CN102161699B - Clam polypeptide and preparation method thereof - Google Patents
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Abstract
The invention belongs to the field of biological pharmacy, particularly relating to a clam polypeptide and a preparation method thereof. The N-tail end sequence of the clam polypeptide is IDEIQNTGGGTNFR, and the molecular weight of the clam polypeptide is 15KDa. The preparation method comprises the following steps: carrying out fractional precipitation, ultrafiltration, ion-exchange column chromatography, hydrophobic chromatography and opposite phase chromatography on a clam liquid by ammonium sulphate to obtain the polypeptide of which the molecular weight is 15KDa. The invention also relates to an application of the polypeptide in preventing and/ or treating malignant tumors. The preparation method is characterized in that the polypeptide is extracted from the clam and has higher inhibitory activity on various tumor cells. The polypeptide is continuously optimized into a polypeptide substance of which the molecular weight is 15KDa. The antitumor activity of the clam polypeptide is detected with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric method, and the compound can obviously inhibit the growth of various tumor cells but does not have an inhibition action on normal cells. The characteristic that the polypeptide can selectively inhibit the proliferation of tumor cells enables the polypeptide to have the potential to become a new anti-cancer drug.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of clam polypeptide and preparation method thereof.
Background technology
The novelty of the uniqueness of halobiontic diversity, ocean environment and the marine bioactivity structure of matter makes the ocean become the resource treasure-house of original new drug and functional/protective foods.Since the seventies in last century, people have isolated tens thousand of kinds of novel actives from marine organisms, comprise broad varietys such as peptide class, protein-based, polyose, alkaloids, terpene, Macrocyclic polyester class.Wherein, the peptide class is the hugest compounds of quantity.Now prove physiologically active such as that multiple marine peptide class has is antitumor, anti-AIDS, antimycotic, antiviral and immunomodulatory.
Malignant tumour is the disease of a class serious threat human health and life.At present, the chemotherapy of tumors technology is progressive to some extent, the tumour patient survival time obviously prolongs, particularly the treatment to leukemia, malignant lymphoma etc. has had breakthrough, but treatment the most serious, that account for the solid tumor of malignant tumour more than 90% also fails to reach satisfied effect to the harm humans life and health, and ubiquity efficient low, the poor selectivity of treatment, toxic side effect greatly, easily produces problems such as drug resistance of tumor cell.Therefore, the antitumor drug of seeking efficient, low toxicity, high specificity is still the task of top priority of pharmacological agent.
Polypeptide drug is little because of its molecular weight, activity is high, low toxin, causes the extensive concern of Chinese scholars day by day.Didemnin B is that Rinehart group separated antiviral and cyclic peptide compounds cytotoxic activity of having of acquisition in 1981 from Ascidian Trididemnum solidum, entering the I phase clinical experiment stage in 1984, is first marine drug that enters clinical study.Pharmacological experiments shows that this compound has the intensive anti-tumor activity, to L1210 leukemia cell's IC
50Reach 2ng/mL, it also has significant anti-P388 leukemia and B16 melanoma activity simultaneously.Dolastatin 10 is that Pettit group separates the linear pentapeptide that obtains from mollusk sea hare Dolabellaauricularia, has developed multiple Dolastatins derivative at present.The Dolastatins compounds can suppress microtubule polymerization, promotes its depolymerization, and then disturbs the mitotic division of tumour cell, is the growth of tumour cell inhibitor in the novel marine organisms source of a class.Experiment in vitro finds that Dolastatin 10 can induce polyclonal cellular generation apoptosis.More interesting is, finds that recently the medicine that acts on microtubule can obviously reduce tumor vascular volume of blood flow, and wherein Dolastatin 10 can reduce the volume of blood flow of tumor vessel 90%.Its concrete mechanism is still clear, may be relevant with the tumor vascular microtubule function of its influence, and this provides new tool and new approaches for the treatment of solid tumor undoubtedly.
Summary of the invention
The object of the present invention is to provide a kind of clam polypeptide and preparation method thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of clam polypeptide: described clam polypeptide N-end sequence is IDEIQNTGGGTNFR, and its molecular weight is 15KDa, and iso-electric point is 8.85.
Described clam polypeptide prepares as follows:
1) get fresh clam body fluid, centrifugal, supernatant carries out ammonium sulfate precipitation, staticly settles 12 hours at 4 ℃ at every turn;
2) gained activeconstituents precipitation in the step 1) is used water dissolution, use 50KDa and 5KDa ultrafiltration membrance filter respectively, stand-by;
3) get step 2) in the active part of molecular weight between 5-50KDa cross CM-Sepharosefast flow cationic exchange coloum, with containing the buffer solution elution that concentration is the NaCl of 0M, 0.1M, 0.5M or 2M respectively, collection contains the elution peak that concentration is the damping fluid of 0.1M NaCl, and is stand-by; Described damping fluid is 0.05M NaAc, the pH5.5 damping fluid;
4) step 3) gained active ingredient is crossed Phenyl Sepharose 6fast flow drainage column, contained the elution peak that concentration is the damping fluid of 1M NaCl with containing the buffer solution elution that concentration is the NaCl of 2.5M, 2M, 1M or 0M respectively, collecting, stand-by; Described damping fluid is 0.05M NaAc, the damping fluid of pH5.5;
5) the Resource15Q anion-exchange column of step 4) gained elution fraction with fast protein liquid chromatography (FPLC) separated, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 100% of total effluent volume in the initial elutriant, B liquid rises with the speed of per minute 1.5%, flow velocity is 1ml/min, when B liquid account for total effluent volume 5.5% the time active ingredient appears, being the N-end sequence is IDEIQNTGGGTNFR, and its molecular weight is the one-component clam polypeptide of 15KDa, and wherein A liquid is 0.02M Tris, pH9.0, B liquid is 1M NaCl, 0.02M Tris, pH9.0.
Supernatant carries out ammonium sulfate precipitation in the described step 1), is fresh clam body fluid centrifugal, collects supernatant liquor, slowly adds ammonium sulfate to 30% saturation ratio in supernatant; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 70% saturation ratio; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 95% saturation ratio, centrifugal collecting precipitation.Centrifugal condition is 10000 * g in the described step 1), centrifugal 20 minutes.
The preparation method of clam polypeptide:
1) get fresh clam body fluid, centrifugal, supernatant carries out ammonium sulfate precipitation, staticly settles 12 hours at 4 ℃ at every turn;
2) gained activeconstituents precipitation in the step 1) is used water dissolution, use 50KDa and 5KDa ultrafiltration membrance filter respectively, stand-by;
3) get step 2) in the active part of molecular weight between 5-50KDa cross CM-Sepharosefast flow cationic exchange coloum, with containing the buffer solution elution that concentration is the NaCl of 0M, 0.1M, 0.5M or 2M respectively, collection contains the elution peak that concentration is the damping fluid of 0.1M NaCl, and is stand-by; Described damping fluid is 0.05M NaAc, the pH5.5 damping fluid;
4) step 3) gained active ingredient is crossed Phenyl Sepharose 6fast flow drainage column, contained the elution peak that concentration is the damping fluid of 1M NaCl with containing the buffer solution elution that concentration is the NaCl of 2.5M, 2M, 1M or 0M respectively, collecting, stand-by; Described damping fluid is 0.05M NaAc, the damping fluid of pH5.5;
5) the Resource15Q anion-exchange column of step 4) gained elution fraction with fast protein liquid chromatography (FPLC) separated, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 100% of total effluent volume in the initial elutriant, B liquid rises with the speed of per minute 1.5%, flow velocity is 1ml/min, when B liquid account for total effluent volume 5.5% the time active ingredient appears, being the N-end sequence is IDEIQNTGGGTNFR, and its molecular weight is the one-component clam polypeptide of 15KDa, and wherein A liquid is 0.02M Tris, pH9.0, B liquid is 1M NaCl, 0.02M Tris, pH9.0.
Supernatant carries out ammonium sulfate precipitation in the described step 1), is fresh clam body fluid centrifugal, collects supernatant liquor, slowly adds ammonium sulfate to 30% saturation ratio in supernatant; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 70% saturation ratio; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 95% saturation ratio, centrifugal collecting precipitation.Centrifugal condition is 10000 * g in the described step 1), centrifugal 20 minutes.
The advantage that the present invention had:
1, polypeptide drug have that molecular weight is little, non-immunogenicity, simple in structure, active high, low toxin, therefore good application prospects is arranged at clinicing aspect.
2, the present invention extracts polypeptide from clam, and it has the active material of very high inhibition to kinds of tumor cells.Be the polypeptides matter of 15KDa by continuing to optimize it for a part amount size.Detect its anti-tumor activity with the MTT colorimetry, find that this compound can significantly suppress the growth of kinds of tumor cells, but normal cell is not had restraining effect.The characteristics that this polypeptide can selectivity suppresses tumor cell proliferation make it very potentially become new cancer therapy drug.
Description of drawings
The SDS-PAGE electrophoretic analysis figure of the clam polypeptide that Fig. 1 provides for the embodiment of the invention.
Embodiment
Embodiment 1
The preparation of clam body fluid tumor protein p53
(1) gets fresh clam, open shell and collect its body fluid, through 10000 * g centrifugal 20 minutes, remove precipitation.Slow adding has ground to form ammonium sulfate to 30% saturation ratio of fine powdered in making progress clearly according to the ammonium sulfate precipitation agent with scale, 4 ℃ left standstill 12 hours, centrifugal 20 minutes of 10000 * g, collecting precipitation and supernatant respectively, in supernatant, add ammonium sulfate to 70% saturation ratio once more, 4 ℃ left standstill 12 hours, centrifugal 20 minutes of 10000 * g, collecting precipitation and supernatant, in supernatant, add ammonium sulfate to 95% saturation ratio as stated above once more, 4 ℃ left standstill 12 hours, centrifugal 20 minutes of 10000 * g, collecting precipitation.
(2) precipitation at active ingredient place is dissolved with pure water, micro-filtration is crossed 5KDa and 50KDa ultra-filtration membrane then it is divided into three parts by molecular weight to remove insolubles.
(3) getting the active part of molecular weight between 5-50KDa dialyses to pure water, then damping fluid is fully dialysed, the sample that dialysis is good is crossed CM-Sepharose fast flow cationic exchange coloum, uses the buffer solution elution of the NaCl that contains different concns 0M, 0.1M, 0.5M, 2M respectively.Used damping fluid is 0.05M NaAc, the pH5.5 damping fluid.Each elution peak is dialysed to pure water, and lyophilize respectively takes a morsel with an amount of pure water dissolving, behind BCA method mensuration protein concentration, detect the each component activity with the MTT colorimetry, find that active ingredient is arranged in the elutriant that contains 0.1M NaCl, active constituent is placed-20 ℃ of preservations.
(4) active ingredient in the step (3) is fully dialysed with the damping fluid that contains 2.5M NaCl, the sample that dialysis is good is crossed Phenyl Sepharose 6fast flow drainage column, uses the buffer solution elution of the NaCl that contains different concns 2M, 1M, 0M respectively.Used damping fluid is 0.05M NaAc, the pH5.5 damping fluid.Each elution peak is dialysed to pure water, and lyophilize respectively takes a morsel with an amount of pure water dissolving, behind BCA method mensuration protein concentration, detect the each component activity with the MTT colorimetry, find that active ingredient is arranged in the elutriant that contains 1M NaCl, active constituent is placed-20 ℃ of preservations.
(5) with active ingredient in the step (4) with the dissolving of A liquid after, separate with the Resource 15Q anion-exchange column of fast protein liquid chromatography (FPLC), the employing gradient elution method is carried out the active ingredient wash-out.Elutriant is A liquid and B liquid, and A liquid accounts for 100% of total effluent volume in the initial elutriant, and B liquid rises with the speed of per minute 1.5%, and flow velocity is 1ml/min.Each elution peak is dialysed to pure water, and lyophilize respectively takes a morsel with an amount of pure water dissolving, behind BCA method mensuration protein concentration, the MTT colorimetry detects the each component activity, find when B liquid account for total effluent volume 5.5% the time active ingredient appears, active ingredient is placed-20 ℃ of preservations.Used A liquid is 0.02M Tris, and pH9.0, B liquid are 1MNaCl, 0.02M Tris, pH9.0.
(6) active ingredient in the step (5) is dissolved with A liquid, detect purity through high performance liquid chromatography (HPLC) C-18 reversed-phase column.Elutriant is: A liquid is the 0.1%TFA+99.9% ultrapure water, and B liquid is the 0.1%TFA+99.9% acetonitrile, carries out gradient elution.Initial concentration is an A liquid 90%, and B liquid 10%, B liquid concentration per minute rise 1%, and flow velocity is 0.5ml/min, when B liquid account for total effluent volume 43.5% the time elution peak appears.Elution peak is a simple spike, illustrates that this component is an one-component.This component N-end sequence is determined as IDEIQNTGGGTNFR, and iso-electric point is determined as PI=8.85 by isoelectric focusing electrophoresis.
(7) this one-component being carried out SDS-PAGE identifies.Get this one-component and carry out electrophoresis, at the albumen Marker of 14.4-116KDa in contrast with molecular weight ranges.5% concentrated glue adopts 80V voltage, and 15% separation gel adopts 120V voltage.Electrophoresis dyes with coomassie brilliant blue staining liquid after finishing.After 6 hours, high-visible until protein band with the destainer decolouring.Found that single protein band (referring to Fig. 1) is arranged about 15KDa.
Embodiment 2: clam polypeptide extracorporeal suppression tumor cell activity
The anti-tumor activity experiment:
Human lung carcinoma cell (A549), human liver cancer cell (BEL-7402), human cervical carcinoma's epithelial cell (Hela), human colon cancer cell (HCT-116), human breast cancer cell (MCF-15), human pancreatic cancer cell (ASPC-1) can be used to estimate the active strong and weak of antitumor drug, easy and simple to handle, favorable reproducibility.Experimental technique (mtt assay):
Tetrazolium (MTT) is a kind of dyestuff that can accept hydrogen atom.Desaturase relevant with NADH in the plastosome of viable cell can change into xanchromatic MTT insoluble hepatic Jia Za (formazan) in cell, dead cell does not then have this function.Formazan crystalline growing amount is directly proportional with viable count, and this crystalline DMSO solution has maximum absorption band in 570 nanometers, so, can estimate the influence of medicine on cell proliferation by detecting formazan crystalline amount.
Various cell strains are according to the ordinary method cultivation of going down to posterity.The cell of taking the logarithm vegetative period is regulated cell density to 5 * 10
4Individual cells/ml is inoculated in the 96 porocyte culture plates by every hole 180 microlitres, in 37 ℃, cultivates 24h in the incubator of 5% carbonic acid gas.Sample is the clam polypeptide that the foregoing description prepares gained, and it sets 5 concentration gradients, is 15,30,45,60,75 μ g/ml, and each concentration is established four parallel holes.Positive controls is 5-FU, and negative control group is not for containing the substratum of sample.Every hole adds sample or contrasts 20 microlitres.Cultivate after 48 hours, every hole adds MTT 50 microlitres that concentration is 5mg/ml, continues to cultivate 4 hours, removes supernatant liquor.Every hole adds DMSO 150 microlitres, and concussion is 15 minutes on micro-oscillator, after dissolving fully to crystallization, measures the light absorption value (OD value) of each hole in 570 nanometers with microplate reader.The average OD value of getting four parallel holes is IR%=(OD by formula
Negative control-OD
Sample)/OD
Negative controlThe inhibiting rate of * 100% calculation sample on cell proliferation (IR%).Experimental result shows, active ingredient can strongly inhibited human lung carcinoma cell (A549) when 45ug/ml concentration, the propagation of human liver cancer cell (BEL-7402), human cervical carcinoma's epithelial cell (Hela), human colon cancer cell (HCT-116), human breast cancer cell (MCF-15), human pancreatic cancer cell (ASPC-1), inhibiting rate reaches 40%-80%, but people's normal breast epithelial cell (MCF-10A), mouse fibroblast cell (NIH/3T3) are not all had restraining effect, illustrate that this material can optionally suppress the propagation of tumour cell.
Claims (4)
1. clam polypeptide, it is characterized in that: described clam polypeptide N-end sequence is IDEIQNTGGGTNFR, and its molecular weight is 15KDa, and iso-electric point is 8.85;
Described clam polypeptide prepares as follows:
1) get fresh clam body fluid, centrifugal, supernatant carries out ammonium sulfate precipitation, staticly settles 12 hours at 4 ℃ at every turn;
2) gained activeconstituents precipitation in the step 1) is used water dissolution, use 50KDa and 5KDa ultrafiltration membrance filter respectively, stand-by;
3) get step 2) in the active part of molecular weight between 5-50KDa cross CM-Sepharose fast flow cationic exchange coloum, with containing the buffer solution elution that concentration is the NaCl of 0M, 0.1M, 0.5M or 2M respectively, collection contains the elution peak that concentration is the damping fluid of 0.1M NaCl, and is stand-by; Described damping fluid is 0.05M NaAc, the pH5.5 damping fluid;
4) step 3) gained active ingredient is crossed Phenyl Sepharose6fas t flow drainage column, contained the elution peak that concentration is the damping fluid of 1M NaCl with containing the buffer solution elution that concentration is the NaCl of 2.5M, 2M, 1M or 0M respectively, collecting, stand-by; Described damping fluid is 0.05M NaAc, the damping fluid of pH5.5;
5) the Resource15Q anion-exchange column of step 4) gained elution fraction with fast protein liquid chromatography (FPLC) separated, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 100% of total effluent volume in the initial elutriant, B liquid rises with the speed of per minute 1.5%, flow velocity is 1ml/min, when B liquid account for total effluent volume 5.5% the time active ingredient appears, being the N-end sequence is IDEIQNTGGGTNFR, and its molecular weight is the one-component clam polypeptide of 15KDa, and wherein A liquid is 0.02M Tris, pH9.0, B liquid is 1M NaCl, 0.02M Tris, pH9.0;
Supernatant carries out ammonium sulfate precipitation in the described step 1), is fresh clam body fluid centrifugal, collects supernatant liquor, slowly adds ammonium sulfate to 30% saturation ratio in supernatant; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 70% saturation ratio; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 95% saturation ratio, centrifugal collecting precipitation.
2. by the described clam polypeptide of claim 1, it is characterized in that: centrifugal condition is 10000 * g in the described step 1), centrifugal 20 minutes.
3. the preparation method of the described clam polypeptide of claim 1, it is characterized in that: the clam polypeptide prepares as follows:
1) get fresh clam body fluid, centrifugal, supernatant carries out ammonium sulfate precipitation, staticly settles 12 hours at 4 ℃ at every turn;
2) gained activeconstituents precipitation in the step 1) is used water dissolution, use 50KDa and 5KDa ultrafiltration membrance filter respectively, stand-by;
3) get step 2) in the active part of molecular weight between 5-50KDa cross CM-Sepharosefast flow cationic exchange coloum, with containing the buffer solution elution that concentration is the NaCl of 0M, 0.1M, 0.5M or 2M respectively, collection contains the elution peak that concentration is the damping fluid of 0.1M NaCl, and is stand-by; Described damping fluid is 0.05M NaAc, the pH5.5 damping fluid;
4) step 3) gained active ingredient is crossed Phenyl Sepharose6fast flow drainage column, contained the elution peak that concentration is the damping fluid of 1M NaCl with containing the buffer solution elution that concentration is the NaCl of 2.5M, 2M, 1M or 0M respectively, collecting, stand-by; Described damping fluid is 0.05M NaAc, the damping fluid of pH5.5;
5) the Resource15Q anion-exchange column of step 4) gained elution fraction with fast protein liquid chromatography (FPLC) separated, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 100% of total effluent volume in the initial elutriant, B liquid rises with the speed of per minute 1.5%, flow velocity is 1ml/min, when B liquid account for total effluent volume 5.5% the time active ingredient appears, being the N-end sequence is IDEIQNTGGGTNFR, and its molecular weight is the one-component clam polypeptide of 15KDa, and wherein A liquid is 0.02M Tris, pH9.0, B liquid is 1M NaCl, 0.02M Tris, pH9.0;
Supernatant carries out ammonium sulfate precipitation in the described step 1), is fresh clam body fluid centrifugal, collects supernatant liquor, slowly adds ammonium sulfate to 30% saturation ratio in supernatant; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 70% saturation ratio; Centrifugal, collect supernatant liquor, in supernatant, slowly add ammonium sulfate to 95% saturation ratio, centrifugal collecting precipitation.
4. the preparation method of the described clam polypeptide of claim 3, it is characterized in that: centrifugal condition is 10000 * g in the described step 1), centrifugal 20 minutes.
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| CN106916206A (en) * | 2015-12-25 | 2017-07-04 | 无限极（中国）有限公司 | Hard clam polypeptide and preparation method and application |
| CN106916868A (en) * | 2015-12-25 | 2017-07-04 | 无限极（中国）有限公司 | A kind of white clam polypeptide with anti-oxidant in vivo and neuroprotective function and preparation method and application |
| CN107522770A (en) * | 2017-10-25 | 2017-12-29 | 贵州大学 | Aspongopus medicinal ingredient isolation and purification method |
| CN114134191B (en) * | 2020-09-04 | 2023-12-12 | 琛蓝(美国)营养制品股份有限公司 | Preparation method and application of anti-inflammatory kidney-protecting clam peptide |
| CN113817792B (en) * | 2021-11-25 | 2022-04-01 | 山东方明药业集团股份有限公司 | Application of marine biological polypeptide in preparation of medicine for treating osteoporosis |
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| 张玉艳.文蛤抗肿瘤多肽的分离纯化及其作用机理的研究.《中国博士学位论文全文数据库医药卫生科技辑》.2010,(第10期),全文. |
| 文蛤多肽的分离纯化及抗肿瘤机制研究;王翠翠;《中国博士学位论文全文数据库医药卫生科技辑》;20110815(第08期);全文 * |
| 文蛤抗肿瘤多肽的分离纯化及其作用机理的研究;张玉艳;《中国博士学位论文全文数据库医药卫生科技辑》;20101015(第10期);全文 * |
| 王翠翠.文蛤多肽的分离纯化及抗肿瘤机制研究.《中国博士学位论文全文数据库医药卫生科技辑》.2011,(第08期),全文. |
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