CN102174096B - Ciona intestinalis polypeptide and preparation method thereof - Google Patents
Ciona intestinalis polypeptide and preparation method thereof Download PDFInfo
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- CN102174096B CN102174096B CN 201010620668 CN201010620668A CN102174096B CN 102174096 B CN102174096 B CN 102174096B CN 201010620668 CN201010620668 CN 201010620668 CN 201010620668 A CN201010620668 A CN 201010620668A CN 102174096 B CN102174096 B CN 102174096B
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Abstract
The invention belongs to the filed of pharmaceutical sciences and particularly relates to ciona intestinalis polypeptide and a preparation method thereof. The ciona intestinalis polypeptide has the N- terminal sequence of YQPDKFMLRGELRNN, the molecular weight of 5931Da and the isoelectric point of 7.25. The preparation method of the ciona intestinalis polypeptide comprises the following steps: precipitating fresh ciona intestinalis by using 10-90% acetone, dialyzing and desalting active components, and purifying by adopting ion-exchange chromatography, gel filtration chromatography and HPLC (high-performance liquid chromatography) to obtain the ciona intestinalis polypeptide which has the molecular weight of 5931Da. The in vivo and in vitro activity experiments verify and confirm that theapplication of the ciona intestinalis polypeptide to malignant tumor prevention and treatment.
Description
Technical field
The present invention relates to the pharmaceutical science field, specifically a kind of Ciona polypeptide and preparation thereof.
Background technology
Marine drug is the big hot fields of one in the present drug research, wherein noticeable with the achievement in research of antitumor drug, the various countries scientist from marine animal and plant separation and purification go out multiple novel structure and have the antineoplastic compound of specific activity, be a great problem that the separation and purification of ocean anti-tumor active substance faces but the content of active substance in marine animal and plant is low, separation and purification is difficult.Separate the separation and purification equipment that a highly active compound needs great deal of raw materials, rational test course and advanced person that obtains.Be not raw material separation and purification polypeptides matter and be used for prevention and the report for the treatment of tumor disease seeing with the Ciona up to now.
Summary of the invention
The object of the invention is to provide a kind of Ciona polypeptide and preparation thereof.
For achieving the above object, the technical solution adopted in the present invention is:
A kind of Ciona polypeptide: Ciona polypeptide N-end sequence is YQPDKFMLRGELRNN, and molecular weight is 5931Da, and iso-electric point is 7.25.
Described Ciona polypeptide obtains as follows:
1) fresh Ascidian is precipitated extraction 1-3 time by organic solvent with 4-20 ℃, throw out carries out ultrafiltration by filter membrane and holds back the material greater than 5KDa, and is stand-by;
2) hold back the material greater than 5KDa with above-mentioned, carry out the DEAE anion exchange chromatography with the NaCl damping fluid of 0.05-0.5M as elutriant, collect 0.15M NaCl buffer solution elution component, stand-by;
3) with the elution fraction of above-mentioned collection through Superdex75 molecular sieve gel chromatography, do elutriant with ultrapure water, flow velocity is 0.8-1.2ml/min, collects elution fraction, and is stand-by;
4) with above-mentioned elution fraction through the separation and purification of HPLC C-18 reversed-phase column, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 90% of total effluent volume in the initial elutriant, and B liquid accounts for 10% of total effluent volume, and the speed of B liquid per minute 1% rises, flow velocity is 0.5-0.8ml/min, when B liquid account for total effluent volume 28.4% the time active ingredient appears, namely obtaining the N-end sequence is YQPDKFMLRGELRNN, molecular weight is the one-component Ciona polypeptide of 5931Da; Wherein A liquid is the TFA of A liquid cumulative volume 0.1% and the ultrapure water of A liquid cumulative volume 99.9%, and B liquid is the TFA of B liquid cumulative volume 0.1% and the acetonitrile of B liquid cumulative volume 99.9%.
It is the aqueous acetone solution of 10%-90% that described step 1) adopts volumn concentration during by organic solvent deposit, and each sedimentation time is 12 hours.
It is the aqueous acetone solution of 60%-80% that described step 1) adopts volumn concentration during by organic solvent deposit.Described polypeptide is that N-end sequence and its homology are at the variant or derivatives thereof more than 95%.
The preparation method of Ciona polypeptide: 1) fresh Ascidian is precipitated extraction 1-3 time by organic solvent with 4-20 ℃, throw out carries out ultrafiltration by filter membrane and holds back the material greater than 5KDa, and is stand-by;
2) hold back the material greater than 5KDa with above-mentioned, carry out the DEAE anion exchange chromatography with the NaCl damping fluid of 0.05-0.5M as elutriant, collect 0.15M NaCl buffer solution elution component, stand-by;
3) with the elution fraction of above-mentioned collection through Superdex75 molecular sieve gel chromatography, do elutriant with ultrapure water, flow velocity is 0.8-1.2ml/min, collects elution fraction, and is stand-by;
4) with above-mentioned elution fraction through the separation and purification of HPLC C-18 reversed-phase column, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 90% of total effluent volume in the initial elutriant, and B liquid accounts for 10% of total effluent volume, and the speed of B liquid per minute 1% rises, flow velocity is 0.5-0.8ml/min, when B liquid account for total effluent volume 28.4% the time active ingredient appears, namely obtaining the N-end sequence is YQPDKFMLRGELRNN, molecular weight is the one-component Ciona polypeptide of 5931Da; Wherein A liquid is the TFA of A liquid cumulative volume 0.1% and the ultrapure water of A liquid cumulative volume 99.9%, and B liquid is the TFA of B liquid cumulative volume 0.1% and the acetonitrile of B liquid cumulative volume 99.9%.
It is the aqueous acetone solution of 10%-90% that described step 1) adopts volumn concentration during by organic solvent deposit, and each sedimentation time is 12 hours.It is the aqueous acetone solution of 60%-80% that described step 1) adopts volumn concentration during by organic solvent deposit.
The advantage that the present invention has:
1. the polypeptide that relates among the present invention is a kind of material of marine source, because the singularity of the marine eco-environment, make marine organisms have the unexistent active substance of some land biology, these active substances often have characteristics such as novel structure, high reactivity, resistance be low, can be new drug research and exploitation supply a pattern structure and medical precursor.
2. this polypeptide has passed through 70 ℃ of heat in leaching process, still keeps active this polypeptide of constant explanation to have thermostability after the heating, makes in the future the shelf-life that can prolong medicine behind the medicine.
Description of drawings:
Fig. 1 is the polypeptide PI5931 that the embodiment of the invention 2 is extracted.(CA USA) measures its molecular weight in the mode that strengthens for Applied Biosystems, Foster City with the Q Trap ionspray mass spectrograph of ABI company.Mass spectrograph is equipped with TurbolonSpray source and operates with cation mode
Fig. 2 is the Tricine-SDS-Page electrophoretic analysis figure of the polypeptide PI5931 of the embodiment of the invention 2 extractions.
Embodiment
The preparation method of embodiment 1 Ciona polypeptide:
1) acetone precipitation prepares crude extract: get fresh Ascidian and clean, pulverize, disperse refiner homogenate, heat 30min down at 70 ℃, then with 10000rpm, centrifugal 20min, get supernatant, add the acetone of three times of supernatant liquor volumes in the supernatant liquor, obtaining volumn concentration is 70% acetone soln.4 ℃ of standing over night; With the centrifugal 20min of 10000rpm, get precipitation after the standing over night, the precipitation dissolving back filter membrane by 5KDa is again carried out ultrafiltration and held back, and keeps the part greater than 5KDa, and is stand-by.
2) DEAE Sepharose
TMFast Flow anion exchange chromatography:
At first, be the dialysis tubing of 3.5kD with the crude extract molecular retention amount of packing into, adopt pure water tentatively to dialyse, then it is fully dialysed to initial buffered soln; The sample that dialysis is good takes out, and the centrifugal removal insolubles of 10000rpm is prepared upper prop; Initial buffered soln is 0.02M Tris-Cl, pH8.0.
Secondly, get an amount of DEAE Sepharose
TMFast Flow filler (production of GE company) adds in the ultrapure water, after usefulness glass stick gentle agitation is even, and the glass stick drainage, the chromatography column of 16 * 26 specifications of packing into 3-4 column volume of initial buffered soln balance, can use after the baseline stability.
Again, behind the sample upper prop, with the flushing of initial buffered soln, treat that hanging column albumen does not penetrate fully after, the beginning wash-out, elutriant is Tris-Cl, NaCl solution, namely on the basis of initial damping fluid, prepare 0.05M, 0.1M, 0.15M, 0.2M, 0.3M and 0.5MNaCl solution respectively, then carry out stepwise elution, flow velocity is 5ml/ minute, and whole chromatography process adopts 280nm to detect the protein elution peak.
At last, freeze-drying after will fully being dialysed with pure water by six resulting components of different concns concentration NaCl wash-out is dissolved with pure water; Each constituent mass concentration is 200ug/ml, and the MTT test detects the each component activity, and 0.15M NaCl elution fraction is active ingredient, active constituent is placed-20 ℃ of preservations, and is stand-by.
3) superdex 75 molecular sieve gel chromatographies:
At first, get an amount of superdex 75 molecular sieve gel fillers (production of GE company), clear water soaked 24 hours, after even with the glass stick gentle agitation, glass stick drainage, the chromatography column of 1.6 * 80cm specification of packing into, with 3-4 column volume of pure water balance, can use after the baseline stability.
Secondly, with above-mentioned 0.15M NaCl elution fraction upper prop, with the speed of 1ml/min flushing chromatography column, whole chromatography process adopts 280nm to detect the protein elution peak with clear water, altogether four elution peaks, each elution peak is a component, with each component lyophilize, after employing BCA method was measured protein concentration, the MTT test detected the each component activity, the 4th elution fraction is active ingredient, namely intercepts 1 hour 40 minutes to 1 hour 55 minutes elutriant.Active constituent is placed-20 ℃ of preservations.
4) with above-mentioned elution fraction through HPLC C-18 reversed-phase column (chromatography column of 4.6 * 250mm specification) separation and purification, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 90% of total effluent volume in the initial elutriant, B liquid accounts for 10% of total effluent volume, the speed of B liquid per minute 1% rises, flow velocity is 0.5ml/min, when B liquid account for total effluent volume 28.4% the time active ingredient appears, identify that through mass spectrum namely obtaining the N-end sequence is YQPDKFMLRGELRNN, molecular weight is the one-component Ciona polypeptide (referring to Fig. 1) of 5931Da; It is PI=7.25 that active ingredient is measured iso-electric point by isoelectric focusing electrophoresis.Wherein A liquid is the TFA of A liquid cumulative volume 0.1% and the ultrapure water of A liquid cumulative volume 99.9%, and B liquid is the TFA of B liquid cumulative volume 0.1% and the acetonitrile of B liquid cumulative volume 99.9%.
5) the Tricine-SDS-Page electrophoretic analysis of Acidian polypeptide is identified: the concentrated glue of configuration 4% and 16.5% separation gel.Got C-18 reversed-phase column component point sample, be contrast with low molecular weight protein (LMWP) Marker, carry out the Tricine-SDS-Page protein electrophoresis, concentrate glue and adopt 50V voltage, separation gel adopts 60V voltage to carry out electrophoresis, dye with coomassie brilliant blue staining liquid behind the electrophoresis, can find big or small about a 6KD bright band in sample lane, the molecular weight about 6KD (referring to Fig. 2) of this polypeptide is described.
The mensuration of embodiment 2 antitumor spectras:
Cell: human cervical carcinoma's epithelial cell (Hela), human liver cancer cell (BEL-7402), human colon cancer cell (HCT-116), human breast cancer cell (MCF-7), human lung carcinoma cell (A549), human glioma cell (U8T).Above cell is ocean institute of Chinese Academy of Sciences marine drug laboratory and provides.
Medicine and reagent: Ciona polypeptide: ocean institute of Chinese Academy of Sciences marine drug laboratory provides; 5-fluor-uracil (5-FU) Nantong elite pharmaceutical Co. Ltd product, lot number is 100402; MTT (SIGMA, USA); DMSO (SIGMA, USA); Trypsin SIGMA, USA); RPMI1640 (GIBCO, Invitragen Co., USA); DMEM (GIBCO, Invitragen Co., USA); The top grade foetal calf serum (GIBCO, Invitragen Co., USA); Superfine foetal calf serum Fetal BovineSerum (Hyclone).
Instrument: Ultralow Temperature Freezer (Nature, USA), cell culture incubator (SANYO, Japan), inverted microscope (NIKON, Japan), microplate reader (Bio-Tek Instruments, Inooski, VT, USA), PH counts (Thermo orion, USA), Bechtop (FLC-Harbin Dong Lian instrument plant).
Then detect antitumous effect by mtt assay, the tumor cell line of getting various people goes down to posterity according to the ordinary method cultivation, collects the logarithmic phase cell, regulates concentration of cell suspension 5 * 10
4About individual/ml.Cell suspension is added in 96 well culture plates, and each hole adds 180 μ l.After placing 37 ℃ of constant incubators to cultivate 24h, experimental group adds each concentration above-described embodiment gained polypeptide extract 20 μ l/ hole, and with 5-FU as positive controls, establish 4 parallel holes for every group, and establish clear water respectively and contrast to return to zero.37 ℃ of incubators, behind the cultivation 48h, with liquid-transfering gun liquid in 96 orifice plates is cleaned every hole, back and add MTT (1mg/ml) 30 μ l, put CO
2Incubator is cultivated 4h for 37 ℃, supernatant discarded, and every hole adds DMSO 150 μ l, puts shaking table and shakes up 30min, detects under 492nm with microplate reader, utilizes the SPSS statistical software, calculates cell mortality, asks for IC
5(referring to table 1)
0
Experimental result shows, through the SPSS software statistics, the Ciona polypeptide all has restraining effect in various degree to being used for 6 kinds of tumour cells of test, and be certain dose-dependence, the Ciona polypeptide of different concns is to inhibiting rate, the IC of various tumour cells
50Value sees table 1 for details.
The Ciona polypeptide of table 1 different concns is to the inhibiting rate of 5 kinds of tumour cells
Experimental result shows, active ingredient can strongly inhibited human liver cancer cell (BEL-7402) when 50ug/ml concentration, human cervical carcinoma's epithelial cell (Hela), human colon cancer cell (HCT-116), human breast cancer cell (MCF-7), human glioma cell (U8T), human lung carcinoma cell (A549) growth, inhibiting rate reaches 50%-75%, and the increment of the enough anticancer of embodiment of the invention gains mass-energy is described.
Claims (4)
1. Ciona polypeptide, it is characterized in that: Ciona polypeptide N-end sequence is YQPDKFMLRGELRNN, and molecular weight is 5931Da, and iso-electric point is 7.25;
Described Ciona polypeptide obtains as follows:
1) fresh Ascidian is precipitated extraction 1-3 time by organic solvent with 4-20 ℃, throw out carries out ultrafiltration by filter membrane and holds back the material greater than 5KDa, and is stand-by;
Described organic solvent is the aqueous acetone solution of 10%-90% for adopting volumn concentration, and each sedimentation time is 12 hours;
2) hold back the material greater than 5KDa with above-mentioned, carry out the DEAE anion exchange chromatography with the NaCl damping fluid of 0.05-0.5M as elutriant, flow velocity is 5ml/min, collects 0.15M NaCl buffer solution elution component, and is stand-by; The chromatography column of described DEAE anion-exchange column is 16 * 26 specifications;
3) with the elution fraction of above-mentioned collection through Superdex75 molecular sieve gel chromatography, do elutriant with ultrapure water, flow velocity is 0.8-1.2ml/min, collects 1 hour 40 minutes to 1 hour 55 minutes elution fractions, and is stand-by; The chromatography column of described Superdex75 molecular sieve gel chromatography is 1.6 * 80cm specification;
4) with above-mentioned elution fraction through the separation and purification of HPLC C-18 reversed-phase column, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 90% of total effluent volume in the initial elutriant, and B liquid accounts for 10% of total effluent volume, and the speed of B liquid per minute 1% rises, flow velocity is 0.5-0.8ml/min, when B liquid account for total effluent volume 28.4% the time active ingredient appears, namely obtaining the N-end sequence is YQPDKFMLRGELRNN, molecular weight is the one-component Ciona polypeptide of 5931Da; Wherein A liquid is the TFA of A liquid cumulative volume 0.1% and the ultrapure water of A liquid cumulative volume 99.9%, and B liquid is the TFA of B liquid cumulative volume 0.1% and the acetonitrile of B liquid cumulative volume 99.9%; The chromatography column of described HPLC C-18 reversed-phase column is 4.6 * 250mm specification.
2. by the described Ciona polypeptide of claim 1, it is characterized in that: it is the aqueous acetone solution of 60%-80% that described step 1) adopts volumn concentration during by organic solvent deposit.
3. the preparation method of the described Ciona polypeptide of claim 1 is characterized in that:
1) fresh Ascidian is precipitated extraction 1-3 time by organic solvent with 4-20 ℃, throw out carries out ultrafiltration by filter membrane and holds back the material greater than 5KDa, and is stand-by;
Described organic solvent is the aqueous acetone solution of 10%-90% for adopting volumn concentration, and each sedimentation time is 12 hours;
2) hold back the material greater than 5KDa with above-mentioned, carry out the DEAE anion exchange chromatography with the NaCl damping fluid of 0.05-0.5M as elutriant, flow velocity is 5ml/min, collects 0.15M NaCl buffer solution elution component, and is stand-by; The chromatography column of described DEAE anion-exchange column is 16 * 26 specifications;
3) with the elution fraction of above-mentioned collection through Superdex75 molecular sieve gel chromatography, do elutriant with ultrapure water, flow velocity is 0.8-1.2ml/min, collects 1 hour 40 minutes to 1 hour 55 minutes elution fractions, and is stand-by; The chromatography column of described Superdex75 molecular sieve gel chromatography is 1.6 * 80cm specification;
4) with above-mentioned elution fraction through the separation and purification of HPLC C-18 reversed-phase column, elutriant is A liquid and B liquid, carry out gradient elution, A liquid accounts for 90% of total effluent volume in the initial elutriant, and B liquid accounts for 10% of total effluent volume, and the speed of B liquid per minute 1% rises, flow velocity is 0.5-0.8ml/min, when B liquid account for total effluent volume 28.4% the time active ingredient appears, namely obtaining the N-end sequence is YQPDKFMLRGELRNN, molecular weight is the one-component Ciona polypeptide of 5931Da; Wherein A liquid is the TFA of A liquid cumulative volume 0.1% and the ultrapure water of A liquid cumulative volume 99.9%, and B liquid is the TFA of B liquid cumulative volume 0.1% and the acetonitrile of B liquid cumulative volume 99.9%; The chromatography column of described HPLC C-18 reversed-phase column is 4.6 * 250mm specification.
4. by the preparation method of the described Ciona polypeptide of claim 3, it is characterized in that: it is the aqueous acetone solution of 60%-80% that described step 1) adopts volumn concentration during by organic solvent deposit.
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| US5998374A (en) * | 1997-02-28 | 1999-12-07 | The Regents Of University Of California | Clavaspirins |
| CN100355775C (en) * | 2006-01-20 | 2007-12-19 | 南方医科大学 | Polypeptide of ascidicea and preparation thereof |
| CN1962689A (en) * | 2006-11-23 | 2007-05-16 | 中国科学院海洋研究所 | Ciona polypeptide with resistance to angiogenesis and tumour and its preparation method |
| CN101250222A (en) * | 2008-03-05 | 2008-08-27 | 深圳市圣西马生物技术有限公司 | Ascidian antibacterial peptide, precursor peptide, coding gene and use thereof |
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