The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of a tuna dark meat protein anti-cervical cancer polypeptide, which comprises the following steps:

1) Pretreatment of tuna dark meat: Homogenize tuna dark meat, heat it to 95-100°C, keep the temperature for 10-15min, then lower it to room temperature, add isopropyl alcohol according to the ratio of material to liquid 1g:4-5mL, and degrease at room temperature for 22 minutes ~24 h, then centrifuge at 4°C and 10000 rpm for 15-20 min to remove isopropanol, and collect the dark meat solids of defatted tuna, which is the dark meat protein of defatted tuna;

2) Enzymatic hydrolysis of defatted tuna dark meat protein: using defatted tuna dark meat protein as raw material, add Gly-NaOH buffer (0.05 mol/L, pH 9 to 10) at a solid-to-liquid ratio of 1 g: 20 to 25 mL, and mix The temperature of the mixture was raised to 45-55 °C, preheated for 5-10 min, and protease was added according to 1.5-2.5% of the mass of the dark flesh of the defatted tuna, and the enzymatic hydrolysis temperature was 45-55 °C. The solution was heated to 90-95°C and kept at this temperature for 10-15 min, centrifuged at 10,000 g for 20-25 min, and the supernatant was taken, which is the enzymatic hydrolysis product;

3) Preparation of tuna dark meat protein anti-cervical cancer polypeptide: the prepared enzymolysis product is subjected to ultrafiltration treatment with a 3 kDa ultrafiltration membrane, and the fraction with a molecular weight less than 3 kDa is collected to obtain an ultrafiltration enzymatic hydrolysate, and then the enzymatic hydrolysis solution is sequentially The anti-cervical cancer polypeptide was obtained by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) purification.

Preferably, the tuna in step 1) is bonito ( Katsuwonus pelamis ).

Preferably, the protease in the step 2) is alkaline protease, and the enzyme activity is ≥2.0×10 ⁵ U/g.

As an improvement, the specific processes of gel filtration chromatography, cell membrane chromatography and RP-HPLC purification in the step 3) are:

Gel filtration chromatography: The above-mentioned ultrafiltration enzymatic hydrolyzate was prepared into a solution of 15-25 mg/mL with pH 6.5-7.5 phosphate buffer, and subjected to Sephadex G-25 column chromatography (2.6 × 80 cm ) was separated, eluted with pH 6.5-7.5 phosphate buffer, and the eluted fractions were collected according to the absorbance curve at 220 nm. Among them, the fraction that significantly inhibited the proliferation of human cervical cancer cell line HeLa cells was the Gel chromatography enzymatic digest.

Purification by Cell Membrane Chromatography: The above gel chromatography enzymatic hydrolysate was prepared into a solution of 40-50 μg/mL with three-distilled water, added to a cell membrane chromatography column for purification, and the eluted fractions were collected according to the absorbance curve at 220 nm. , the component that has a significant inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells is the cell membrane chromatography-purified enzymatic hydrolysate.

RP-HPLC purification: The above-mentioned cell membrane chromatography-purified enzymatic hydrolysate was made into a solution of 80-100 μg/mL with three-distilled water, and purified by RP-HPLC. According to the inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells, 1 was obtained. A highly active anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW), the molecular weight detected by ESI-MS is 902.88 Da.

Preferably, the cell membrane chromatography conditions are: the injection volume is 5-10 μL; the cell membrane chromatography column (150 mm × 4.6 mm, 5 μm); the column temperature is 37 °C; the mobile phase: phosphate buffer (25 mmol/L, pH 7.4); UV detection wavelength 220nm; flow rate: 0.2 mL/min.

Preferably, the RP-HPLC conditions are: the injection volume is 15-20 μL; the chromatographic column is Zorbax C18 (250 mm × 4.6 mm, 5 μm); the mobile phase is 35% acetonitrile; and the ultraviolet detection wavelength is 220 nm.

Further preferably, the cell membrane used in the cell membrane chromatography column is Chinese white rabbit erythrocyte membrane, and the carrier used to fix the cell membrane is silica gel (5 μm, 200Å).

The tuna dark meat protein anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) prepared by the invention has a significant inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells, and the IC ₅₀ is 0.16 mg/mL; Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) has the advantages of safety, no toxic side effects and strong activity, and can be used for preparing medicines for preventing and treating cervical cancer.

Description of drawings

Fig. 1 is a Sephadex G-25 column chromatography chromatogram of the ultrafiltration enzymatic hydrolyzate (≤3 kDa fraction) of the present invention.

Fig. 2 is a cell membrane chromatogram of the enzymatic hydrolyzate (Fr.A3) prepared by Sephadex G-25 of the present invention.

Fig. 3 is the RP-HPLC chromatogram of the cell membrane chromatography-purified enzymatic hydrolyzate (Fr.A3-1) of the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments.

A preparation method of tuna dark meat protein anti-cervical cancer polypeptide, the preparation process is as follows: tuna dark meat → degreasing → enzymatic hydrolysis → ultrafiltration → gel filtration chromatography → cell membrane chromatography purification → high performance liquid chromatography purification → anti-cervical cancer peptide.

Example:

1) Pretreatment of tuna dark meat: tuna dark meat homogenate, heated to 95°C, incubated for 10 min, then lowered to room temperature, added isopropanol according to the ratio of material to liquid 1g: 5mL, degreased at room temperature for 24 hours, and then kept at room temperature for 4 hours. ℃, 10000 rpm centrifugation for 20 min to remove isopropanol, and collect the dark meat solids of defatted tuna, which is the dark meat protein of defatted tuna;

2) Enzymatic hydrolysis of defatted tuna dark meat protein: using defatted tuna dark meat protein as raw material, add Gly-NaOH buffer (0.05 mol/L, pH 9.5) at a solid-to-liquid ratio of 1 g : 25 mL to obtain a mixed solution; The temperature of the solution was raised to 50 °C and preheated for 10 min. Protease was added according to 2% of the mass of the dark flesh of the defatted tuna. The enzymatic hydrolysis temperature was 50 °C. After enzymatic hydrolysis for 4 h, the solution was heated to 95 °C and kept at this temperature for 10 min. Then, centrifuge at 10000g for 25 min, and take the supernatant, which is the enzymatic hydrolysis product;

3) Preparation of tuna dark meat protein anti-cervical cancer polypeptide: the prepared enzymolysis product is subjected to ultrafiltration treatment with a 3 kDa ultrafiltration membrane, and the fraction with a molecular weight less than 3 kDa is collected to obtain an ultrafiltration enzymatic hydrolysate, and then the enzymatic hydrolysis solution is sequentially The anti-cervical cancer polypeptide was obtained by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) purification.

①Gel filtration chromatography: The above ultrafiltration enzymatic hydrolysis solution was prepared into a 20 mg/mL solution with pH 7.0 phosphate buffer, and separated by Sephadex G-25 column chromatography (2.6 × 80 cm). Elution was carried out with pH 7.0 phosphate buffer, and the eluted fractions were collected according to the absorbance curve at 220 nm. Among them, the fraction that had a significant inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells was gel chromatography enzymatic hydrolysis. Fr.A3 (see Figure 1).

② Purification by cell membrane chromatography: The above gel chromatography enzymatic hydrolysate Fr.A3 was prepared into a solution of 50 μg/mL with distilled water, and added to the cell membrane chromatography column for purification (the injection volume was 5 μL; Chinese white rabbit red blood cell membrane chromatography Column (150 mm × 4.6 mm, 5 μm); column temperature: 37 °C; mobile phase: phosphate buffer (25 mmol/L, pH 7.4); UV detection wavelength: 220 nm; flow rate: 0.2 mL/min), according to 220 The eluted fractions were collected from the absorbance curve under nm, and the fraction that had a significant inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells was Fr.A3-1, a purified enzymatic hydrolysate by cell membrane chromatography (see Figure 2).

③ RP-HPLC purification: The above gel chromatography enzymatic hydrolyzate Fr.A3-1 was prepared into a solution of 100 μg/mL with ultrapure water, and purified by RP-HPLC (the injection volume was 15 μL; the chromatographic column was Zorbax C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was 35% acetonitrile; the UV detection wavelength was 220 nm), according to the proliferation inhibition of human cervical cancer cell line HeLa cells, a highly active anti-cervical cancer polypeptide was obtained (see image 3).

④Structure detection: The polypeptide with the strongest inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells was collected and detected as a single peak, and the amino acid sequence was determined by a protein/peptide sequence analyzer to be Gln-Tyr-Asp-Glu-Tyr-Trp ( QYDEYW), and the molecular weight detected by ESI-MS was 902.88 Da.

The tuna dark meat protein anti-cervical cancer polypeptide Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) prepared above was subjected to cell proliferation inhibition experiments. The experimental results show that: Gln-Tyr-Asp-Glu-Tyr-Trp (QYDEYW) has a significant inhibitory effect on the proliferation of human cervical cancer cell line HeLa cells, with an IC ₅₀ of 0.16 mg/mL.

Finally, it should be noted that the above list is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang Ocean University

<120> Preparation method of tuna dark meat protein 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 (8)

1. The preparation method of the cervical cancer resisting polypeptide of the tuna dark meat protein comprises 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 10000rpm 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 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 ℃ for 4-5 h, heating the solution to 90-95 ℃, keeping the temperature for 10-15 min after enzymolysis, centrifuging for 20-25 min at 10000g, and taking supernatant fluid, namely an enzymolysis product;

3) preparing cervical cancer resisting polypeptide from tuna dark meat protein, namely 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-HP L C) in sequence to obtain the cervical cancer resisting polypeptide;

the specific process of the gel filtration chromatography, the cell membrane chromatography and the reversed-phase high performance liquid chromatography (RP-HP L C) purification in the step 3) comprises the steps of gel filtration chromatography, namely preparing the ultrafiltration enzymolysis liquid into 15-25 mg/m L solution by using phosphate buffer solution with pH of 6.5-7.5, separating by using sephadex G-25 column chromatography, eluting by using phosphate buffer solution with pH of 6.5-7.5, and collecting elution components according to an absorbance curve under 220nm, wherein the component which has a remarkable inhibition effect on the proliferation of the He L a cells of the human cervical cancer cell strains is gel chromatography enzymolysis products;

purifying the cell membrane chromatography, namely preparing the gel chromatography zymolyte into a solution of 40-50 mu g/m L by using triple distilled water, adding the solution into a cell membrane chromatography column for purification, and collecting elution components according to an absorbance curve at 220nm, wherein the component which has a remarkable inhibition effect on proliferation of human cervical cancer cell strains He L a is the cell membrane chromatography 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, collecting the polypeptide with the strongest proliferation inhibition effect on the He L a cells of the human cervical cancer cell strain, detecting the polypeptide as a single peak, and determining that the amino acid sequence is Gln-Tyr-Asp-Glu-Tyr-Trp by using a protein/polypeptide sequence analyzer, wherein the molecular weight is 902.88Da by ESI-MS detection.

2. The method according to claim 1, wherein the tuna in the step 1) is bonito.

3. The method according to claim 1, wherein the protease in step 2) is an alkaline protease having an enzymatic activity of 2.0 to 2.0 × 10⁵U/g。

4. The preparation method of claim 1, wherein the cell membrane chromatography conditions comprise a sample amount of 5-10 μ L, a column temperature of 37 ℃, a mobile phase of phosphate buffer solution, an ultraviolet detection wavelength of 220nm, and a flow rate of 0.2m L/min.

5. The preparation method of claim 1, wherein the RP-HP L C is prepared by using a sample amount of 15-20 μ L, a chromatographic column of Zorbax C18, a mobile phase of 35% acetonitrile and an ultraviolet detection wavelength of 220 nm.

6. The method according to claim 1, wherein the cell membrane used in the cell membrane chromatographic column is a red cell membrane of a Chinese rabbit, and the cell membrane-fixing carrier used is silica gel having a specification of 5 μm and 200 Å.

7. The method according to claim 1, wherein the cell membrane column has a size of 150mm × 4.6.6 mm, 5 μm.

8. The method according to claim 5, wherein the phosphate buffer solution is 25 mmol/L, pH7.4, and the Zorbax C18 chromatographic column has a size of 250mm × 4.6.6 mm, 5 μm.

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