CN101200496A - Method for preparing angiogenesis inhibition factor - Google Patents
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- CN101200496A CN101200496A CNA2006101552550A CN200610155255A CN101200496A CN 101200496 A CN101200496 A CN 101200496A CN A2006101552550 A CNA2006101552550 A CN A2006101552550A CN 200610155255 A CN200610155255 A CN 200610155255A CN 101200496 A CN101200496 A CN 101200496A
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- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 7
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- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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Abstract
The present invention provides a preparation method of angiogenesis inhibitory factor. Dasyatis cartilage is firstly crashed, then extracted, roughly separated and purified, finally desalinated, frozen and dried, thus obtaining the dasyatis cartilage angiogenesis inhibitory factor, shortened as DCAIF-I; wherein, the purification adopts Hitrap DEAEFF ion-exchange column chromatography, Superdex 75 10/300 GL ion-exchange column chromatography and Shim-packVP-ODS HPLC ion-exchange column chromatography. The preparation method of angiogenesis inhibitory factor provided by the present invention provides a method of extracting the angiogenesis inhibitory factor from the dasyatis cartilage. Through experiment, the DCAIF-I obtained in the method can effectively inhibit the angiogenesis and has a certain concentration dependence. The preparation method is a method in which the angiogenesis inhibitory factor can be effectively and optimally distilled from the dasyatis cartilage.
Description
Technical field
That the present invention relates to is a kind of preparation method of Angiostatin, and being specifically has the method for protein that suppresses angiogenic action a kind of the preparation from red ray fish cartilage.
Background technology
The growth of primary tumor, invasion and shift the vasculogenesis all depend on tumour, suppress angiogenic growth, tumor vessel as the novel targets for the treatment of cancer, just might control growth of tumor and transfer effectively.Many investigators have found to have the active factor of the inhibition tumor growth that molecular weight do not wait to exist, all have certain angiogenesis inhibiting activity, and wherein the overwhelming majority comes from shark suft bone.As Chinese invention patent 98111074.6 " shark-cartrilage blood-vessel generation inhibitory factor and separation purification method thereof " a kind of shark-cartrilage blood-vessel generation inhibitory factor has been proposed promptly, this factor " is made through separation and purification by shark suft bone; its SDS-PAGE electrophoresis silver dyes and shows a band, and its molecular weight is 18000." separation purification method of this factor be " with shark suft bone through guanidine hydrochloride solution extracting 45-50 hour, the centrifugal 25-35 of 800rpm minute, with the acetone organic solvent fractionation precipitation of 45-65%; With Resource Q ion exchange chromatography, obtain 3 elution peaks, wherein, buffer A:20mM Tris-Cl, pH8.5, Buffer B:1M NaCI, 20mM Tris-Cl, pH7.6 collects the 3rd peak, carry out Sephacryl S-300 sieve chromatography after concentrating, obtain 3 elution peaks, collect the 3rd peak, carry out SDS-PAGE after concentrating and prepare electrophoresis, wash-out obtains 4 peaks, regathers the 3rd peak, concentrate dry freeze, promptly get white powder material SCAIF-I.”
Though red ray also belongs to chondrichthyes, its cartilage total resources more helps being used for preparing the cartilage Angiostatin far above shark.But, cause not forming as yet at present the preparation method of red ray cartilage Angiostatin because activated protein composition and textural difference in red ray and the shark suft bone are more.
Summary of the invention
At above-mentioned defective, technical problem to be solved by this invention is to set up a technology of extracting the cartilage Angiostatin from red ray cartilage, to propose a kind of preparation method of effective, optimized red ray cartilage Angiostatin.
The preparation method of Angiostatin provided by the invention pulverizes red ray cartilage earlier, again through extracting, rough segmentation, purifying, and last desalination, lyophilize obtains red ray cartilage Angiostatin, and its english abbreviation name is designated as DCAIF-I, wherein
Extracting is: the red ray cartilage that will pulverize earlier is in 1: (g: ratio mL) is positioned in the extract 4-6,2-(the N-morphine quinoline) ethyl sulfonic acid (MES), (weight/volume percent) ethylenediamine tetraacetic acid (EDTA) (EDTA) of 0.02% and the Guanidinium hydrochloride of 1.0-2.0mol/L that contain 0.05-0.1mol/L in the extract, subsequently the pH value is transferred to 5.4-6.2, in the 15-30 ℃ of 72h that vibrates down, again under 4 ℃, 10000r/min is centrifugal, abandons residue, and getting supernatant liquor is red ray cartilage extract;
Rough segmentation is: earlier red ray cartilage extract is obtained the red ray cartilage Angiostatin crude extract of molecular weight at 3-200KD through ultrafiltration, again red ray cartilage Angiostatin crude extract is made fractionation precipitation with acetone, the precipitation that obtains is that 7.0-7.6, concentration are that trihydroxy methyl aminomethane-hydrochloric acid (Tris-HCl) buffered soln of 0.02mol/L is dialysed with the pH value, obtains red ray cartilage Angiostatin raw product through lyophilize again;
Purifying is: get the Angiostatin raw product and carry out desalination with Hitrap 26/10 Desalting post earlier, carry out chromatography with Hitrap DEAE FF ion exchange column again, obtain 10 tangible detached peakses, collect the 1st peak; Carry out chromatography with Superdex75 10/300 GL post again, obtain 4 tangible detached peakses, collect the 1st peak; Carry out chromatography with Shim-pack VP-ODSHPLC post again, obtain 3 tangible detached peakses, collect the 1st peak.
The preparation method of Angiostatin provided by the invention, wherein when extracting, said under 4 ℃, centrifugal with 10000r/min, abandon residue and be divided into for two steps, promptly be earlier under 4 ℃, with the centrifugal 25min of 10000r/min, abandon residue, continue under 4 ℃ with supernatant liquor again, with the centrifugal 25min of 10000r/min, abandon residue again.
The preparation method of Angiostatin provided by the invention, wherein when rough segmentation, said is that acetone soln with the different concns of one-level concentration between 20%-80% carries out fractionation precipitation to red ray cartilage Angiostatin crude extract with acetone as fractionation precipitation, and all precipitations are merged the back dialyses.
The preparation method of Angiostatin provided by the invention wherein when purifying is, is 3-6ml/min with the elution speed of Hitrap DEAE FF ion-exchange chromatography; Be 0.5-1.2ml/min with the elution flow rate of Shim-packVP-ODS HPLC column chromatography with the elution speed of Superdex 75 10/300GL column chromatographies be 0.5-1.2ml/min, wherein moving phase is 35%～50% acetonitrile+0.05% trifluoroacetic acid (TFA).
The preparation method of Angiostatin provided by the invention, the DCAIF-I of gained is shown as a band through sodium laurylsulfonate-polyacrylamide gel (SDS-PAGE) electrophoresis-Xylene Brilliant Cyanine G (Coomassie blue) staining of 12%, molecular weight is 62KD, and iso-electric point is about 4.0～5.4.
The preparation method of Angiostatin provided by the invention, gained DCAIF-I makes to suppress the mensuration of chick chorioallantoic membrane (CAM) vasculogenesis, measuring method is with reference to He Guoan, Luo Jinxian, Zhang Tianyuan etc.: " improved chick chorioallantoic membrane technique-no air chamber is hatched method " [is recorded in " Zhongshan University's journal (natural science edition) ", 2003 the 2nd (the 42nd volume): 126-128 page or leaf], the filter paper size is 3mm * 3mm during mensuration; The application of sample preincubation time is 4 days; Application of sample amount: DCAIF-I is respectively 0.1g/L, 0.4g/L, 0.8g/L; 0.01mol/L the negative contrast of the phosphate buffered saline buffer of pH 7.6 (PBS); 0.4g/L the positive contrast of chondroitin sulfate (CS), the application of sample amount is 10 μ L.Continue hatching 24h behind the application of sample, to be the center to snack made with traditional Chinese medicines, 5mm is divided into 3 zones with CAM with the radius interval, counts each regional blood vessel number, and its statistic analysis result sees the following form:
Compare the difference between each dosage group and the control group, the result shows, PBS negative control group angiogenic growth is good, blood vessel quantity is many and close, CS positive controls angiogenic growth reduces in a large number by comparison, and caliber is thin and branch is few, and the DCAIF-I treatment group presents similar result to the CS positive controls, 3 interior blood vessel quantity in numeration zone all significantly reduce, and along with the increase blood vessel quantity of application of sample amount reduces more obviously.Experiment finds that also the angiogenesis inhibiting activity of DCAIF-I treatment group is stronger, and the effect that the DCAIF-I treatment group suppresses vasculogenesis is obvious, and the blood vessel occurrence of large-area on the CAM is faded, and branch ruptures, and vessel density reduces.Along with the increase of DCAIF-I amount, the quantity of the last blood vessel of CAM reduces more obvious.0.1g/L DCAIF-I the inhibiting rate of CAM is reached 56%, to suppress effect suitable with the vasculogenesis of the CS of 0.4g/L.Show that DCAIF-I can significantly suppress the CAM new vessel and generate, it is strong that the DCAIF-I blood vessel suppresses the more existing angiostatin CS of activity, and the inhibition effect has certain concentration dependent.
The preparation method of Angiostatin provided by the present invention has proposed a method of extracting Angiostatin from red ray cartilage, and prepared DCAIF-I can effectively suppress vasculogenesis through test, and certain concentration dependent arranged.This preparation method be a kind of effectively, optimization, the brief method of from red ray cartilage, extracting Angiostatin.
Description of drawings
Fig. 1, the detached peaks figure that is obtained when carrying out ion-exchange with Hitrap DEAE FF ion exchange column, wherein transverse axis is elution time (min), and longitudinal axes is absorbance (mAU), and curve A 2801 is an elution curve;
Fig. 2, the detached peaks figure that is obtained when carrying out chromatography with Superdex 75 10/300GL posts, wherein transverse axis is elution time (min), and longitudinal axes is absorbance (mAU), and curve A 2802 is an elution curve;
Fig. 3, the detached peaks figure that is obtained when carrying out chromatography with Shim-pack VP-ODS HPLC post, wherein transverse axis is elution time (min), and longitudinal axes is absorbance (V), and curve A 2803 is an elution curve;
Fig. 4, usefulness SDS-PAGE electrophoresis-coomassie brilliant blue staining Faxian diagrammatic sketch, the longitudinal axis is molecular weight (KD), and wherein swimming lane 1 (1) is the DCAIF-I electrophoretic band, and swimming lane 2 (2) is a standard molecular weight protein;
Fig. 5, work suppress the comparison diagram as a result of the mensuration of CAM vasculogenesis, wherein 3 is the photo of an example in the PBS negative control group, 4 is the photo of an example in the CS positive controls, 5 is the photo of an example in the 1.0g/L DCAIF-I test group, 6 is the photo of an example in the 4.0g/L DCAIF-I test group, and 7 is the photo of an example in the 8.0g/L DCAIF-I test group.
Embodiment
The preparation method of the Angiostatin that present embodiment provides, its process is
1, pulverizes red ray cartilage.
2, red ray cartilage has particle 1000g and 1000mL extract to mix, 8mol MES, 20g EDTA160mol Guanidinium hydrochloride are arranged in the extract, pH value with this miscellany transfers to 6 subsequently, in 20 ℃ of 72h that vibrate down, centrifugal 25min under 4 ℃, 10000r/min rotating speed abandons residue again, continues centrifugal 25min under 4 ℃, 10000r/min rotating speed with the gained supernatant liquor again, abandon residue again, obtain supernatant liquor.
3, the 2nd step gained supernatant liquor is obtained the red ray cartilage Angiostatin crude extract of molecular weight at 3-200KD through ultrafiltration, use 25% successively, 35%, 40%, 50%, 60%, the acetone of 70%, 80% (weight/volume percent) carries out fractionation precipitation to the red ray cartilage of gained crude extract, after the precipitation of gained is mixed, dialyse with the Tris-HCl buffered soln that the pH value is 7.3, concentration is 0.02mol/L, obtain the DCAIF-I raw product through lyophilize again;
4, get the DCAIF-I raw product
4.1, carry out desalination with Hitrap 26/10 Desalting post;
4.2, carry out chromatography with Hitrap DEAE FF ion exchange column, elution speed is 5ml/min, obtains 10 tangible detached peakses, collects the 1st peak;
4.3, carry out chromatography with Superdex 75 10/300GL posts, elution speed is 0.9ml/min, obtains 4 obvious detached peakses, collects the 1st peak;
4.4, carry out chromatography with Shim-pack VP-ODS HPLC post, elution flow rate is 0.9ml/min, wherein moving phase is 40% acetonitrile+0.05%TFA, obtains 3 obvious detached peakses, collects the 1st peak.
5, carry out desalination with Hitrap 26/10 Desalting post.
6, lyophilize obtains the DCAIF-I crystal.
The DCAIF-I of gained is shown as a band through SDS-PAGE electrophoresis-coomassie brilliant blue staining method of 12%, and molecular weight is 62KD.
Claims (4)
1. the preparation method of an Angiostatin is characterized in that earlier red ray cartilage being pulverized, again through extracting, rough segmentation, purifying, and last desalination, lyophilize obtains red ray cartilage Angiostatin, and its english abbreviation name is designated as DCAIF-I, wherein
Extracting is: the red ray cartilage that will pulverize earlier is in 1: the ratio of 4-6 is positioned in the extract, this ratio is weight and volume ratio, unit is g: mL, MES, the weight/volume percent that contains 0.05-0.1mol/L in the said extract is 0.02% EDTA and the Guanidinium hydrochloride of 1.0-2.0mol/L, subsequently the pH value transferred to 5.4-6.2, in 15-30 ℃ of vibration 72h down, again under 4 ℃, 10000r/min is centrifugal, abandons residue, and getting supernatant liquor is red ray cartilage extract;
Rough segmentation is: earlier red ray cartilage extract is obtained the red ray cartilage Angiostatin crude extract of molecular weight at 3-200KD through ultrafiltration, again red ray cartilage Angiostatin crude extract is made fractionation precipitation with acetone, the precipitation that obtains is that 7.0-7.6, concentration are that the Tris-HCl buffered soln of 0.02mol/L is dialysed with the pH value, obtains red ray cartilage Angiostatin raw product through lyophilize again;
Purifying is: get the Angiostatin raw product and carry out desalination with Hitrap 26/10 Desalting post earlier, carry out chromatography with Hitrap DEAE FF ion exchange column again, obtain 10 tangible detached peakses, collect the 1st peak; Carry out chromatography with Superdex75 10/300GL post again, obtain 4 tangible detached peakses, collect the 1st peak; Carry out chromatography with Shim-pack VP-ODSHPLC post again, obtain 3 tangible detached peakses, collect the 1st peak.
2. the preparation method of Angiostatin as claimed in claim 1, it is characterized in that when extracting, said under 4 ℃, centrifugal with 10000r/min, abandon residue and be divided into for two steps, promptly be earlier under 4 ℃, with the centrifugal 25min of 10000r/min, abandon residue, continue under 4 ℃ with supernatant liquor again, with the centrifugal 25min of 10000r/min, abandon residue again.
3. the preparation method of Angiostatin as claimed in claim 1, its feature is when rough segmentation, said is that acetone soln with the different concns of one-level concentration between 20%-80% carries out fractionation precipitation to red ray cartilage Angiostatin crude extract with acetone as fractionation precipitation, and all precipitations are merged the back dialyses.
4. the preparation method of Angiostatin as claimed in claim 1, its feature are 3-6ml/min with the elution speed of HitrapDEAE FF ion-exchange chromatography when purifying is; Elution speed with Superdex 75 10/300GL column chromatographies is 0.5-1.2ml/min; Elution flow rate with Shim-pack VP-ODS HPLC column chromatography is 0.5-1.2ml/min, and wherein moving phase is 35%～50% acetonitrile+0.05%TFA.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104873952A (en) * | 2015-05-11 | 2015-09-02 | 浙江海洋学院 | Application of dasyatis akajei cartilage polypeptide angiogenesis inhibition factor |
| CN104877009A (en) * | 2015-05-11 | 2015-09-02 | 浙江海洋学院 | Dasyatis akajei cartilage polypeptide angiogenesis inhibitor |
| CN104928338A (en) * | 2015-05-11 | 2015-09-23 | 浙江海洋学院 | Method for quickly preparing dasyatis akajei cartilage polypeptide angiogenesis inhibitor |
-
2006
- 2006-12-12 CN CNA2006101552550A patent/CN101200496A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104873952A (en) * | 2015-05-11 | 2015-09-02 | 浙江海洋学院 | Application of dasyatis akajei cartilage polypeptide angiogenesis inhibition factor |
| CN104877009A (en) * | 2015-05-11 | 2015-09-02 | 浙江海洋学院 | Dasyatis akajei cartilage polypeptide angiogenesis inhibitor |
| CN104928338A (en) * | 2015-05-11 | 2015-09-23 | 浙江海洋学院 | Method for quickly preparing dasyatis akajei cartilage polypeptide angiogenesis inhibitor |
| CN104877009B (en) * | 2015-05-11 | 2020-08-11 | 浙江海洋学院 | Dasyatis akajei cartilage polypeptide angiogenesis inhibiting factor |
| CN104928338B (en) * | 2015-05-11 | 2020-09-08 | 浙江海洋学院 | Method for rapidly preparing dasyatis akajei cartilage polypeptide angiogenesis inhibiting factor |
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