CN103408650A - Cereus sinensis verrill soluble cell peptide CCT-I and separating and purifying method and application thereof - Google Patents
Cereus sinensis verrill soluble cell peptide CCT-I and separating and purifying method and application thereof Download PDFInfo
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Abstract
The invention discloses a cereus sinensis verrill soluble cell peptide CCT-I and a separating and purifying method and an application thereof. The separating and purifying method comprises the following steps: after cereus sinensis verrill tentacle crude toxin is salted out and fractionally precipitated with ammonium sulfate, active sites are separated and purified through Sephadex G-100 and superdex 75 gel filtration chromatography columns in sequence, and the soluble cell peptide is obtained through separation from a cereus sinensis verrill tentacle at the first time and is finally authenticated as the novel cereus sinensis verrill soluble cell peptide that has not been reported through SDS-PAGE and LC-MS, wherein two peptides (alpha and beta) in the novel cereus sinensis verrill soluble cell peptide are similar in molecular weight of about 80 kDa, and the HPLC test shows that the two peptides are high in purify and similar in polarity. The half soluble cell rate is tested to be 5.75 micrograms*mL<-1>, and the MTT process test displays that the CCT-I has strong antitumor activity in vitro, and can be used for preparing tumor-treating drug.
Description
Technical field
The invention belongs to natural medicine technical field in Chinese medicine, relate to the molten cell polypeptide CCT-I of Chinese celestial shadow sea anemone and separation purification method and application.
Background technology
Statistical information shows, China is from the eighties in last century, the sickness rate of malignant tumour is obvious ascendant trend, the sickness rate of kinds of tumor is with the speed increment of 5-10%, annual Incidence number is 1,600,000, the patient who dies from malignant tumour every year is 1,300,000 people, and the number of tumour patient is 5,400,000 at present.Lung cancer in big and medium-sized cities, breast cancer incidence are the highest; Rural areas is the highest with cancer of the stomach, Incidence of esophageal cancer.Visible, the market potential of China's antitumour drug is huge.The correlative study work for the treatment of malignant tumour constantly makes progress, and the antitumor drug commonly used that WHO announces is 49 kinds.Because the R&D cycle of antitumor drug is long, the most domestic enterprise product is only imitated external listing kind at present, has the domestic antitumor drug of independent intellectual property right urgently to develop.
Sea anemone (sea anemone) has another name called extra large chrysanthemum, belongs to Coelenterata (Coelenterata) Anthozoa (Anthozoa) Actiniaria (actiniaria) animal.Extensively be distributed in the torrid zone and warm band marine site, mainly anchor on marine rock or in silt, the sea anemone that China marine site distributes has yellow Anthopleura (Anthopleura xanthogrammica), Anthopleura pacifica (A.pacifica), taeniae rock sea anemone (Haliplanella luciae Hand), Chinese celestial shadow sea anemone (Cereus sinensis Verrill) etc.Sea anemone has the effects such as nourishing and strengthening vital, astringing to arrest diarrhea, removing dampness and killing parasites, among the peoplely is mainly used to treat hemorrhoid, prolapse of the anus, rich parasitosis and ringworm of the body etc.Modern study shows from sea anemone, separating the synocytotoxin obtained to have stronger anti-tumor activity more, such as StI, EqtII, EqtIIA179C, EqtIIK20C and EqtIIR126C etc. have stronger restraining effect to different tumour cells.China coast sea anemone aboundresources, particularly along with Jiangsu Prov. Inst. of Marine Aquatic Products carries out artificial breeding and cultivates successfully the celestial shadow sea anemone of China, this makes and utilizes the sea anemone resource to carry out the industrialization comprehensive exploitation to become possibility, the celestial shadow sea anemone of China (Cereus sinensis Verrill) is under the jurisdiction of the celestial shadow Helenopolypus of Sagartiidae, be the poisonous sea anemone of a kind of edible that China coast extensively distributes, the separation and purification of its synocytotoxin, composition and property research there is not yet report.
The separation and purification difficulty of congestin is larger, and its major cause has the following aspects: the first, in the thick poison of sea anemone, contain many kinds of protein ingredients, and the character between some of them albumen (as: molecular weight, iso-electric point, hydrophobicity etc.) is very approaching; The second, congestin mostly is protein matter, easily is subject to the impact of external environment factors vary and causes its activity decreased even to be lost; The 3rd, sea anemone not of the same race, due to the difference of its growing environment etc., cause contained toxin composition and character in its physiological structure and body also to have larger difference, this also makes the not separation and purification work of congestin of the same race be difficult to directly imitate, and can only grope just may obtain by loaded down with trivial details experiment.
Summary of the invention
The objective of the invention is the above-mentioned deficiency for prior art, the separation purification method of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of a kind of China is provided.
Another object of the present invention is to provide the molten cell polypeptide CCT-I of the celestial shadow sea anemone of China that the method separation and purification obtains.
Another purpose of the present invention is to provide the application of the molten cell polypeptide CCT-I of Chinese celestial shadow sea anemone.
The separation method of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of China mainly comprises the following steps:
1) by China's celestial shadow sea anemone tentacle crude venom after ammonium sulfate segmentation salt precipitation, obtain tri-ammonium sulfate saturation ratio sections of 0-40%, 40%-60% and 60-90%, then with pH=7.0, contain 0.15molL
-1The 10mmolL of sodium-chlor
-1The PBS damping fluid redissolves, stand-by;
2) above-mentioned 60-90% ammonium sulfate saturation ratio section precipitation is redissolved to liquid through containing Sephadex G-100 gel chromatographic columns, adopt elutriant to carry out separation and purification with the 0.5mL/min flow velocity, the detection wavelength is 280nm, collect the peak value pipe at each peak shown on detector, the elutriant of collecting concentrates with the super filter tube of 3kDa under 4 ℃ of conditions, collect peak 1 concentrated solution stand-by; Described elutriant is that pH=7.0 contains 0.15molL
-1The 10mmolL of sodium-chlor
-1Phosphoric acid buffer;
3) get above-mentioned steps 2) in peak 1 concentrated solution through gel chromatographic columns superdex75, adopt elutriant to carry out separation and purification with flow velocity 0.3mL/min flow velocity, the detection wavelength is 280nm, collect the peak value pipe at each peak shown on detector, the elutriant of collecting concentrates with the super filter tube of 3kDa under 4 ℃ of conditions, collect Peak Activity 1 concentrated solution, and with 3.5kDa dialysis tubing dialyzed overnight, dialysis tubing liquid outward is pure water, after lyophilize, obtain described molten cell polypeptide CCT-I; Described elutriant is that pH=7.0 contains 0.15molL
-1The 10mmolL of sodium-chlor
-1The PBS damping fluid.
Wherein, prepared by the following method by described China celestial shadow sea anemone tentacle crude venom: the China that will cut celestial shadow sea anemone tentacle-80 ℃ carries out 2～3 freeze thawing, after under 20 ℃～28 ℃, suitably thawing, with scissors, shred, and with after the abundant homogenate of glass homogenizer, add ultrapure water, sealed membrane is sealed and is placed in 4 ℃ of refrigerators, place 1.5～2h, every 15min stirs once during this time, then at 4 ℃, centrifugal 1～1.5h under 10000～12000r/min condition, take out supernatant liquor, centrifugal 0.8～1h under similarity condition again, collect supernatant liquor and obtain Chinese celestial shadow sea anemone tentacle crude venom.
The preferred 1:4 of solid-liquid ratio of the celestial shadow sea anemone tentacle of the China described in the celestial shadow sea anemone tentacle crude venom preparation process of China and ultrapure water～6, further preferred 1:5.
Described China celestial shadow sea anemone tentacle crude venom through the method for ammonium sulfate segmentation salt precipitation is: accurately measure slightly poison of 100mL sea anemone, with reference to 0 ℃ of saturation table of ammonium sulfate, take and be ground to ammonium sulfate in small, broken bits, slowly join in 100mL sea anemone crude venom, then in 4 ℃ of refrigerator hold over night.Under 4 ℃, 10000～12000rmin
-1Centrifugal 35～45min, take out supernatant, and precipitation contains 0.15molL with pH=7.0
-1The 10mmolL of sodium-chlor
-1The PBS damping fluid redissolves, and according to the method described above sea anemone being saltoutd is 0～40%, 40%～60% and 60%～90% 3 ammonium sulfate saturation ratio section.
The molten cell polypeptide CCT-I of the celestial shadow sea anemone of China prepared by described separation method.
Described molten cell polypeptide CCT-I mainly all is comprised of at two polypeptide of 80kDa molecular weight.
The application of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of described China in preparing cancer therapy drug, the preferably application in the medicine for preparing anti-cervical cancer, anti-lung cancer or anti-liver cancer.
Beneficial effect:
The present invention is by after the fractional precipitation of the thick poisons ammonium sulfate precipitation of China's celestial shadow sea anemone tentacle, reactive site is passed through to Sephadex G-100 and two kinds of gel filtration chromatography column separating purifications of superdex75 successively, finally first from China's celestial shadow sea anemone tentacle, separating and obtain the molten cell polypeptide of a class, and be accredited as the molten cell polypeptide of novel sea anemone that a class has no report, called after CCT-I by SDS-PAGE electrophoresis and LC-MS.Two polypeptide (α β) that wherein include, molecular weight is close, is the 80kDa left and right, detects through HPLC, and purity is higher, and two polypeptide polarity are close.And measured the molten cell rate of its half and be about 5.75 μ gmL
-1, through the mtt assay detection display, CCT-I has strong anti tumor activity in vitro, can be used for preparing the medicine for treating tumor thing.
The accompanying drawing explanation
The Sephadex G-100 gel filtration chromatography figure of the thick malicious 60%-90% ammonium sulfate precipitation section redissolution liquid of the celestial shadow sea anemone of Fig. 1 the present invention China, peak 1 is Peak Activity.
Sephadex G-100 gel filtration chromatography figure is crossed at peak 1 in Fig. 2 Sephadex G-100 gel chromatography again.
The superdex75 gel filtration chromatography figure of peak 1 concentrated solution in Fig. 3 Sephadex G-100 gel chromatography, peak 1 is Peak Activity.
SDS-PAGE electrophorogram in Fig. 4 the present invention, wherein M is standard minute quality Marker; 1 is the peak 1 in Sephadex G-100 gel chromatography; 2 is CCT-I.
Fig. 5 is that the celestial shadow sea anemone synocytotoxin CCT-I of the present invention China detects the elution curve of purity through HPLC.
The dissolved cell activity figure of the celestial shadow sea anemone synocytotoxin CCT-I of Fig. 6 China.
Fig. 7 A figure is the HPLC elution profile of the present invention celestial shadow sea anemone synocytotoxin CCT-I-α of China after the Trypsin enzymolysis; B figure is the mass spectrum of the celestial shadow sea anemone synocytotoxin CCT-I-α of the present invention China; C figure is the HPLC elution profile of the present invention celestial shadow sea anemone synocytotoxin CCT-I-β of China after the Trypsin enzymolysis; D figure is the mass spectrum of the celestial shadow sea anemone synocytotoxin CCT-I-β of the present invention China.
The microscopic examination figure of the celestial shadow sea anemone synocytotoxin CCT-I inhibition tumor cell Hela growth of Fig. 8 China.
Wherein, a figure is contrast, and in b figure, CCT-I concentration is 3.125 μ gmL
-1, in c figure, CCT-I concentration is 6.25 μ gmL
-1, in d figure, CCT-I concentration is 12.5 μ gmL
-1, in e figure, CCT-I concentration is 25 μ gmL
-1, in f figure, CCT-I concentration is 50 μ gmL
-1.
The microscopic examination figure of the celestial shadow sea anemone synocytotoxin CCT-I inhibition tumor cell A549 growth of Fig. 9 China.
Wherein, a figure is contrast, and in b figure, CCT-I concentration is 3.125 μ gmL
-1, in c figure, CCT-I concentration is 6.25 μ gmL
-1, in d figure, CCT-I concentration is 12.5 μ gmL
-1, in e figure, CCT-I concentration is 25 μ gmL
-1, in f figure, CCT-I concentration is 50 μ gmL
-1.
The microscopic examination figure of the celestial shadow sea anemone synocytotoxin CCT-I inhibition tumor cell HepG2 growth of Figure 10 China.
Wherein, a figure is contrast, and in b figure, CCT-I concentration is 3.125 μ gmL
-1, in c figure, CCT-I concentration is 6.25 μ gmL
-1, in d figure, CCT-I concentration is 12.5 μ gmL
-1, in e figure, CCT-I concentration is 25 μ gmL
-1, in f figure, CCT-I concentration is 50 μ gmL
-1.
Embodiment:
Below with embodiment, further illustrate essentiality content of the present invention, but content of the present invention is not limited to this.
The separation of the molten cell polypeptide of the Chinese celestial shadow sea anemone of embodiment 1
The first step, the thick poison preparation of sea anemone.The China of live body celestial shadow sea anemone tentacle is cut, be chilled in rapidly in-80 ℃ of refrigerators, before preparation, the sea anemone tentacle that the taking-up freeze thawing is 2 times, after under room temperature, suitably thawing, accurately take 20g, shred with scissors, and with after the abundant homogenate of glass homogenizer, by solid-liquid ratio 1:5, add a certain amount of ultrapure water, sealed membrane is sealed and is placed in 4 ℃ of refrigerators, places 2h, every 15min stirs once during this time, notes stirring gently in order to avoid producing a large amount of foams.Then at 4 ℃, centrifugal 1h under the 10000r/min condition, take out supernatant liquor, then under similarity condition centrifugal 1h, the gained supernatant liquor is the sea anemone crude venom.By the Bradford method, detect protein concentration, and detect thick malicious dissolved cell activity with SD rat blood reactive system.
Second step, ammonium sulfate precipitation is processed.Accurately measure above-mentioned crude venom 100mL, with reference to 0 ℃ of saturation table of ammonium sulfate, take a certain amount of ammonium sulfate in small, broken bits that is ground to, slowly join (whole experimentation all carries out on ice water bath environment and magnetic stirring apparatus) in 100mL sea anemone crude venom, then in 4 ℃ of refrigerator hold over night.Under 4 ℃, 10000rmin
-1Centrifugal 40min, take out supernatant, a certain amount of 10mmolL of precipitation
-1(PBS pH=7.0 contains 0.15molL to phosphate buffered saline buffer
-1Sodium-chlor) redissolve.With reference to aforesaid method, it is 0～40%(I), 40%～60%(II) and 60%～90%(III) 3 section that sea anemone is saltoutd.By the Bradford method, detect the respectively section of saltouing protein concentration, and detect the respectively dissolved cell activity of the section of saltouing redissolution liquid with SD rat blood reactive system.
The 3rd step, Sephadex G-100 gel filtration chromatography.Obtain as stated above the 60%th～90%(III) section of the saltouing PBS redissolution liquid that dissolved cell activity is stronger.The Sephadex G-100 gel-filtration column (10mmolL of 3.7cm * 100cm)
-1(PBS pH=7.0 contains 0.15molL to phosphate buffered saline buffer
-1Sodium-chlor) balance, be splined on this post by sample after fully.Use same buffer solution elution, detect absorbancy at 280nm, collect the peak value pipe (Fig. 1) at each peak, after the 3kDa super filter tube is concentrated, with SD rat blood reactive system, detect the dissolved cell activity at each peak, result shows that the dissolved cell activity of Peak Activity 1 is the strongest, collect Peak Activity 1(Fig. 2), stand-by.
The 4th step, Superdex75 gel permeation chromatography post.Superdex75 gel-filtration column (the same 10mmolL of 1.6cm * 100cm)
-1(PBS pH=7.0 contains 0.15molL to phosphate buffered saline buffer
-1Sodium-chlor) balance, peak 1 concentrated solution of activeconstituents Sephadex G-100 gel-filtration obtained after fully is splined on this post.Use same buffer solution elution, at 280nm, detect absorbancy, collect the peak value pipe (Fig. 3) at each peak, after the 3kDa super filter tube is concentrated, with SD rat blood reactive system, detect the dissolved cell activity at each peak, result shows that the dissolved cell activity of Peak Activity 1 is the strongest, collects Peak Activity 1, and with 3.5kDa dialysis tubing dialyzed overnight, the outer liquid of dialysis tubing is pure water, after lyophilize, CCT-I powder-20 ℃ preservation, stand-by.
The detection that embodiment 2 hemolytic activities detect
The first step, the blood sample preparation.Get the SD rat of healthy adult, heart extracting blood, blood are stored in anticoagulant tube, and 4 ℃ save backup.
Second step, the 2% red blood cell suspension preparation of SD rat.From anticoagulant tube, taking out a certain amount of SD rat blood, use 10mmolL
-1(PBS pH=7.0 contains 0.15molL to phosphate buffered saline buffer
-1Sodium-chlor) washing, 1500rmin under 4 ℃ of conditions
-1Centrifugal 5min, supernatant discarded, repeat 4～5 times, until the as clear as crystal redfree of supernatant, red corpuscle is diluted to 2% red blood cell suspension with PBS, 4 ℃ save backup.
The 3rd step, hemolytic activity detects.The CCT-I solution of getting 200 μ L different concns joins in 200 μ L2% red blood cell suspensions, then with PBS, is settled to 1mL, 37 ℃, hatches 30min, 1500rmin under 4 ℃ of conditions
-1Centrifugal 5min, at the A of 540nm mensuration supernatant
540Value, 3 groups, the parallel work of each concentration, negative control is for adding 200 μ L PBS, and positive control is for adding 200 μ L0.5%Triton X-100.Calculate the molten cell rate of each sample:
Molten cell rate=(sample A
540-negative control A
540)/(positive control A
540-negative control A
540) * 100%
Result shows that the molten cell rate of half of CCT-I is about 5.5 μ gmL
-1(Fig. 6).
The hemolytic activity of each peak value pipe elutriant is also to measure as stated above.
The evaluation of the molten cell polypeptide CCT-I of the Chinese celestial shadow sea anemone of embodiment 3
The first step, SDS-PAGE electrophoresis check CCT-I purity, estimation molecular weight.Sample and 5 * sample-loading buffer mix (v:v=4:1) and boil 5min in 95 ℃, then with 5% concentrated glue, separate with 12% separation gel, finally adopt the fixing dyeing of coomassie brilliant blue R_250, by with standard protein, carrying out definite molecular weight recently.The standard protein that standard protein Marker contains has: Phosphoric acid esterase b97.2kDa, bovine serum albumin 66.4kDa, ovalbumin 44.3kDa, carbonic anhydrase 29.0kDa, trypsin inhibitor 20.1kDa and N,O-Diacetylmuramidase 14.3kDa.In result demonstration CCT-I, mainly comprise two protein bands, purity is high, and molecular weight is all in 80kDa left and right (Fig. 4).
Second step, HPLC check CCT-I purity.CCT-I is dissolved in to 10% acetonitrile (acetonitrile: 0.1%TFA=1:9), after aperture is 0.22 μ m filtering with microporous membrane, use HPLC(Thermo Ultimate3000) check purity, chromatographic column is C18 post (4.6 * 25mm), elution requirement is linear elution, be specially in 0-60min, (acetonitrile: 0.1%TFA=1:9-13:12), the detection wavelength is 280nm to acetonitrile by 10%-52%.In result demonstration CCT-I, mainly contain two polypeptide, polarity is close, and purity high (Fig. 5).
The 3rd step, LC-MS identifies α and two polypeptide of β in CCT-I in conjunction with the method in search NBCI storehouse.
1) HPLC condition,
Moving phase: A liquid H
2O(mass spectrum level), 0.1%TFA
B liquid acetonitrile (mass spectrum level), 0.1%TFA
2uL/min after flow velocity: 200uL/min(shunting)
Gradient: 120min (5%B to35%B in40min, to95%in20min, balance 20min)
Sample size: 20uL
Pillar model: CTICAP5150100
2) MS condition,
Model: LTQ XL Thermo
Ion source: NSI; Capillary voltage: 3.5KV;
Ion source temperature: 260 ℃; Desolventizing temperature degree: 260 ℃;
Desolventizing airshed: 20unit/min detector voltage: 200V
3) retrieval NBCI parameter,
Retrieval software: Proteomics Discovery1.2 (Thermo)
Searching algorithm: Sequest
Database: the client provides protein to originate, Actiniaria(NCBI)
Peak error 1Da
Data screening: Xcorr > 1.9 if Charge=1
Xcorr>2.2 if Charge=2
Xcorr>3.75 if Charge=3
4) sample preparation.After with scalpel, two protein bands of SDS-PAGE running gel in the above-mentioned the first step being cut, then adhesive tape is cut into to 1mm
3Little, be placed in 1.5ml Eppendorf pipe; With the 200ul distilled water, wash 2 times each 10min; Add to examine and dye destainer (50mM NH4HCO3/CH3CN) 200ul, ultrasonic decolouring 15min, blot, and repeats 3 times, until decolouring fully; Add CH
3CN100ul dewaters to micelle and bleaches, and vacuum is drained 10min; Add 200ul10mM DTT/25mM NH
4HCO
3, 37 ℃ of water-bath 1h; Be chilled to room temperature, blot, add fast 200ul50mM IAA/25mM NH
4HCO
3, be placed in darkroom 45min; With following solution, carry out ultrasonic cleaning successively: 25mM NH
4HCO
3(2 times), 25mM NH
4HCO
3+ 50%CH
3CN(2 time) and CH
3CN (1 time), each 10min.CH
3CN dewaters to micelle and bleaches, and vacuum is drained 10min; By 0.1 μ g/ μ L trypsin solution 25mM NH
4HCO
3Doubly, every pipe adds 2-3 μ L to dilution 10-20, once centrifugal a little, allows enzyme liquid fully contact with micelle, places 30min for 4 ℃.Treat that enzyme liquid is absorbed fully by micelle, add 25mM NH4HCO3 to cumulative volume 300 μ L, 37 ℃ are spent the night; The enzymolysis supernatant liquor is collected in centrifugal collection, is placed in another 1.5ml Eppendorf pipe; The residue micelle, with the ultrasonic 15min of extraction buffer (67%CH3CN, 5%TFA, 28%ddH2O), repeats 3 times, and enzymolysis solution is mixed, and vacuum is super dry; Add 5%B liquid and redissolve, use successively above-mentioned 1), 2) and 3) conditional detects.Result show do not retrieve with CCT-I in the albumen that mates fully of two bands, in this explanation CCT-I, two albumen (α & β) are newfound albumen (Fig. 7).
The China prepared by the aforesaid method molten cell polypeptide of celestial shadow sea anemone is from the celestial shadow sea anemone tentacle of coelenteron class animal China, separating the molten cell polypeptide of novel sea anemone that the class obtained has no report, and molecular weight is in the 80kDa left and right, and has higher degree.
The molten cell polypeptide CCT-I of the Chinese celestial shadow sea anemone of embodiment 4 inhibition tumor cell growth
The Hela taken the logarithm vegetative period, A549 and HepG2 tumour cell, with corresponding substratum, tumour cell being diluted to density is 2 * 10
4/ mL, then be inoculated in 96 orifice plates, every hole 100 μ L, and in 37 ℃, 5%CO
2, after in the CO2gas incubator of saturated humidity, cultivating 24h, every hole adds the CCT-I sample of 20 μ L with corresponding substratum dilution, and concentration is respectively 3.125,6.25,12.5,25 and 50 μ gmL
-1, each experimental group arranges 5 multiple holes, and negative control is for adding the corresponding substratum of 20 μ L.Continue to cultivate the microscopic examination figure grown for Chinese celestial shadow sea anemone synocytotoxin CCT-I inhibition tumor cell after 48h and see Fig. 8～Figure 10, by the restraining effect of mtt assay working sample to two strain tumour cells.EC
50It is the drug level when 50% tumour cell is produced to restraining effect.
The Chinese celestial shadow sea anemone synocytotoxin CCT-I antitumor cell growth of table 1
As seen from Table 1, the celestial shadow sea anemone synocytotoxin CCT-I of China can effectively suppress the growth of Human cervical cancer cell lines (Hela), lung cancer cell line (A549) and hepatoma cell line (HepG2), and this shows that Chinese celestial shadow sea anemone synocytotoxin CCT-I has the effect of very strong antitumor cell growth.
Claims (8)
1. the separation method of the molten cell polypeptide CCT-I of Chinese celestial shadow sea anemone is characterized in that mainly comprising the following steps:
1) by China's celestial shadow sea anemone tentacle crude venom after ammonium sulfate segmentation salt precipitation, obtain tri-ammonium sulfate saturation ratio sections of 0-40%, 40%-60% and 60-90%, then with pH=7.0, contain 0.15molL
-1The 10mmolL of sodium-chlor
-1The PBS damping fluid redissolves, stand-by;
2) above-mentioned 60-90% ammonium sulfate saturation ratio section precipitation is redissolved to liquid through containing Sephadex G-100 gel chromatographic columns, adopt elutriant to carry out separation and purification with the 0.5mL/min flow velocity, the detection wavelength is 280nm, collect the peak value pipe at each peak shown on detector, the elutriant of collecting concentrates with the super filter tube of 3kDa under 4 ℃ of conditions, collect peak 1 concentrated solution stand-by; Described elutriant is that pH=7.0 contains 0.15molL
-1The 10mmolL of sodium-chlor
-1Phosphoric acid buffer;
3) get above-mentioned steps 2) in peak 1 concentrated solution through gel chromatographic columns superdex75, adopt elutriant to carry out separation and purification with flow velocity 0.3mL/min flow velocity, the detection wavelength is 280nm, collect the peak value pipe at each peak shown on detector, the elutriant of collecting concentrates with the super filter tube of 3kDa under 4 ℃ of conditions, collect Peak Activity 1 concentrated solution, and with 3.5kDa dialysis tubing dialyzed overnight, dialysis tubing liquid outward is pure water, after lyophilize, obtain described molten cell polypeptide CCT-I; Described elutriant is that pH=7.0 contains 0.15molL
-1The 10mmolL of sodium-chlor
-1The PBS damping fluid.
2. the separation method of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of China according to claim 1, it is characterized in that: prepared by described China celestial shadow sea anemone tentacle crude venom by the following method: the China that will cut celestial shadow sea anemone tentacle-80 ℃ carries out 2～3 freeze thawing, after under 20 ℃～28 ℃, suitably thawing, with scissors, shred, and with after the abundant homogenate of glass homogenizer, add ultrapure water, sealed membrane is sealed and is placed in 4 ℃ of refrigerators, place 1.5～2h, every 15min stirs once during this time, then at 4 ℃, centrifugal 1～1.5h under 10000～12000r/min condition, take out supernatant liquor, centrifugal 0.8～1h under similarity condition again, collect supernatant liquor and obtain Chinese celestial shadow sea anemone tentacle crude venom.
3. the separation method of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of China according to claim 2, is characterized in that the solid-liquid ratio of the celestial shadow sea anemone tentacle of the China described in Chinese celestial shadow sea anemone tentacle crude venom preparation process and ultrapure water is 1:4～6.
4. the separation method of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of China according to claim 1, it is characterized in that: described China celestial shadow sea anemone tentacle crude venom through the method for ammonium sulfate segmentation salt precipitation is: accurately measure slightly poison of 100mL sea anemone, with reference to 0 ℃ of saturation table of ammonium sulfate, take and be ground to ammonium sulfate in small, broken bits, slowly join in 100mL sea anemone crude venom, then in 4 ℃ of refrigerator hold over night.Under 4 ℃, 10000～12000rmin
-1Centrifugal 35～45min, take out supernatant, and precipitation contains 0.15molL with pH=7.0
-1The 10mmolL of sodium-chlor
-1The PBS damping fluid redissolves, and according to the method described above sea anemone being saltoutd is 0～40%, 40%～60% and 60%～90% 3 ammonium sulfate saturation ratio section.
5. the molten cell polypeptide CCT-I of the celestial shadow sea anemone of China prepared according to the described separation method of any one in claim 1～4.
6. molten cell polypeptide CCT-I according to claim 5, is characterized in that described molten cell polypeptide CCT-I mainly all is comprised of at two polypeptide of 80kDa molecular weight.
7. the application of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of China claimed in claim 5 in preparing cancer therapy drug.
8. application according to claim 7, is characterized in that the application of the molten cell polypeptide CCT-I of the celestial shadow sea anemone of described China in the medicine for preparing anti-cervical cancer, anti-lung cancer or anti-liver cancer.
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| CN105030838A (en) * | 2015-05-31 | 2015-11-11 | 浙江海洋学院 | Preparing method of sea anemone crude extract and anti-tumor application of sea anemone crude extract |
| CN112458138A (en) * | 2020-11-30 | 2021-03-09 | 海南医学院 | Preparation method and application of actinia violaceus enzymatic hydrolysis polypeptide |
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| US20020077454A1 (en) * | 2000-05-09 | 2002-06-20 | Xianxhang Yu | Therapeutic pore-forming peptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020077454A1 (en) * | 2000-05-09 | 2002-06-20 | Xianxhang Yu | Therapeutic pore-forming peptides |
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| ANDERLUH G.等: "Cytolytic peptide and protein toxins from sea anemones (Anthozoa:Actiniaria)", 《TOXICON》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105030838A (en) * | 2015-05-31 | 2015-11-11 | 浙江海洋学院 | Preparing method of sea anemone crude extract and anti-tumor application of sea anemone crude extract |
| CN112458138A (en) * | 2020-11-30 | 2021-03-09 | 海南医学院 | Preparation method and application of actinia violaceus enzymatic hydrolysis polypeptide |
| CN112458138B (en) * | 2020-11-30 | 2023-05-05 | 海南医学院 | Preparation method and application of sea anemone enzymatic polypeptide |
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