CN100548314C - Snake venom cytotoxin and preparation method thereof and application - Google Patents
Snake venom cytotoxin and preparation method thereof and application Download PDFInfo
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- CN100548314C CN100548314C CNB2008101062550A CN200810106255A CN100548314C CN 100548314 C CN100548314 C CN 100548314C CN B2008101062550 A CNB2008101062550 A CN B2008101062550A CN 200810106255 A CN200810106255 A CN 200810106255A CN 100548314 C CN100548314 C CN 100548314C
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Abstract
The invention discloses a kind of snake venom cytotoxin and preparation method thereof and application.The method for preparing snake venom cytotoxin disclosed in this invention comprises the steps: that (1) carry out ion-exchange chromatography with snake venom solution, and first absworption peak of collecting on the absorbance curve that 280nm in the eluent detects wavelength and second absorbs peak-to-peak effluent; (2) effluent that step (1) is obtained carries out exclusion chromatography, and as mobile phase, second absworption peak on the absorbance curve of 280nm detection wavelength obtains snake venom cytotoxin in the collection eluent with water.Phoorsa calmy poison cell toxin of the present invention can obviously be induced people's laryngeal cancer cell Hep-2 apoptosis, and when snake venom cytotoxin concentration was 0.01mg/ml, it induced the apoptotic ability of Hep-2 to be better than the Bleomycin A5 that concentration is 100 μ g/ml; And the undue toxicity experimental results show that this cytotoxic side effect is light.Phoorsa calmy poison cell toxin of the present invention can be used as a kind of anticancer disease drug, and the biological anticancer medicine is become a reality.
Description
Technical field
The present invention relates to a kind of snake venom cytotoxin and preparation method thereof and application.
Background technology
At present, the cancer therapy drug of Shi Yonging is mainly based on chemical medicine clinically, i.e. the chemotherapeutic of often saying.This class medicine common drawback is that side effect is serious, generally can cause vomiting that the patient is serious and alopecia etc.Therefore, the less biological anticancer medicine of side effect becomes the hot topic of research in recent years, mainly concentrates on the research of snake venom, centipede toxin etc.
Contain the cytotoxin that can destroy the tumor cell structure in the snake venom of some Serpentis kind, relevant report was arranged before this, for example in the Agkistrodon acutus snake venom of U.S.'s copperhead snake venom, China Wan Nan, all found to have the cytotoxin of antitumaous effect, these discoveries also are in the academic research stage at present, do not carry out medicinal exploitation.But extracting the cytotoxin obtain having antitumaous effect in phoorsa calmy poison is not reported before this.
Summary of the invention
An object of the present invention is to provide a kind of method for preparing snake venom cytotoxin.
The method for preparing snake venom cytotoxin (snake venom extract) provided by the present invention comprises the steps:
(1) snake venom solution is carried out ion-exchange chromatography, used ion-exchange group is the diethylamino ethyl in the described ion-exchange chromatography, to contain Tris-HCl buffer sodium chloride, that pH value is 8.2, concentration is 0.05mol/L is that mobile phase is carried out the sodium chloride linear gradient elution, the sodium chloride concentration gradient is 0-0.5mol/L, and first absworption peak and second in the collection eluent on the absorbance curve of 280nm detection wavelength absorbs peak-to-peak effluent;
(2) effluent that step (1) is obtained carries out exclusion chromatography, with water as mobile phase, used chromatography media is polyacrylamide gel Bio-Gel-P-60, the diameter of used chromatography pillar is that 4.5cm, post bed height are 120cm, second absworption peak on the absorbance curve of 280nm detection wavelength obtains snake venom cytotoxin in the collection eluent.
In described ion-exchange chromatography, used chromatography media can be DEAE-Sephadex A-50; The flow velocity of described mobile phase can be 0.8-1.0ml/min.
In described exclusion chromatography, the flow velocity of described mobile phase can be 40ml/h.
Described snake venom solution can also carry out following processing before carrying out ion-exchange chromatography: use earlier saturated ammonium sulphate, collect supernatant, more described supernatant is dialysed.
Described snake venom is specifically as follows the snake venom of phoorsa Serpentis.
The snake venom cytotoxin of above-mentioned arbitrary described method preparation also belongs to protection scope of the present invention.
With above-mentioned snake venom cytotoxin is that the medicine that prevents and/or treats cancer of active component also belongs to protection scope of the present invention.
The application of above-mentioned snake venom cytotoxin in the anticancer disease drug of preparation.
Wherein, described cancer in particular can be laryngeal carcinoma.
The present invention obtains cytotoxin by biochemical means from huge squama adder snake venom, this cytotoxin can be by destroying the basic structure cell killing of cell, and the affinity of some cancerous cell is higher than normal cell far away.Experimental result shows, phoorsa calmy poison cell toxin of the present invention can obviously be induced people's laryngeal cancer cell Hep-2 apoptosis, when snake venom cytotoxin concentration was 0.01mg/ml, it induced the apoptotic ability of Hep-2 to be better than the Bleomycin A5 that concentration is 100 μ g/ml; And the undue toxicity experimental results show that this material does not have i.e. this cytotoxic side effect of toxicity light to white mice.Phoorsa calmy poison cell toxin of the present invention can be used as a kind of anticancer disease drug, and the biological anticancer medicine is become a reality.
Description of drawings
Fig. 1 is an ion-exchange chromatography collection of illustrative plates as a result, and wherein, vertical coordinate is an absorbance, abscissa for the pipe number.
Fig. 2 is an exclusion chromatography collection of illustrative plates as a result, and wherein, vertical coordinate is an absorbance, abscissa for the pipe number.
The specific embodiment
Employed experimental technique among the following embodiment is conventional method if no special instructions.
The preparation of embodiment 1, snake venom cytotoxin
Be raw material with phoorsa calmy poison lyophilized powder (purchasing Yu Jing city Kai Tai pharmaceutcal corporation, Ltd) in the present embodiment.
The experimental procedure of extracting is as follows:
1, snake venom dissolving: with 8g phoorsa calmy poison lyophilized powder be dissolved in 100ml Tris-HCl buffer (pH8.2,0.05mol/L) in.
2, remove foreign protein: add 6ml saturated ammonium sulfate solution in the snake venom solution that step 1 obtains, placed at ambient temperature after the mixing 1 hour, centrifugal 10 minutes of 3000rpm discards precipitation, keeps supernatant solution.
3, dialysis desalination: whole supernatant that step 2 is obtained are enclosed in the bag filter of holding back the 14000D molecular weight, then bag filter are put into distilled water dialysis 15 hours, change water once in per 3 hours, the volume 1200ml of distilled water.The about 180ml of the dialysis resulting dialysis solution in back.
4, ion-exchange chromatography: the Tris-HCl buffer balance DEAE-SephadexA-50 chromatographic column of usefulness pH 8.2,0.05mol/L 15 hours, flow velocity is 1.0ml/min; The dialysis solution that the dialysis that step 3 is obtained obtains moves on to the DEAE-Sephadex A-50 chromatographic column after the balance, treat supernatant move to fully the glue face following after, above the glue face, add Tris-HCl buffer (PH8.2,0.05mol/l) 50ml; (pH8.2 is 0.05mol/l) for initial liquid, (pH8.2 0.05mol/l) carries out the sodium chloride linear gradient elution for stop buffer with the Tris-HCl buffer that contains 0.5mol/L sodium chloride with the Tris-HCl buffer.Used eluent is 4000ml altogether; The flow velocity of eluent is that (pace of change that is the concentration of sodium chloride is 1 * 10 to 0.8-1.0ml/min
-4Mol/L/min-1.25 * 10
-4Mol/L/min).
Collect with automatic fraction collector, every pipe 15ml, by UV spectrophotometer measuring chromatographic solution absorbance, the detection wavelength is 280nm, makes point-absorbance curve, as shown in Figure 1.Collect first absworption peak and second and absorb peak-to-peak part (16-29 pipe flow fluid), the about 210ml of the effluent of collection.
5, exclusion chromatography: the 210ml effluent that step 4 is obtained carries out exclusion chromatography.Separate (handle and irritate post) with the BIO-GEL-P-60 gel chromatography according to the gel description, chromatography column length 150cm, diameter 4.5cm, the glue bed height is not less than 120cm, with purified water as mobile phase, flow velocity 40ml/h; Collect chromatographic solution with automatic fraction collector, the 15ml/ pipe, by ultraviolet spectrophotometer monitoring chromatographic solution absorbance, the detection wavelength is 280nm; Make point-absorbance curve, as shown in Figure 2.Collect second absworption peak (26-34 pipe flow fluid) on the absorbance curve, the about 135ml of the effluent of collection.
6, recording collection liquid eggs white matter concentration with Folin-phenol reagent process (lowry method) is 0.08mg/ml.
Be diluted to the solution that protein content is 0.01mg/ml with collecting liquid, promptly get phoorsa calmy poison cell toxin stock solution 1080ml.
By the molecular weight of SDS-polyacrylamide gel electrophoresis detection snake venom cytotoxin, testing result shows that the gained cytotoxin presents a band, and molecular weight is 31000D.
The functional verification of embodiment 2, phoorsa calmy poison cell toxin
One, the anticancer disease functional verification of phoorsa calmy poison cell toxin
With people's laryngeal cancer cell Hep-2 cell strain (Liaoning Medical University's head and neck institute of Surgical Research provides) is target cell, observes snake venom cytotoxin of the present invention and Bleomycin A5 inducing cancer cell and transfers the efficient contrast of dying.Experimental technique reference literature (the meaningful king's snowy peak of Tai Bleomycin A5 is induced experimentation " Liaoning Medical University's journal " 2004Aug.25 (4) the 5-7 page or leaf of people's laryngeal carcinoma H ep-2 natural death of cerebral cells), specific as follows:
The cultivation of people's Human Laryngeal Cancer Cell Hep-2 cell strain:, place 37 ℃, contain 5%CO with the complete culture solution (hyclone 10%, penicillin 100u/ml, streptomycin 100ug/ml) of cell inoculation in RPMI-1640
2Cultivate in the incubator.
After getting the phoorsa calmy poison cell toxin stock solution 20ml lyophilization of embodiment 1 preparation, standby with the redissolution of 20mlRPMI-1640 culture fluid, the concentration of cytotoxin (protein) is 0.01mg/ml.
Experiment is divided into three processing: contain concentration and be in the RPMI-1640 culture fluid of the above-mentioned snake venom cytotoxin of 0.01mg/ml as experimental group, the RPMI-1640 culture fluid of Bleomycin A5 that contains concentration and be 100 μ g/ml is as positive controls, and the RPMI-1640 culture medium is as the blank group.Above-mentioned Hep-2 cell is inoculated in above-mentioned three processing respectively, and inoculum density is all identical, places 37 ℃ then, contains 5%CO
2Cultivated 24 hours in the incubator.Each processing all repeats 10 times.
Adopt PI singly to dye method, carry out flow cytometer and detect, thereby calculate apoptosis rate (apoptosis rate=apoptotic cell number/living cells number+apoptotic cell number).
The result is as shown in table 1, and the numerical value in the table 1 is mean+SD.The result shows that the phoorsa calmy poison cell toxin of gained of the present invention can obviously be induced the Hep-2 apoptosis, and when snake venom cytotoxin concentration was 0.01mg/ml, it induced the apoptotic ability of Hep-2 to be better than the Bleomycin A5 that concentration is 100 μ g/ml.
Table 1, apoptosis situation
* P＜0.01 with the blank group relatively; Δ P＜0.01 with positive controls relatively
Two, phoorsa calmy poison cell toxin undue toxicity investigates
Select 5 of healthy Kunming kind one-level white mice, body weight 18～22g, male and female are not limit.Behind the filtering with microporous membrane of phoorsa calmy poison cell toxin stock solution through 0.2 micron pore size with embodiment 1 gained, the sterile working gives little of the caudal vein injection, every white mice injection 1ml, normal diet breeding observing 48 hours.5 white mice do not see any unusual after 48 hours as a result.Above-mentioned consumption to white mice approximately is more than 75 times of HTD, and white mice is no abnormal, so phoorsa calmy poison cell toxin undue toxicity is qualified, and safety is reliable.
Claims (4)
1, a kind of method for preparing snake venom cytotoxin comprises the steps:
(1) snake venom dissolving: 8g phoorsa calmy poison lyophilized powder is dissolved in the Tris-HCl buffer of 100ml pH8.2,0.05mol/L;
(2) remove foreign protein: add 6ml saturated ammonium sulfate solution in the snake venom solution that step (1) obtains, placed at ambient temperature after the mixing 1 hour, centrifugal 10 minutes of 3000rpm discards precipitation, keeps supernatant solution;
(3) dialysis desalination: whole supernatant that step (2) is obtained are enclosed in the bag filter of holding back the 14000D molecular weight, then bag filter is put into distilled water dialysis 15 hours, changed water once in per 3 hours, the volume 1200ml of distilled water, the resulting dialysis solution in dialysis back is 180ml;
(4) ion-exchange chromatography: the Tris-HCl buffer balance DEAE-Sephadex A-50 chromatographic column of usefulness pH8.2,0.05mol/L 15 hours, flow velocity is 1.0ml/min; The dialysis solution that the dialysis that step (3) is obtained obtains moves on to the DEAE-Sephadex A-50 chromatographic column after the balance, treat supernatant move to fully the glue face following after, above the glue face, add the Tris-HCl buffer 50ml of pH8.2,0.05mol/L; Tris-HCl buffer with pH8.2,0.05mol/L is initial liquid, the Tris-HCl buffer that contains 0.5mol/L sodium chloride with pH8.2,0.05mol/L is that stop buffer carries out the sodium chloride linear gradient elution, used eluent is 4000ml altogether, the flow velocity of eluent is 0.8-1.0ml/min, and promptly the pace of change of the concentration of sodium chloride is 1 * 10
-4Mol/L/min-1.25 * 10
-4Mol/L/min;
Collect with automatic fraction collector, every pipe 15ml is by UV spectrophotometer measuring chromatographic solution absorbance, the detection wavelength is 280nm, makes point-absorbance curve, collects first absworption peak and second and absorbs peak-to-peak part, i.e. 16-29 pipe flow fluid, the effluent of collection is 210ml;
(5) exclusion chromatography: the 210ml effluent that step (4) is obtained carries out exclusion chromatography, separate with the BIO-GEL-P-60 gel chromatography, chromatography column length 150cm, diameter 4.5cm, the glue bed height is not less than 120cm, with purified water as mobile phase, flow velocity 40ml/h; Collect chromatographic solution with automatic fraction collector, the 15ml/ pipe, by ultraviolet spectrophotometer monitoring chromatographic solution absorbance, the detection wavelength is 280nm; Make point-absorbance curve, collect second absworption peak on the absorbance curve, promptly 26-34 pipe flow fluid is collected effluent 135ml, obtains snake venom cytotoxin.
2, the snake venom cytotoxin for preparing with the described method of claim 1.
3, a kind of medicine that prevents and/or treats laryngeal carcinoma, its active component is the snake venom cytotoxin described in the claim 2.
4, the application of the described snake venom cytotoxin of claim 2 in the medicine of the anti-laryngeal carcinoma of preparation.
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Owner name: JINZHOU AOHONG PHARMACEUTICAL CO.,LTD. Free format text: FORMER OWNER: YU HONGRU Effective date: 20091030 |
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Effective date of registration: 20091030 Address after: No. 10, Fuzhou street, Taihe District, Liaoning, Jinzhou Patentee after: Aohong Pharmaceutical Co., Ltd., Jinzhou Address before: Liaoning Rui Lai street 10, Taihe District, Liaoning, Jinzhou Province China Medical Science and Technology Co., Ltd. Patentee before: Yu Hongru |
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Address after: 121013 Songshan street, Taihe District, Jinzhou, Liaoning Province, No. 55 Patentee after: Aohong Pharmaceutical Co., Ltd., Jinzhou Address before: 121013 10 Fuzhou street, Taihe District, Liaoning, Jinzhou Patentee before: Aohong Pharmaceutical Co., Ltd., Jinzhou |