A sea anemone polypeptide, and methods of making and using the same
By isolating and purifying sea anemone polypeptides from sea anemones, the problem of isolating and purifying antitumor active substances from marine plants and animals has been solved. Sea anemone polypeptides that are effective against a variety of tumor cells have been prepared, with significant inhibitory effects and thermal stability, and are suitable for new drug research.
Patent Information
- Application Number
- CN202610505482.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-16
- Publication Date
- 2026-06-26
AI Technical Summary
It is difficult to isolate and purify antitumor active substances from marine plants and animals in the existing technology. In particular, there are no reports on the isolation and purification of sea anemone polypeptides, and the content of active substances is low, making it difficult to use them for the prevention and treatment of tumor diseases.
A sea anemone polypeptide with the N-terminal sequence YPDQKMFELFWLEENNA was prepared by a method of homogenization of fresh sea anemone, precipitation with acetone aqueous solution, ultrafiltration membrane ultrafiltration, dialysis, DEAE anion exchange column chromatography, Superdex 75 molecular sieve gel chromatography, and HPLC C18 reverse phase column separation and purification. The polypeptide had a molecular weight of 6027 Da and an isoelectric point of 7.25.
The prepared sea anemone polypeptide has a significant inhibitory effect on a variety of tumor cells, but no inhibitory effect on normal cells. It is thermally stable and can specifically inhibit the proliferation of cancer cells, providing a model for new drug research and development.
Smart Images
Figure CN122277669A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical science, specifically to a sea anemone polypeptide, its preparation method, and its application. Background Technology
[0002] The ocean is the source of life. Its unique high-salinity, low-temperature environment produces many bioactive substances specific to marine organisms. Obtaining natural products from marine organisms with therapeutic effects is currently a major focus of drug research, with anti-tumor drugs being particularly noteworthy. Scientists worldwide have isolated and purified numerous novel and specifically active anti-tumor compounds from marine plants and animals. However, the low concentration and difficulty in isolation and purification of these active substances in marine flora and fauna make the isolation and purification of marine anti-tumor active substances a significant challenge. Isolating a highly active compound requires large quantities of raw materials, a rational experimental route, and advanced separation and purification equipment. To date, there are no reports of using sea anemones as a raw material to isolate and purify polypeptides for the prevention and treatment of tumors. Summary of the Invention
[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sea anemone polypeptide, its preparation method, and its application. The method involves homogenizing fresh sea anemone, precipitating with acetone aqueous solution, ultrafiltration, dialyzing the crude extract, performing DEAE anion exchange column chromatography, Superdex 75 molecular sieve gel chromatography, and ultrafiltration with a 50 Da ultrafiltration membrane. The final product is then analyzed by HPLC. 18 The anemone polypeptide was purified by reverse-phase column chromatography; the obtained polypeptide showed significant inhibitory effects on various tumor cells, but did not inhibit the growth of normal cells.
[0004] To solve the problems of the prior art, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a sea anemone polypeptide, characterized in that the N-terminal sequence of the sea anemone polypeptide is YPDQKMFELFWLEENNA, the molecular weight is 6027 Da, and the isoelectric point is 7.25.
[0005] A second aspect of this invention provides a method for preparing the aforementioned sea anemone polypeptide, comprising the following steps: 1) Homogenize fresh sea anemones, precipitate them in steps at 4°C overnight using a 15%-70% acetone aqueous solution, collect the 70% acetone precipitate by centrifugation, and use an ultrafiltration membrane to filter out substances with a content greater than 5 kDa to obtain the crude extract. 2) The crude extract was placed into a dialysis bag with a molecular weight cutoff of 3.5 kD and subjected to preliminary dialysis with pure water, followed by thorough dialysis with an initial buffer solution; the initial buffer solution was 0.02 M Tris-Cl, pH 8.0, 0.05 M NaCl. 3) DEAE Sepharose™ Fast Flow anion exchange column chromatography Remove the dialyzed sample, centrifuge at 10,000 rpm to remove insoluble matter, dissolve in a small amount of 0.02 M sodium phosphate buffer, and load onto a DEAE exchange column. After loading the sample onto the column, wash with starting buffer solution. Once the non-cognate proteins have completely permeated the column, begin elution. Elute stepwise with buffers of 0.02 M Tris-Cl, pH 8.0, 0.05, 0.1, 0.15, 0.2, 0.3, and 0.5 M NaCl at a flow rate of 5-7 ml / min. Detect the activity of each component using an MTT assay. Collect the eluted fraction in the 0.02 M Tris-Cl, pH 8.0, and 0.15 M NaCl buffers as the active fraction, and store it at -20°C for later use. 4) Superdex 75 molecular sieve gel chromatography: The above eluent fractions were subjected to Superdex 75 molecular sieve gel chromatography for ultrapure elution at a flow rate of 0.9-1.5 mL / min. The eluent fractions were collected after 1 hour 40 minutes to 1 hour 50 minutes. The active components were stored at -20℃ for later use. 5) The above elution group was ultrafiltered through a 50 Da ultrafiltration membrane. The upper layer retained components were dissolved and analyzed by HPLC. 18 Reverse-phase column chromatography was used for separation and purification. Eluents A and B were used in a gradient elution process. Initially, eluent A comprised 90% of the total eluent volume, and eluent B comprised 10%. Eluent B was added at a rate of 1% per minute, with a flow rate of 0.6-0.8 mL / min. The active component appeared when eluent B comprised 21.4% of the total eluent volume, yielding a single-component sea anemone polypeptide with the N-terminal sequence YPDQKMFELFWLEENNA and a molecular weight of 6027 Da. Eluent A consisted of 0.1% TFA and 99.9% ultrapure water, while eluent B consisted of 0.1% TFA and 99.9% acetonitrile. The HPLC C... 18 The reversed-phase chromatography column has a size of 4.6 × 250 mm.
[0006] Preferably, step 3) includes the following steps: Take out the dialyzed sample, centrifuge at 10000 rpm to remove insoluble matter, and prepare for column loading; take an appropriate amount of DEAE Sepharose™ Fast Flow packing material, add it to ultrapure water, gently stir with a glass rod, guide the flow with a glass rod, and load it into a 16×26 chromatography column. Equilibrate with 3-4 column volumes of starting buffer solution. Use the column only after the baseline has stabilized; after loading the sample onto the column, rinse with starting buffer solution. Once the non-column proteins have completely permeated, begin elution. The eluent is a NaCl solution containing Tris-Cl, i.e., prepare 0.05M, 0.1M, 0.15M, 0.2M, 0.3M, and 0.5M NaCl solutions based on the starting buffer solution. Then perform stepwise elution at a flow rate of 5 ml / min. The entire chromatography process is performed at 280 nm. The protein elution peaks were detected. Finally, the fractions obtained by elution with six different concentrations of NaCl were thoroughly dialyzed with pure water, lyophilized, and dissolved in pure water. The mass concentration of each fraction was 200 μg / ml. The activity of each fraction was detected by MTT assay. The fraction eluted with a buffer of 0.02 M Tris-Cl, pH 8.0, and 0.15 M NaCl was the active fraction. The active fraction was stored at -20°C for later use.
[0007] Preferably, step 4) includes the following steps: First, take an appropriate amount of Superdex 75 molecular sieve gel packing material, soak it in water for 24 hours, stir it gently with a glass rod, guide the flow with the glass rod, and load it into a 1.6×80cm chromatography column. Equilibrate with pure water for 3-4 column volumes. The column can only be used after the baseline stabilizes. Second, load the elution fraction from the above-mentioned 0.02M Tris-Cl, pH 8.0, 0.15M NaCl buffer onto the column. Rinse the chromatography column with water at a rate of 1 ml / min. Detect the protein elution peak at 280 nm throughout the chromatography process, obtaining four elution peaks, each representing a fraction. Freeze-dry each fraction, determine the protein concentration using the BCA method, and then detect the activity of each fraction using the MTT assay. The fourth elution fraction is the active fraction, i.e., the eluent taken at 1 hour 40 minutes to 1 hour 50 minutes. Store the active fraction at -20℃.
[0008] A third aspect of this invention provides the application of the aforementioned sea anemone polypeptide in the preparation of antitumor drugs. The polypeptide obtained using the above preparation method exhibits significant inhibitory effects on various tumor cell types, but does not inhibit the growth of normal cells.
[0009] The beneficial effects of this invention are as follows: The polypeptide involved in this invention is a marine-derived substance. Due to the unique characteristics of the marine ecological environment, marine organisms possess certain active substances not found in terrestrial organisms. These active substances often exhibit novel structures, high activity, and low drug resistance. Results showed that the sea anemone polypeptide exhibited varying degrees of inhibitory effects on six types of tumor cells used in the experiment, including gastric cancer cells (SGC-7901), lung cancer cells (H460), breast cancer cells (MCF7), liver cancer cells (HepG2), pancreatic cancer cells (Panc1), and colon cancer cells (HCT8), showing a dose-dependent relationship. The inhibition rate against tumor cells was above 80%, but no inhibitory effect was observed on normal liver cells (L-02). This indicates that the polypeptide obtained in the embodiments of this invention can specifically inhibit the proliferation of cancer cells. The polypeptide was heated to 80°C during extraction, and its activity remained unchanged after heating, indicating that the polypeptide has thermal stability and can extend the shelf life of future pharmaceutical products. It can provide model structures and pharmaceutical progeny for new drug research and development. Attached Figure Description
[0010] Figure 1 This is the Tricine-SDS-Page electrophoresis pattern of the polypeptide HK6027 extracted in Example 1 of this invention. Detailed Implementation
[0011] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.
[0012] Example 1: A method for preparing sea anemone polypeptide 1) Preparation of crude extract by acetone precipitation.
[0013] Fresh sea anemones were washed, homogenized, and heated at 80°C for 30 min. The mixture was then centrifuged at 10,000 rpm for 20 min to obtain the supernatant. Three times the volume of acetone was added to the supernatant to obtain a 70% acetone solution. The solution was allowed to stand overnight at 4°C. The precipitate was then collected and redissolved. It was then ultrafiltered through a 5 kDa membrane, retaining the fraction larger than 5 kDa to obtain the crude extract for later use.
[0014] 2) Dialysis.
[0015] The crude extract was placed into a dialysis bag with a molecular cutoff of 3.5 kD and initially dialyzed with pure water, followed by thorough dialyzing with an initial buffer solution of 0.02 M Tris-Cl, pH 8.0.
[0016] 3) DEAE Sepharose™ Fast Flow anion exchange column chromatography.
[0017] Remove the dialyzed sample and centrifuge at 10,000 rpm to remove insoluble matter, preparing for column loading. Take an appropriate amount of DEAESepharose™ Fast Flow packing material (manufactured by GE), add it to ultrapure water, gently stir with a glass rod, guide the flow with the glass rod, and load it into a 16×26 chromatography column. Equilibrate with 3-4 column volumes of starting buffer solution. Use the column only after the baseline has stabilized. After loading the sample onto the column, wash with starting buffer solution. Once the non-column proteins have completely permeated, begin elution. The eluent is a Tris-hydrochloric acid buffer containing 0.05-0.5 M NaCl, pH 8.0. Prepare 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.3 M, and 0.5 M NaCl solutions based on the starting buffer. Perform stepwise elution at a flow rate of 5 ml / min. Detect the protein elution peak at 280 nm throughout the entire chromatography process. Finally, the fractions obtained by elution with six different concentrations of NaCl were thoroughly dialyzed with pure water, then freeze-dried and dissolved in pure water; the mass concentration of each fraction was 200 ug / ml, and the activity of each fraction was detected by MTT assay. The fraction eluted with 0.15M NaCl was the active fraction, which was stored at -20℃ for later use.
[0018] 4) Superdex 75 molecular sieve gel chromatography: First, take an appropriate amount of Superdex 75 molecular sieve gel packing material (manufactured by GE), soak it in water for 24 hours, stir gently with a glass rod until homogeneous, guide the flow with the glass rod, and pack it into a 1.6×80cm chromatography column. Equilibrate with pure water for 3-4 column volumes. Use the column only after the baseline has stabilized. Second, load the above 0.15M NaCl eluent onto the column, and wash the column with water at a rate of 1 ml / min. Detect the protein elution peak at 280 nm throughout the chromatography process, obtaining four elution peaks, each representing a fraction. Freeze-dry each fraction, determine the protein concentration using the BCA method, and then detect the activity of each fraction using the MTT assay. The fourth elution fraction is the active fraction, i.e., the eluent collected at 1 hour 40 minutes to 1 hour 50 minutes. Store the active fraction at -20℃.
[0019] 5) The above eluent was ultrafiltered through a 50 Da ultrafiltration membrane. The upper layer retained components were dissolved and purified using an HPLC C-18 reversed-phase column (4.6 × 250 mm column). Gradient elution was performed using solutions A and B. Initially, solution A comprised 90% of the total eluent volume, and solution B comprised 10%. Solution B was added at a rate of 1% per minute at a flow rate of 0.5 ml / min. The active component appeared when solution B comprised 21.4% of the total eluent volume. Mass spectrometry identification yielded a single component, an anemone polypeptide, with the N-terminal sequence YPDQKMFELFWLEENNA and a molecular weight of 6027 Da. (The molecular weight was determined using an ABI Q Trap ion spray mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a Turbolon Spray ion spectrometer.) (Source operated in cation mode); the isoelectric point of the active component was determined by isoelectric focusing electrophoresis, which showed to be PI=7.25. Solution A consisted of 0.1% TFA and 99.9% ultrapure water by volume, and solution B consisted of 0.1% TFA and 99.9% acetonitrile by volume.
[0020] 6) Tricine-SDS-Page electrophoresis analysis and identification of sea squirt polypeptides: Prepare a 4% stacking gel and a 16.5% separating gel. Take C... 18 Samples were spotted onto a reverse-phase column, using a low molecular weight protein marker as a control, and Tricine-SDS-Page protein electrophoresis was performed. The stacking gel was charged at 50V, and the separating gel at 60V. After electrophoresis, the sample was stained with Coomassie Brilliant Blue. A bright band of approximately 6 kDa was observed in the sample lane, indicating that the polypeptide's molecular weight is around 6 kDa (see [link to article]). Figure 1 ).
[0021] Example 2: Determination of antitumor spectrum: Cells: Human gastric cancer cells (SGC-7901), human large cell lung cancer cells (NCI-H460), human breast cancer cells (MCF-7), human liver cancer cells (Hep G2), human pancreatic cancer cells (PANC-1), human colon cancer cells (HCT-8), and human normal hepatocytes (L-02). All cells were provided by the laboratory of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.
[0022] Drugs and reagents: Sea anemone polypeptide: provided by the Food and Drug Safety Laboratory of Shandong University of Political Science and Law; 5-fluorouracil (5-FU): product of Jiangsu Nantong Jinghua Pharmaceutical Co., Ltd., batch number 100402; MTT (SIGMA, USA); DMSO (SIGMA, USA); Trypsin (SIGMA, USA); RPMI1640 (GIBCO, Invitragen Co., USA); DMEM (GIBCO, Invitragen Co., USA); Superior grade fetal bovine serum (GIBCO, Invitragen Co., USA); Premium grade fetal bovine serum (Hyclone).
[0023] Instruments: Ultra-low temperature freezer (Nature, USA), cell culture incubator (SANYO, Japan), inverted microscope (NIKON, Japan), microplate reader (Bio-Tek Instruments, Inooski, VT, USA), pH meter (Thermo Orion, USA), clean bench (FLC-Harbin Donglian Instrument Factory).
[0024] The antitumor effect was detected by the MTT assay. Various human tumor cell lines were cultured and passaged using standard methods. Cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 5 × 10⁻⁶. 4 Approximately 180 μl / ml of cell suspension was added to each well of a 96-well plate. After incubation at 37°C for 24 h, 20 μl / well of the polypeptide extract obtained in the above examples was added to each well of the experimental group, with 5-FU as a positive control. Each group had 4 parallel wells, and a water control was set up for zeroing. After incubation at 37°C for 48 h, the liquid in the 96-well plate was washed with a pipette, and 30 μl of MTT (1 mg / ml) was added to each well. The plate was incubated at 37°C for 4 h in a CO2 incubator. The supernatant was discarded, and 150 μl of DMSO was added to each well. The plate was shaken for 30 min, and the cells were detected at 492 nm using a microplate reader. The cell death rate was calculated using SPSS statistical software, and the IC50 was determined. 50 (See Table 1.)
[0025] Inhibition rate and IC50 of different concentrations of sea anemone polypeptide on tumor cells 50 Values are detailed in Table 1.
[0026] Table 1: Inhibitory effects of different concentrations of sea anemone polypeptide on tumor cell growth
[0027] Note: N / A indicates that no inhibitory effect was measured.
[0028] Experimental results showed that, according to SPSS software analysis, the sea anemone polypeptide exhibited varying degrees of inhibitory effects on six types of tumor cells used in the experiment, including gastric cancer cells (SGC-7901), lung cancer cells (H460), breast cancer cells (MCF7), liver cancer cells (HepG2), pancreatic cancer cells (Panc1), and colon cancer cells (HCT8), in a dose-dependent manner. Treatment with 10 μg / mL resulted in an inhibition rate of over 80% against tumor cells, but no inhibitory effect was observed on normal liver cells L-02. This indicates that the polypeptide obtained in this embodiment of the invention can specifically inhibit the proliferation of cancer cells.
[0029] The above description is merely an example of embodiments of the present invention and is not intended to limit the present invention in any way. The scope of protection of the present invention is defined by the claims and is not limited to the specific embodiments described above. Any simple modifications or equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the scope of protection of the present invention.
Claims
1. A sea anemone polypeptide, characterized in that, The N-terminal sequence of the sea anemone polypeptide is YPDQKMFELFWLEENNA, the molecular weight is 6027 Da, and the isoelectric point is 7.
25.
2. The method for preparing sea anemone polypeptide according to claim 1, characterized in that... Includes the following steps: 1) Homogenize fresh sea anemones, precipitate them in steps at 4°C overnight using a 15%-70% acetone aqueous solution, collect the 70% acetone precipitate by centrifugation, and use an ultrafiltration membrane to filter out substances with a content greater than 5 kDa to obtain the crude extract. 2) The crude extract was placed into a dialysis bag with a molecular weight cutoff of 3.5 kD and subjected to preliminary dialysis with pure water, followed by thorough dialysis with an initial buffer solution; the initial buffer solution was 0.02 M Tris-Cl, pH 8.0, 0.05 M NaCl. 3) DEAE Sepharose™ Fast Flow anion exchange column chromatography Remove the dialyzed sample, centrifuge at 10,000 rpm to remove insoluble matter, dissolve in a small amount of 0.02 M sodium phosphate buffer, and load onto a DEAE exchange column. After loading the sample onto the column, wash with starting buffer solution. Once the non-cognate proteins have completely permeated the column, begin elution. Elute stepwise with buffers of 0.02 M Tris-Cl, pH 8.0, 0.05, 0.1, 0.15, 0.2, 0.3, and 0.5 M NaCl at a flow rate of 5-7 ml / min. Detect the activity of each component using an MTT assay. Collect the eluted fraction in the 0.02 M Tris-Cl, pH 8.0, and 0.15 M NaCl buffers as the active fraction, and store it at -20°C for later use. 4) Superdex 75 molecular sieve gel chromatography: The above eluent fractions were subjected to Superdex 75 molecular sieve gel chromatography for ultrapure elution at a flow rate of 0.9-1.5 mL / min. The eluent fractions were collected after 1 hour 40 minutes to 1 hour 50 minutes. The active components were stored at -20℃ for later use. 5) The above elution group was ultrafiltered through a 50 Da ultrafiltration membrane. The upper layer retained components were dissolved and analyzed by HPLC. 18 Reverse-phase column chromatography was used for separation and purification. Eluents A and B were used in a gradient elution process. Initially, eluent A comprised 90% of the total eluent volume, and eluent B comprised 10%. Eluent B was added at a rate of 1% per minute, with a flow rate of 0.6-0.8 mL / min. The active component appeared when eluent B comprised 21.4% of the total eluent volume, yielding a single-component sea anemone polypeptide with the N-terminal sequence YPDQKMFELFWLEENNA and a molecular weight of 6027 Da. Eluent A consisted of 0.1% TFA and 99.9% ultrapure water, while eluent B consisted of 0.1% TFA and 99.9% acetonitrile. The HPLC C... 18 The reversed-phase chromatography column has a size of 4.6 × 250 mm.
3. The method for preparing sea anemone polypeptide according to claim 1, characterized in that... Step 3) includes the following steps: Remove the dialyzed sample, centrifuge at 10000 rpm to remove insoluble matter, and prepare for column loading; take an appropriate amount of DEAE Sepharose™ FastFlow packing material, add it to ultrapure water, gently stir with a glass rod, guide the flow with the glass rod, and load it into a 16×26 chromatography column. Equilibrate with starting buffer for 3-4 column volumes. Use only after the baseline has stabilized; after loading the sample onto the column, rinse with starting buffer. Once the non-column proteins have completely permeated, begin elution. The eluent is a NaCl solution containing Tris-Cl. Specifically, prepare 0.05M, 0.1M, 0.15M, 0.2M, 0.3M, and 0.5M NaCl solutions based on the starting buffer. Then perform stepwise elution at a flow rate of 5 ml / min. The entire chromatography process is performed at 280 nm. The protein elution peaks were detected. Finally, the fractions obtained by elution with six different concentrations of NaCl were thoroughly dialyzed with pure water, lyophilized, and dissolved in pure water. The mass concentration of each fraction was 200 μg / ml. The activity of each fraction was detected by MTT assay. The fraction eluted with a buffer of 0.02 M Tris-Cl, pH 8.0, and 0.15 M NaCl was the active fraction. The active fraction was stored at -20°C for later use.
4. The method for preparing sea anemone polypeptide according to claim 1, characterized in that... Step 4) includes the following steps: First, take an appropriate amount of Superdex 75 molecular sieve gel packing material, soak it in water for 24 hours, stir gently with a glass rod until homogeneous, guide the flow with the glass rod, and load it into a 1.6×80cm chromatography column. Equilibrate with pure water for 3-4 column volumes. Use the column only after the baseline has stabilized. Second, load the elution fraction from the above-mentioned 0.02M Tris-Cl, pH 8.0, 0.15M NaCl buffer onto the column. Rinse the column with water at a rate of 1 ml / min. Detect the protein elution peak at 280 nm throughout the chromatography process, obtaining four elution peaks, each representing a fraction. Freeze-dry each fraction, determine the protein concentration using the BCA method, and then detect the activity of each fraction using the MTT assay. The fourth elution fraction is the active fraction, i.e., the eluent taken at 1 hour 40 minutes to 1 hour 50 minutes. Store the active fraction at -20℃.
5. The application of the sea anemone polypeptide according to claim 1 in the preparation of antitumor drugs.