CN102125686B - Application of hard clam polypeptide - Google Patents
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- CN102125686B CN102125686B CN 201110036543 CN201110036543A CN102125686B CN 102125686 B CN102125686 B CN 102125686B CN 201110036543 CN201110036543 CN 201110036543 CN 201110036543 A CN201110036543 A CN 201110036543A CN 102125686 B CN102125686 B CN 102125686B
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Abstract
The invention relates to the field of biochemicals and biomedicines, in particular relates to an application of hard clam polypeptide. The hard clam polypeptide is applied to prepare medicaments for preventing and/or treating malignant tumors. According to the invention, a polypeptide Mere15 is isolated from hard clams and has an antitumor effect. The studies show that the polypeptide can induce the cell cycle arrest at the G2/M phase in the lung cancer cell line A 549 in vitro and has a significant cytotoxic activity against other tumor cells. Compared with the chemotherapeutic medicaments in the prior art, the hard clam polypeptide as antitumor peptide medicament has the advantages of less toxic and adverse effects, small possibility of drug resistance and the like.
Description
Technical field
The present invention relates to biochemistry and biomedicine field, specifically a kind of application of hard clam polypeptide.
Background technology
Malignant tumor is that a kind of sickness rate is high, the disease of serious threat human health.Since 20th century middle and late stage, cancer morbidity presents the trend of rising always.In the tumor therapeuticing method of classics, chemotherapeutics plays an important role always, and has obtained sizable progress, but still has the shortcomings such as poor selectivity, toxic and side effects be large.Therefore, clinically in the urgent need to the antitumor drug of development with high, the high specificity of exploitation selectivity, determined curative effect.
The ocean be covered with earth surface long-pending 71%, be the cradle of life, be also the living marine resources storehouse that has a high potential.According to statistics, the biological species number of ocean accounts for 80% of tellurian sum considerably beyond land, amounts to approximately 30 more than 50 ten thousand kinds, and this species diversity has consisted of the basis of marine pharmaceutical resource Chemical Diversity.In addition, marine environment has the advantages that high salt, high pressure, low temperature, low light photograph, oligotrophic etc. are different from terrestrial environment.For adapting to this special ecological environment, marine organisms not only come in every shape, and have produced the metabolic system different from terrestrial life and body defending system.In its metabolism and growth process, produced and accumulated the active substance of a large amount of novel structures, function uniqueness.Therefore, the novelty of the uniqueness of halobiontic multiformity, marine environment and the marine bioactivity structure of matter makes the ocean become the source that enriches of original new drug.
Conch Meretricis seu Cyclinae Meretrix meretrix Linnaeus is the Veneridae Mollusca, is the important seashells resource of China, utilizes Conch Meretricis seu Cyclinae treatment tumor to have a long history in China.Shennong's Herbal record: " Conch Meretricis seu Cyclinae master malignant boil ", have the effect of hard masses softening and resolving, strengthening vital QI to eliminate pathogenic factors, can moisten five lungs, only quench one's thirst, open taste, be used for the treatment of pulmonary tuberculosis, hypertension, tumor, tinnitus due to deficiency of the kidney.Also there is Conch Meretricis seu Cyclinae to remove the record of tumor in the essentials of Matea Medica of the Wang Ang of the Qing Dynasty.In recent years, Chinese scholar has been carried out a series of researchs for Conch Meretricis seu Cyclinae.Cold ripple etc. with the Conch Meretricis seu Cyclinae homogenate through ethanol extracting, molecular sieve filtration chromatography, the polypeptide that it is 3147.41Da that purification obtains a molecular weight, this polypeptide is in the external propagation that can significantly suppress stomach cancer cell BGC-823.China Medicine University opens platinum and has obtained glycopeptide MGP0405 by method purification from Conch Meretricis seu Cyclinae such as ultrafiltration, macroporous absorption chromatography, gel permeation chromatographies, and it has inhibitory action to human nasopharyngeal carcinoma KB cell.
Summary of the invention
The object of the invention is to provide a kind of application of hard clam polypeptide.
For achieving the above object, the technical solution used in the present invention is:,
A kind of application of hard clam polypeptide: hard clam polypeptide prevents and/or treats application in malignant tumor medicine in preparation.
Described hard clam polypeptide prevents and/or treats application in human lung carcinoma cell, human liver cancer cell, human cervical carcinoma's epithelial cell, human colon cancer cell, human breast cancer cell or human pancreatic cancer cell medicine in preparation.Described hard clam polypeptide comes inhibition tumor cell propagation by the inducing cell Cycle Arrest.Described hard clam polypeptide comes the inducing cell Cycle Arrest by suppressing microtubule polymerization.
Described hard clam polypeptide prepares as follows:
1) get fresh Conch Meretricis seu Cyclinae body fluid, centrifugal, supernatant carries out ammonium sulfate precipitation, staticly settles 12 hours at 4 ℃ at every turn;
2) with step 1) in gained active component precipitation use water dissolution, use respectively 50KDa and 5KDa ultrafiltration membrance filter, stand-by;
3) get step 2) in the active part of molecular weight between 5-50KDa cross CM-Sepharosefast flow cation exchange column, with containing respectively the buffer solution elution that concentration is the NaCl of 0M, 0.1M, 0.5M or 2M, collection contains the eluting peak that concentration is the buffer of 0.1M NaCl, and is stand-by; Described buffer is 0.05M NaAc, the pH5.5 buffer;
4) with step 3) the gained active component crosses Phenyl Sepharose 6 fast flow drainage columns, contains with containing respectively the buffer solution elution that concentration is the NaCl of 2.5M, 2M, 1M or 0M, collecting the eluting peak that concentration is the buffer of 1M NaCl, and is stand-by; Described buffer is 0.05M NaAc, the buffer of pH5.5;
5) with step 4) the gained elution fraction separates with the Resource15Q anion-exchange column of fast protein liquid chromatography (FPLC), eluent is A liquid and B liquid, carry out gradient elution, in initial eluent, A liquid accounts for 100% of total effluent volume, B liquid rises with per minute 1.5% speed, flow velocity is 1ml/min, when B liquid account for total effluent volume 5.5% the time active component appears, being the N-end sequence is IDEI QNTGGGTNFR, its molecular weight is the one-component hard clam polypeptide of 15KDa, wherein A liquid is 0.02M Tris, pH9.0, B liquid is 1M NaCl, 0.02M Tris, pH9.0.
Described step 1) in, supernatant carries out ammonium sulfate precipitation, is fresh Conch Meretricis seu Cyclinae body fluid centrifugal, collects supernatant, slowly adds ammonium sulfate to 30% saturation in supernatant; Centrifugal, collect supernatant, slowly add ammonium sulfate to 70% saturation in supernatant; Centrifugal, collect supernatant, slowly add ammonium sulfate to 95% saturation, centrifugal collecting precipitation in supernatant.Described step 1) in, centrifugal condition is 10000 * g, centrifugal 20 minutes.
The present invention has advantages of:
1, the high salt of marine environment, high pressure, low temperature, low light photograph, oligotrophic characteristics are active substances that marine organisms had produced and accumulated a large amount of novel structures, function uniqueness.
2, Conch Meretricis seu Cyclinae is as a kind of nutritious seashells food, and its foreseeable safety has extensively and the potentiality of long-term prescription it.
3, the present invention separates the polypeptide Mere15 that obtains having antitumor action from Conch Meretricis seu Cyclinae, experiment confirm, G2/M phase cell cycle arrest occurs at the external human lung carcinoma cell line A549 that can induce in this polypeptide, and other kinds of tumor cells are all had significant cytotoxic activity.Compare with existing chemotherapeutics, antineoplastic polypeptide class medicine has the advantages such as molecular weight is little, non-immunogenicity, simple in structure, activity is high, toxic and side effects is little, difficult generation drug resistance.
Description of drawings
Fig. 1 is the SDS-PAGE electrophoretic analysis figure of the hard clam polypeptide of the embodiment of the present invention 1 preparation.
The affect figure of the antitumor hard clam polypeptide that Fig. 2 provides for the embodiment of the present invention on the lung cell A549 cell cycle, wherein A. does not add the A549 cell that the hard clam polypeptide of embodiment 1 preparation gained is processed; The A549 cell that the hard clam polypeptide of B, 30 μ g/ml embodiment 1 preparation gained is processed; The A549 cell that the hard clam polypeptide of C, 60 μ g/ml embodiment 1 preparation gained is processed.
The affect figure of the antitumor hard clam polypeptide that Fig. 3 provides for the embodiment of the present invention on the lung cell A549 tubulin polymerization, wherein the A549 cell processed for adding embodiment 1 preparation gained hard clam polypeptide of A; The A549 cell that the hard clam polypeptide of B, 30 μ g/ml embodiment 1 preparation gained is processed; The A549 cell that the hard clam polypeptide of C, 60 μ g/ml embodiment 1 preparation gained is processed.
The specific embodiment
The preparation of Conch Meretricis seu Cyclinae body fluid antineoplastic polypeptide
(1) get fresh Conch Meretricis seu Cyclinae, open shell and collect its body fluid, through 10000 * g centrifugal 20 minutes, remove precipitation.Slowly add ammonium sulfate to 30% saturation that grinds to form fine powdered in making progress clearly according to the ammonium sulfate precipitation agent with scale, 4 ℃ standing 12 hours, centrifugal 20 minutes of 10000 * g, respectively collecting precipitation and supernatant, again add ammonium sulfate to 70% saturation in supernatant, 4 ℃ standing 12 hours, centrifugal 20 minutes of 10000 * g, collecting precipitation and supernatant, again add ammonium sulfate to 95% saturation in supernatant as stated above, 4 ℃ standing 12 hours, centrifugal 20 minutes of 10000 * g, collecting precipitation.
(2) precipitation at active component place is dissolved with pure water, then microfiltration is crossed 5KDa and 50KDa ultrafilter membrane it is divided into three parts by molecular weight to remove insoluble matter.
(3) getting the active part of molecular weight between 5-50KDa dialyses to pure water, then buffer is fully dialysed, the sample that dialysis is good is crossed CM-Sepharose fast flow cation exchange column, uses respectively the buffer solution elution of the NaCl that contains variable concentrations 0M, 0.1M, 0.5M, 2M.Buffer used is 0.05M NaAc, the pH5.5 buffer.Each eluting peak is dialysed to pure water, and lyophilization respectively takes a morsel with appropriate pure water dissolving, after measuring protein concentration with the BCA method, active with MTT colorimetric determination each component, find that active component is arranged in the eluent that contains 0.1M NaCl, active constituent is placed in-20 ℃ of preservations.
(4) active component in step (3) is fully dialysed with the buffer that contains 2.5M NaCl, the sample that dialysis is good is crossed Phenyl Sepharose 6fast flow drainage column, uses respectively the buffer solution elution of the NaCl that contains variable concentrations 2M, 1M, 0M.Buffer used is 0.05M NaAc, the pH5.5 buffer.Each eluting peak is dialysed to pure water, and lyophilization respectively takes a morsel with appropriate pure water dissolving, after measuring protein concentration with the BCA method, active with MTT colorimetric determination each component, find that active component is arranged in the eluent that contains 1M NaCl, active constituent is placed in-20 ℃ of preservations.
(5) with active component in 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 component eluting.Eluent is A liquid and B liquid, and in initial eluent, A liquid accounts for 100% of total effluent volume, and B liquid rises with per minute 1.5% speed, and flow velocity is 1ml/min.Each eluting peak is dialysed to pure water, and lyophilization respectively takes a morsel with appropriate pure water dissolving, after measuring protein concentration with the BCA method, MTT colorimetric determination each component is active, find when B liquid account for total effluent volume 5.5% the time active component appears, active component is placed in-20 ℃ of preservations.A liquid used is 0.02M Tris, and pH9.0, B liquid are 1MNaCl, 0.02M Tris, pH9.0.
(6) active component in step (5) is dissolved with A liquid, detect purity through high performance liquid chromatography (HPLC) C-18 reversed-phase column.Eluent is: A liquid is the 0.1%TFA+99.9% ultra-pure water, and B liquid is the 0.1%TFA+99.9% acetonitrile, carries out gradient elution.Initial concentration is A liquid 90%, B liquid 10%, the rising 1% per minute of B liquid concentration, flow velocity is 0.5ml/min, when B liquid account for total effluent volume 43.5% the time eluting peak appears.Eluting peak is simple spike, illustrates that this component is one-component.This component N-end sequence is determined as IDEIQNTGGGTNFR, and isoelectric point, IP 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, with molecular weight ranges at the albumen Marker of 14.4-116KDa in contrast.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, decolour until protein band is high-visible with destaining solution.Found that single protein band (referring to Fig. 1) is arranged about 15KDa.
Mere15 is in the detection of extracorporeal suppression tumor cell propagation
Cell: 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).
Mtt assay: various cell strains are according to the conventional method cultivation of going down to posterity.The take the logarithm cell of trophophase is regulated cell density to 5 * 10
4Individual/ml is inoculated in 96 porocyte culture plates by every hole 180 μ l, in 37 ℃, cultivates 24h in the incubator of 5% carbon dioxide.Sample is that above-described embodiment prepares the gained hard clam polypeptide, and sample is set 5 Concentraton gradient, i.e. 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 culture medium of sample.Every hole adds sample or contrasts 20 μ l.Cultivate after 48 hours, it is the MTT 50 μ l of 5mg/ml that every hole adds concentration, continues to cultivate 4 hours, removes supernatant.Every hole adds DMSO 150 μ l, and concussion is 15 minutes on micro-oscillator, after dissolving fully to crystallization, measures each hole at the light absorption value (OD value) of 570 nanometers with microplate reader.The average OD value of getting four parallel holes is IR%=(OD by formula
Negative right According to-OD
Sample)/OD
Negative controlThe suppression ratio of * 100% calculation sample on cell proliferation (IR%), and calculate its half-inhibition concentration IC
50
Experimental result: Mere15 is to 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) half-inhibition concentration IC
50Referring to table 1.
The half-inhibition concentration IC of table 1Mere15 to A-549, BEL-7402, Hela, HCT-116, MCF-15 and ASPC-1
50
Experimental example 3 flow cytometers detect cell cycle to be changed:
Cell cycle analysis: the A549 cell of the trophophase of taking the logarithm, regulate cell density to 5 * 10
4Individual/ml is inoculated in 6 porocyte culture plates by every hole 2ml, in 37 ℃, cultivates in the incubator of 5% carbon dioxide.Add above-mentioned embodiment after 24h in 6 porocyte culture plates and prepare the gained hard clam polypeptide, its Concentraton gradient is 0,30,60 μ g/ml.The A549 cell of effect after 12h used 0.25% trypsinization, 1000r/min then, and 4 ℃ of centrifugal 5min, making density is 5 * 10
5～1 * 10
6The single cell suspension of individual/ml, the DNA-Prep test kit that uses Beckman Coulter company to produce adds DNA-Prep LPR 100 μ l, mixing several seconds, add again DNA-Prep Stain Reagent500 μ l, room temperature lucifuge 15min after mixing, the up flow type cell instrument detects.
By comparing the untreated fish group cell and adding above-described embodiment to prepare the cell cycle of gained hard clam polypeptide processed group cell, can find that the G2/M phase that above-described embodiment prepares gained hard clam polypeptide processed group obviously increases, this shows that cell proliferation may be arrested in the G2/M phase.And, along with the increase of concentration for the treatment of, the also corresponding increase (referring to Fig. 2) of ratio that the G2/M phase is shared.
The impact of experimental example 4 indirect IF staining methods on the polymerization state of α-tubulin:
Microtubule dyeing: the A549 cell of exponential phase is digested, blow and beat into single cell suspension and counting, be diluted to 5 * 10 with culture medium
4The cell suspension of individual/ml.Acid soak is cleaned and autoclaved coverslip is put into 6 porocyte culture plates bottoms, cell suspension is added in 6 porocyte culture plates, every hole 2mL is placed in 37 ℃ of constant incubators and cultivates.Add above-mentioned embodiment after 24h in 6 porocyte culture plates and prepare the gained hard clam polypeptide, its Concentraton gradient is 0,30,60 μ g/ml.。After 1h, the sucking-off culture medium, every hole adds the paraformaldehyde of 1mL 2%, and room temperature is 10min fixedly.The sucking-off fixative, every hole adds 1mL PBS, the standing 5min of room temperature, repeated washing is once.Add 1mL PBS/FBS (9: 1), the standing 10-20min of room temperature.With 0.1%TritonX-100/PBS/FBS by 1: 500 dilution proportion primary antibodie and two anti-.Primary antibodie is dripped hatch 1h on coverslip, clean 3 times with PBS/FBS, each 5min; Two anti-dripping are hatched 1h on coverslip, clean 3 times with PBS/FBS, each 5min.Be placed in the fluorescence microscopy Microscopic observation.
The indirect IF staining method is found the polymerization state research of α-tubulin, α-tubulin is through primary antibodie and two anti-immunofluorescence dyeings, the tubulin polymerization of untreated fish group cell is normal, and the polymerization that adds above-described embodiment to prepare the tubulin of gained hard clam polypeptide processed group cell is interfered (referring to Fig. 3).
Claims (5)
1. the application of a hard clam polypeptide is characterized in that: the application of hard clam polypeptide in preparation treatment human lung carcinoma cell, human liver cancer cell, human cervical carcinoma's epithelial cell, human colon cancer cell, human breast cancer cell or human pancreatic cancer cell medicine;
Described hard clam polypeptide extracts as follows:
1) get fresh Conch Meretricis seu Cyclinae body fluid, centrifugal, supernatant carries out ammonium sulfate precipitation, staticly settles 12 hours at 4 ℃ at every turn;
2) with step 1) in gained active component precipitation use water dissolution, use respectively 50KDa and 5KDa ultrafiltration membrance filter, stand-by;
3) get step 2) in the active part of molecular weight between 5-50KDa cross CM-Sepharose fast flow cation exchange column, with containing respectively the buffer solution elution that concentration is the NaCl of 0M, 0.1M, 0.5M or 2M, collection contains the eluting peak that concentration is the buffer of 0.1M NaCl, and is stand-by; Described buffer is 0.05M NaAc, pH 5.5 buffer;
4) with step 3) the gained active component crosses Phenyl Sepharose 6 fast flow drainage columns, contains with containing respectively the buffer solution elution that concentration is the NaCl of 2.5M, 2M, 1M or 0M, collecting the eluting peak that concentration is the buffer of 1M NaCl, and is stand-by; Described buffer is 0.05M NaAc, the buffer of pH5.5;
5) with step 4) the gained elution fraction separates with the Resource15Q anion-exchange column of fast protein liquid chromatography (FPLC), eluent is A liquid and B liquid, carry out gradient elution, in initial eluent, A liquid accounts for 100% of total effluent volume, B liquid rises with per minute 1.5% speed, flow velocity is 1ml/min, when B liquid account for total effluent volume 5.5% the time active component appears, being the N-end sequence is IDEIQNTGGGTNFR, its molecular weight is the one-component hard clam polypeptide of 15KDa, wherein A liquid is 0.02M Tris, pH9.0, B liquid is 1M NaCl, 0.02M Tris, pH9.0.
2. by the application of hard clam polypeptide claimed in claim 1, it is characterized in that: described hard clam polypeptide comes inhibition tumor cell propagation by the inducing cell Cycle Arrest.
3. by the application of hard clam polypeptide claimed in claim 1, it is characterized in that: described hard clam polypeptide comes the inducing cell Cycle Arrest by suppressing microtubule polymerization.
4. by the application of hard clam polypeptide claimed in claim 1, it is characterized in that: described step 1), supernatant carries out ammonium sulfate precipitation, is fresh Conch Meretricis seu Cyclinae body fluid centrifugal, collects supernatant, slowly adds ammonium sulfate to 30% saturation in supernatant; Centrifugal, collect supernatant, slowly add ammonium sulfate to 70% saturation in supernatant; Centrifugal, collect supernatant, slowly add ammonium sulfate to 95% saturation, centrifugal collecting precipitation in supernatant.
5. by the application of hard clam polypeptide claimed in claim 4, it is characterized in that: described step 1), centrifugal condition is 10000 * g, centrifugal 20 minutes.
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| CN1451393A (en) * | 2002-04-19 | 2003-10-29 | 清华大学 | Clam extract, and preparing process and use thereof |
| CN101496817A (en) * | 2008-12-30 | 2009-08-05 | 厦门大学 | Application of clam active polypeptide in preparing anti-lung cancer medicament |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1451393A (en) * | 2002-04-19 | 2003-10-29 | 清华大学 | Clam extract, and preparing process and use thereof |
| CN101496817A (en) * | 2008-12-30 | 2009-08-05 | 厦门大学 | Application of clam active polypeptide in preparing anti-lung cancer medicament |
Non-Patent Citations (2)
| Title |
|---|
| 康劲翮等.文蛤多肽体外对人肺癌A549细胞的抑制作用.《台湾海峡》.2009,第28卷(第04期), * |
| 张剑等.文蛤多肽对体外培养宫颈癌Hela细胞的抑制作用.《厦门大学学报(自然科学版)》.2009,第48卷(第05期), * |
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