CN101260151A - Separation and primary structure of sea purse blood vessel generation inhibitive factor 1, and application thereof in preparing blood vessel generation inhibiting medicament and antineoplastic medicam - Google Patents
Separation and primary structure of sea purse blood vessel generation inhibitive factor 1, and application thereof in preparing blood vessel generation inhibiting medicament and antineoplastic medicam Download PDFInfo
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Abstract
The invention discloses a first-level structure of sea purse angiogenesis inhibitive factor 1 which is obtained from the sea purse and strong in resisting the growth and transfer of tumor, that is the combination and sequence of amino acids, wherein the first-level structure of sea purse angiogenesis inhibitive factor is formed by 381 amino acid residues; in the preparation of the sea purse angiogenesis inhibitive factor 1, the column chromatography is conducted according to the following order: 1) Anion Q Sepharose Fast Flow column chromatography; 2) Butyl Sepharose Fast Flow column chromatography; 3) SOURCE RPC column purification. The sea purse angiogenesis inhibitive factor 1 can effectively inhibit the angiogenesis of the chick chorioallantoic membrane and the angiogenesis of the chick chorioallantoic membrane induced by tumor cells, the angiogenesis of tumor tissues, the growth of the Lewis lung cancer of small mice and the transfer of the Lewis lung cancer and the melanoma B16 of small mice. The sea purse angiogenesis inhibitive factor 1 can inhibit expressions of the promoting angiogenesis factor such as the blood vessel endothelial cell growth factor, etc., and the transfer promotion factor CD44v6 and ErBb2 and strengthen expressions of the transfer inhibitive factor nm23. The sea purse angiogenesis inhibitive factor 1 can be used to prepare drugs inhibiting the angiogenesis and preventing tumors.
Description
Separation, the primary structure of the ray Angiostatin 1 of a kind of powerful antitumor growth, transfer is amino acid composition and sequence and the pharmaceutical use that is used to prepare anti-curing oncoma thereof.
Technical field: separation and purification, the primary structure of the ray Angiostatin 1 that the present invention relates to a kind of powerful antitumor growth that comes from ray, shifts are that amino acid is formed and sequence, and suppress application in the medicine of vasculogenesis, anti-curing oncoma in preparation.
Background of invention
Vasculogenesis is from former already present blood vessel, as the process of capillary vessel and postcapillary venule generation neovascularity.The mechanism of control healthy tissues vasculogenesis can not be controlled the vasculogenesis of tumour, and tumor-blood-vessel growth is by tumor angiogenesis factor and the common regulation and control of angiogenesis inhibitor.
95% cancer all forms lump at last, is called solid tumor.The solid malignant cell can obtain nutrition by the dispersion of surrounding tissue in early days under the situation of no angiogenic growth, tumor size generally is no more than 2～3mm
3When volume increases to 2～3mm
3The time, tumour can not rely on disperse to obtain oxygen and nutritive substance merely, if there is not new capillary vessel to generate, tumor tissues will keep dormant state or degenerate.
The formation of neovascularity is the prerequisite of metastases, and the infiltration of tumour and transfer rely on tumor vascular generation.Along with the increase of tumor microvessel density, malignant potential such as tumor invasion transfer also obviously increase.Microvessel density is considered to predict an index of metastases, recurrence and prognosis.Tumor angiogenesis factor as vascular endothelial growth factor (VEGF), Prostatropin (bFGF), platelet derived growth factor (PDGF) etc., can promote the microvascular growth of tumor neogenetic.
Anti-angiogenic therapy is intended to suppress tumor vascular growth, makes tumour can not get enough nutrition and oxygen and atrophy.When the atrophy of tumour radius is arrived less than 2mm, when tumour cell is above apart from capillary vessel 0.2mm, oxygen can't arrive at tumour cell by dispersion, tumour cell can not obtain nutrition and oxygen from tumor vessel, also can't obtain nutrition and oxygen by dispersion, so tumor tissues takes place to degenerate or be in long-term resting stage from surrounding blood vessel.Compare with traditional methods of treatment, it is high and reach plurality of advantages such as target cell easily that angiogenesis inhibitor has an inheritance stability, specificity, and each link of tumor-blood-vessel growth and regulatory factor thereof all can be used as the action target spot of angiogenesis inhibitor.According to the mechanism of action difference, angiogenesis inhibitor can be divided into substantially: suppress basement membrane degradation, inhibition of endothelial cell proliferation function, blocking-up angiogenesis factor, blocking-up endothelial cell specific integration element and nonspecific action mechanism etc.
In recent years, the scientific worker of China also begins to carry out the searching and the research work of angiogenesis inhibitor, has obtained gratifying progress.Institute of materia medica, Chinese Academy of Sciences Shanghai finds to derive from new compound PAA, DYB, the ZH-1 of natural product etc. and has the new vessel of inhibition nucleus formation, and the Endostatin that Zhongshan University is is researching and developing has entered clinical trial.The many pieces of reports that learning value is arranged have also appearred on the periodical.
The scientist of U.S. gene engineering institute successfully cloned VEGF in 1989, produced the antibody A vastln of VEGF again in 1993, and drugs approved by FDA Avastln listing in 2004 is used for first-line treatment transitivity knot, the rectum cancer.Avastln is a kind of new antitumor drug of angiogenesis inhibitor, is a kind of humanized's monoclonal antibody, and its mechanism of action is to suppress new vessel to generate, thereby the blood supply of blocking-up tumor growth suppresses tumor growth and transfer.Be used in combination with traditional chemotherapeutics, can prolong the colorectal carcinoma survival time of patients, the therapy of this " tumour hungry to death " is chosen as 2003 ten big sciences by U.S.'s " science " magazine and breaks through.Wang Ning etc. unite or do not unite Fluracil therapeutic intervention is carried out in the tumor growth and the transfer of human cancer of the stomach nude mice original position implant cast by VEGF monoclonal antibody Avastln, inquire into the influence of Avastln to cancer of the stomach growth, transfer and vasculogenesis.Found that, compare that the Fluracil list obviously reduces with group and drug combination group orthotopic transplantation tumor weight with group, Avastln list with control group. tumour inhibiting rate is respectively 37.52%, 58.76% and 98.51%.Avastln single with group and drug combination group microvessel density all than the obvious minimizing of control group (8.40 ± 1.26 and 7.20 ± 1.23vs 15.30 ± 1.06).Avastln and Fluracil the combined utilization not only growth-inhibiting to tumor in situ are the most obvious, and transfer has significant inhibitory effect to liver.Therefore, it is more remarkable to the result of treatment of cancer of the stomach that Avastln and conventional cell poison the medication combined application of treatment.
Anti-angiogenic therapy treatment solid tumor has been obtained huge progress at present, and with respect to traditional tumor therapeuticing method many advantages is arranged, and produces resistance as being difficult for, and can suppress metastases, is target with the blood vessel, and is effective or the like to kinds of tumors.But still have a lot of problems to need to be resolved hurrily in clinical application: (1) life cycle is long, and dosage is big; (2) heavy dose of long-term prescription strengthens drug toxicity; (3) expense is higher relatively, and the patient is difficult to bear; (4) medicine transformation period weak point in vivo, the big and number of times frequency of the using dosage of medicine.The utmost point need be sought the potent Angiostatin that can overcome above-mentioned shortcoming at present.
Tumor-blood-vessel growth is complex physical, a pathologic process that the many somatomedins of audient are regulated, in these somatomedins, VEGF is the angiogenic growth factor that present known action is the strongest, specificity is the highest, it can increase vascular permeability, promote tumor vessel to form, the propagation of tumor vascular endothelial cell and migration etc. are played a significant role.With the arbitrary link in VEGF and the receptor acting approach thereof is target spot, and blocking VEGF can reach the purpose of containment tumor growth and transfer to tumor vascular effect.VEGF is called vascular permeability factor again, is the homodimer glycoprotein that is made of by the disulfide linkage commissure two identical peptide chains.Its biological effect is to mediate by the specific receptors that is present in endothelial cell surface (VEGFR).
Avastln is the antibody of a kind of anti-VEGF of synthetic.The principle of Avastln is to suppress growth of tumor by anti-VEGF.1 of ray Angiostatin is a kind of natural product of separating from marine organisms, and it not only acts on VEGF, also acts on other a plurality of target spots that relate to vasculogenesis and tumor growth, transfer.
Summary of the invention
The primary structure that the object of the present invention is to provide a kind of ray Angiostatin 1 is that amino acid is formed and sequence, has the biological activity that this seed amino acid is formed and 1 demonstration of the ray Angiostatin of sequence suppresses vasculogenesis, powerful antitumor growth by force and shifts.
Purpose of the present invention still is to provide the method for separation, purifying ray Angiostatin 1.
The present invention also aims to provide the physics and chemistry and the pharmacological properties of ray Angiostatin 1, to be used in the preparation medicine.
A kind of strong inhibition vasculogenesis, the primary structure of the ray Angiostatin 1 of powerful antitumor growth and transfer is made of in the following order 381 amino-acid residues:
MPFGNTHNKWKLNYSAEQEFPDLKHHNNHMAKAL＝
TLD1YKQLRDKETPSGFTLDDV1QTGVDNPGHPF1MTVGCVAGDEE
SYEVFKALFDPV1QDRHGGYKPTDKHKTD＝
LNHENLKGGDDLDPNYVLSSRVRTGRS1KG1ALPPHCSRGERRLVE
KLCLEGLATLTGEFQGKYYPLTTMSDAEQ＝
QQL1DDHFLFDKPVSPLLLASGMARDWPDARG1WHNNDKTFLVW
VNEEDHLRV1SMQKGGNMKEVFRRFCVGLKK＝
1EEFFVKAGRGFMWNEHLGYVLTCPSNLGTGLRGGVHVK1PHLCK
HEKFEEVLKRTRLQKRGTGGVDTAAVGSVY＝
D1SNADRLGFSEVEQVQMVVDGVKLM1EMEKVLEKGKS1DNLMP
AQK
Optimal pH is 7.0-9.0, and stable pH is 4.0-9.0.
Ray, a class selachian, body is flat, rounded or rhombus, smooth no squama.Anal fin disappears, no fish glue, and kind is a lot, as hole ray, sawfish, guitarfish, spot ray, electric ray etc.Ray, holobenthic ray class.Body is flat, and is slightly rounded or square, and it is whiplike that tail is, and telson is arranged.Kind is a lot of, yellow ray, black ray is arranged, red ray etc.China is coastal all to produce.Fishing, limnetic ray, of the same race with ray.The fishing of Longzhou that China finds in recent years promptly belongs to this type of, and is of the same race and be the fresh water ray with the red ray of marine products.The ray class all can be used as the raw material for preparing ray Angiostatin 1, holobenthic ray (ray) You Jia.
The separation and purification of ray Angiostatin 1 is for carrying out following column chromatography successively:
1) Anlon Q Sepharose Fast Flow column chromatography;
2) H1C Butyl Sepharose Fast Flow column chromatography;
3) SOURCE RPC column chromatography.
Above-mentioned inhibition vasculogenesis, the characteristics of ray Angiostatin 1 separation purification method of neoplasm growth and transfer are: the ray tissue adds 0.15mol/L NaCl, 0.01～0.02mol/L TrlsHCl, pH7.0～9.0 damping fluid homogenate, centrifugal (4 ℃ of homogenate, 16000g) 1 hour, collect supernatant liquor and add (the NH of saturation ratio 20～80%
4)
2SO
4Fractional separation.The classification collecting precipitation, after the desalination with 0.01～0.02mol/L, the TrlsHCl damping fluid dissolving of pH8.0～8.8, through Anlon Q chromatography purification, chromatography condition is: mobile phase A is 0.01～0.02mol/L, the Trls-HCl damping fluid of pH7.0～9.0, B liquid adds 1mol/L NaCl for A liquid, flow velocity is 15ml/mln, binary gradient elution, B%
Through Anlon Q column chromatography purification obtained component again through H1C Butyl Sepharose Fast Flow column chromatography, chromatography condition is: mobile phase A liquid is 0.01～0.02mol/L, the Trls-HCl damping fluid of pH7.0～9.0, B liquid is the A liquid that contains 3mol/L NaCl, flow velocity is 10ml/mln, the binary gradient elution, B%
Through H1C Butyl Sepharose Fast Flow column chromatography obtained component again through SOURCE RPC column purification, chromatography condition is: mobile phase A liquid is the aqueous solution that contains 0.1%TFA, and B is the acetonitrile solution that contains 0.1%TFA, and flow velocity is 0.5ml/mln, the binary gradient elution, B%
The high pressure liquid chromatography (HPLC) result of purifying gained ray Angiostatin 1 is simple spike (Fig. 1), and the SDS-PAGE electrophoresis result is single band (Fig. 2).
The biological activity assay result of ray Angiostatin 1 shows: it does not have direct killing effect to tumour cell, significantly suppresses the chick chorioallantoic membrane vasculogenesis of chick chorioallantoic membrane and tumor cell induction, significantly suppresses tumor growth and metastases.
One, the test of pesticide effectiveness
1. the influence of 1 pair of vasculogenesis of ray Angiostatin
1.1 the influence of 1 pair of chick chorioallantoic membrane vasculogenesis of ray Angiostatin
Select cleaning, eggshell homogeneous, air chamber to plant egg uniformly, 1 week of hatching in 19200 types intelligence incubator, open a diameter 1cm aperture at the embryo head end under the aseptic condition, form false air chamber.The filter paper that 100 μ l ray Angiostatins, 1 soup was soaked places on the chorioallantoic membrane in the air chamber, seals with scotch tape.Put in 38 ℃ of constant incubators and cultivate, add soup 100 μ l behind 24h, the 48h respectively again on filter paper, control group then adds the equivalent solvent.Fix 12 minutes respectively with acetone and dehydrated alcohol behind the 72h.Cut the film that is placed with filter paper and put on the slide glass, abandon or adopt filter paper, the branch of 5 visuals field of microscopically picked at random counting visible vessels counts and takes a picture.。The result shows, ray Angiostatin 1 significantly suppresses the chick chorioallantoic membrane vasculogenesis, and be dose-dependence (table 1, Fig. 3).
The restraining effect of 1 pair of chick chorioallantoic membrane vasculogenesis of table 1 ray Angiostatin
1.2 the influence of the low differentiation of 1 couple of people of ray Angiostatin nasopharyngeal carcinoma cell (CNE-2Z) inductive chick chorioallantoic membrane vasculogenesis
Select cleaning, eggshell homogeneous, air chamber to plant egg uniformly, 1 week of hatching in 19200 types intelligence incubator, open a diameter 1cm aperture at the embryo head end under the aseptic condition, form false air chamber.Inoculation CNE-2Z cell (1.9 * 10
6/ only), seal with scotch tape.Put in the incubator and to cultivate after 4 days, the filter paper that an aperture is made a call to by central authorities places on the chorioallantoic membrane in the air chamber, adds ray Angiostatin 1 soup 100 μ l on filter paper, and control group then adds the equivalent solvent, and every day 1 time, administration is 4 times altogether.Remove scotch tape then, fix 12 minutes respectively with acetone and dehydrated alcohol.Cut the chorioallantoic membrane that is placed with filter paper and put on the slide glass, abandon or adopt filter paper, the branch of 5 visuals field of microscopically picked at random counting visible vessels counts and takes a picture.The result shows that ray Angiostatin 1 significantly suppresses the chick chorioallantoic membrane vasculogenesis of CNE-2Z cell induction, and relevant with dosage.(table 2, Fig. 4).
The restraining effect of the chick chorioallantoic membrane vasculogenesis of 1 pair of CNE-2Z cell induction of table 2 ray Angiostatin
1.3 the influence that 1 pair of short angiogenesis factor of ray Angiostatin is expressed
Route of administration is that (20.0 * 14mg/kgd) abdominal injections, solid tumor sample are BALB/c nude mouse Lewis lung cancer tissue to ray Angiostatin 1.Occur yellowish extremely isabelline fine particulate in cytoplasm and the after birth and be colored as positive cell, immunohistochemical methods method detected result shows, the expression of three kinds of short angiogenesis factors such as ray Angiostatin 1 couple of VEGF, bFGF and PDGF has obvious suppression effect (table 3, Fig. 5-7).
The restraining effect that 1 pair of short angiogenesis factor of table 3 ray Angiostatin is expressed
1.4 1 pair of Mice Bearing Lewis Lung Cancer of ray Angiostatin is organized the influence of microvessel density (MVD)
Route of administration is that (20.0 * 14mg/kgd) abdominal injections, solid tumor sample are BALB/c nude mouse Lewis lung cancer tissue to ray Angiostatin 1.Vascular endothelial cell through the CD31 immunohistochemical staining is brown.The groupization of exempting from service method detected result shows that a large amount of hyperplasia of capillary vessel in the control group Lewis lung cancer tissue are enriched in the borderline tumor place, the capillary blood vessel skewness, and form is irregular, and quantity differs greatly, and is heterogeneous, and microvessel count is 18.2 ± 3.2.1 group of ray Angiostatin only between cancer nests matter the minute quantity new capillary vessel is arranged, microvessel count is 6.5 ± 1.5.The microvessel count that the ray Angiostatin is 1 group is starkly lower than control group (Fig. 8).
2. the neoplasm growth of ray Angiostatin 1 and anti metastasis effect
2.1 1 pair of Mice Bearing Lewis Lung Cancer growth of ray Angiostatin and the influence of shifting
The BALB/c nude mice is divided into control group, the positive (endoxan) control group and experimental group at random, and 8 every group, male and female half and half.Every mouse back subcutaneous vaccination 0.2ml (4 * 10
6) the Lewis lung cancer cell.Behind the tumor inoculation the 7th day, experimental group abdominal injection or irritate stomach various dose ray Angiostatin 1 solution, every day 1 time, totally 14 times; Control group abdominal injection or filling stomach equivalent solvent; The positive controls intraperitoneal injection of cyclophosphamide, 1 time weekly, totally 2 times.Sacrificed mouse on the 21st day, and peeled off tumour and weigh (being fixed for immunohistochemical experiment), dissect, observe and record hepatic metastases joint footing with 10% neutral formalin.The result shows that no matter be that abdominal injection or the tumour of irritating 1 group of stomach ray Angiostatin are obviously little than the control group volume, substrate is narrow, easily peels off.Hepatic metastases kitchen range quantity is few, and volume is little.Ray Angiostatin 1 obviously suppresses the growth and the hepatic metastases of Mice Bearing Lewis Lung Cancer, and this effect is relevant with dosage.(table 4-7, Fig. 9).
Table 4 ray Angiostatin 1 abdominal injection is to the restraining effect of nude mouse Lewis lung cancer growth
*Endoxan respectively at tumor inoculation after the 7th day, the 14th day abdominal injection.
Table 5 ray Angiostatin 1 abdominal injection is to the restraining effect of Mice Bearing Lewis Lung Cancer hepatic metastases
*Endoxan respectively at tumor inoculation after the 7th day, the 14th day abdominal injection.
Table 6 ray Angiostatin 1 is irritated the restraining effect of stomach to the growth of nude mouse Lewis lung cancer
*Endoxan respectively at tumor inoculation after the 7th day, the 14th day abdominal injection.
Table 7 ray Angiostatin 1 is irritated stomach to nude mouse Lewis lung cancer effect on liver metastasis
2.2 the BALB/c nude mice that influences that 1 pair of mouse melanin tumor cell of ray Angiostatin (B16) shifts is divided into control group, the positive (endoxan) control group and experimental group at random, and 8 every group, male and female half and half.Every caudal vein inoculation 0.2ml (3 * 10
6) the B16 cell, tumor inoculation same day, experimental group abdominal injection or irritate stomach various dose ray Angiostatin 1 solution, every day 1 time, totally 14 times; Control group abdominal injection or filling stomach equivalent solvent; The positive controls intraperitoneal injection of cyclophosphamide, 1 time weekly, totally 2 times.Sacrificed mouse on the 15th day, and got the lung tissue meter and shift the joint footing.The result shows that ray Angiostatin 1 abdominal injection or filling stomach all significantly suppress nude mouse melanoma cell lung and shift, and relevant with dosage, and its effect is better than endoxan commonly used clinically.Ray Angiostatin 1 and endoxan have synergy (table 8,9, Figure 10).
The influence that table 8 ray Angiostatin 1 abdominal injection shifts nude mouse melanin tumour b16 cell lung
*Endoxan is respectively at tumor inoculation same day, the 7th day abdominal injection in inoculation back.
Table 9 ray Angiostatin 1 is irritated the influence that stomach shifts nude mouse melanin tumour b16 cell lung
*Endoxan is respectively at tumor inoculation same day, the 7th day abdominal injection in inoculation back.
2.3 the influence that 1 pair of Mice Bearing Lewis Lung Cancer transfer factor of ray Angiostatin is expressed
Route of administration is that (20.0 * 14mg/kgd) abdominal injections, solid tumor sample are BALB/c nude mouse Lewis lung cancer tissue to ray Angiostatin 1.Short transfer factor CD44v6 and ErBb2 are expressed in cytolemma and cytoplasm, are colored as the master with after birth, and it is painted a small amount of endochylema to occur; Press down transfer factor nm23 and mainly be expressed in cytoplasm.Occur yellowish extremely isabelline fine particulate in cytoplasm and the after birth and be colored as positive cell, the result of immunohistochemical methods shows, after giving injected in mice ray Angiostatin 1, the expression of CD44v6 and ErBb2 in the Lewis lung cancer cell is reduced, the expression rising (table 10, Figure 11-13) of nm23-H1.
The influence that table 10 ray Angiostatin 1 abdominal injection is expressed the Mice Bearing Lewis Lung Cancer transfer factor
Two, toxicological experiment
Acute toxicity test
Adopt healthy Kunming mouse, body weight 19～22 grams, male and female half and half.The preceding fasting 4h of administration (po) adopts multiple dosing method in the 24h, takes the circumstances into consideration to supply with feed therebetween, can't help water.Dosage is 100 times of (1p, 20mg/kg of ray Angiostatin 1 effective dose; Po 40mg/kg), observed 7 days after the administration.Viewing duration writes down the toxic reaction situation of animal and the distribution of dead animal day by day.Because do not find dead mouse, can not survey LD
50, change then and do " mtd test ", disposable 300 times of (1p, 60mg/kg with ray Angiostatin 1 effective dose; Po, 120mg/kg) 0.5ml and 0.8ml give 20 mouse peritoneal injections respectively or irritate stomach, observe 14 days, and the result does not find mouse toxicity reaction or dead yet.
Three, stability
Adopt Freeze Drying Technique to make ray Angiostatin 1 sample, preserved 1 year in-20 ℃ of refrigerators, its biological activity (suppress vasculogenesis, suppress vitro culture oncocyte and mice transplanted tumor growth) does not have obviously and reduces.
Ray Angiostatin 1 mixes with pharmaceutically useful carrier or dissolves, make tablet, granule, pulvis, capsule etc., or the injection of liquid form, can be used for various entity shape tumours, as the treatment of esophagus cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, colorectal carcinoma, the rectum cancer, cervical cancer, ovarian cancer, mammary cancer, nasopharyngeal carcinoma, oral carcinoma, lip cancer, skin carcinoma, bladder cancer, chorioepithelioma, carcinoma of parotid gland, lymphoma, osteoma, melanoma and various intracranial tumorss etc.
Description of drawings
Fig. 1: the high pressure liquid chromatography (HPLC) figure of ray Angiostatin 1
Fig. 2: the SDS-PAGE electrophorogram of ray Angiostatin 1
Fig. 3: the restraining effect of 1 pair of chick chorioallantoic membrane vasculogenesis of ray Angiostatin
Fig. 4: the restraining effect of the low differentiation of 1 couple of people of ray Angiostatin nasopharyngeal carcinoma cell (CNE-2Z) inductive chick chorioallantoic membrane vasculogenesis
Fig. 5: the restraining effect that 1 pair of vascular endothelial growth factor of ray Angiostatin (VEGF) is expressed
Fig. 6: the restraining effect that 1 pair of Prostatropin of ray Angiostatin (bFGF) is expressed
Fig. 7: the restraining effect that 1 pair of Thr6 PDGF BB of ray Angiostatin (PDGF) is expressed
Fig. 8: the influence of 1 couple of mouse Lewls of ray Angiostatin lung cancer microvessel density (MVD)
Fig. 9: the restraining effect of 1 couple of nude mouse Lewls of ray Angiostatin lung cancer hepatic metastases
Figure 10: the restraining effect that 1 pair of nude mouse melanin tumour b16 of ray Angiostatin cell lung shifts
Figure 11: the restraining effect that the short transfer factor CD44v6 of 1 couple of nude mouse Lewls of ray Angiostatin lung cancer expresses
Figure 12: the restraining effect that the short transfer factor ErBb2 of 1 couple of nude mouse Lewls of ray Angiostatin lung cancer expresses
Figure 13: 1 couple of nude mouse Lewls of ray Angiostatin lung cancer presses down the enhancement that transfer factor nm23-H1 expresses
Claims (3)
1. one kind comes from the powerful antitumor growth of ray, the ray Angiostatin 1 that shifts, and its primary structure is that amino acid is formed and order.Ray Angiostatin 1 is made of in the following order 381 amino-acid residues:
MPFGNTHNKWKLNYSAEQEFPDLKHHNNHMAKAL＝
TLD1YKQLRDKETPSGFTLDDV1QTGVDNPGHPF1MTVGCVAGDEE
SYEVFKALFDPV1QDRHGGYKPTDKHKTD＝
LNHENLKGGDDLDPNYVLSSRVRTGRS1KG1ALPPHCSRGERRLVE
KLCLEGLATLTGEFQGKYYPLTTMSDAEQ＝
QQL1DDHFLFDKPVSPLLLASGMARDWPDARG1WHNNDKTFLVW
VNEEDHLRV1SMQKGGNMKEVFRRFCVGLKK＝
1EEFFVKAGRGFMWNEHLGYVLTCPSNLGTGLRGGVHVK1PHLCK
HEKFEEVLKRTRLQKRGTGGVDTAAVGSVY＝
D1SNADRLGFSEVEQVQMVVDGVKLM1EMEKVLEKGKS1DNLMP
AQK
2. the separating and purifying technology of ray Angiostatin 1.The separation and purification of ray Angiostatin 1 is carried out column chromatography by following order:
1) Anion Q Sepharose Fast Flow column chromatography, chromatography condition is: mobile phase A liquid is 0.01～0.02mol/L, the Tris-HCl damping fluid of pH7.0～9.0, B liquid adds 1mol/L NaCl for A liquid, and flow velocity is 15ml/min, binary gradient elution, B%
2) Butyl Sepharose Fast Flow column chromatography, chromatography condition is: mobile phase A liquid is 0.01～0.02mol/L, and the Tris-HCl damping fluid of pH7.0～9.0, B liquid are the A liquid that contains 3mol/L NaCl, and flow velocity is 10ml/min, binary gradient elution, B%
3) SOURCE RPC column purification, chromatography condition is: mobile phase A liquid is the aqueous solution that contains 0.1%TFA, and B is the acetonitrile solution that contains 0.1%TFA, and flow velocity is 0.5ml/min, adopts the binary gradient elution, B%
3. the application of ray Angiostatin 1 in the medicine of preparation inhibition vasculogenesis and anti-curing oncoma.
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| CN105481964A (en) * | 2014-06-06 | 2016-04-13 | 张勇 | Ray angiogenesis inhibiting factor (1) functional domain variant JG60 and application thereof |
| CN105481962A (en) * | 2014-06-06 | 2016-04-13 | 张勇 | Ray angiogenesis inhibiting factor (1) functional domain variant JG42 and application thereof |
| CN105541985A (en) * | 2014-06-06 | 2016-05-04 | 张勇 | Manta-angiogenesis-inhibitor-1 functional-area variant JG121 and application thereof |
| CN105541984A (en) * | 2014-06-06 | 2016-05-04 | 张勇 | Manta-angiogenesis-inhibitor-1 functional-area variant JG36 and application thereof |
| CN105713081A (en) * | 2014-06-06 | 2016-06-29 | 张勇 | Aptamers K17 of ray angiogenesis inhibiting factor 1 and screening method and application thereof |
| CN105481964B (en) * | 2014-06-06 | 2018-10-09 | 郑理辉 | 1 function region variants JG60 of ray Angiostatin and its application |
| CN105440118B (en) * | 2014-06-06 | 2018-10-16 | 泰州市百英生物科技有限公司 | 1 function region variants JG70 of ray Angiostatin and its application |
| CN105440117B (en) * | 2014-06-06 | 2018-10-26 | 付庆霞 | 1 function region variants JG55 of ray Angiostatin and its application |
| CN105418753B (en) * | 2014-06-06 | 2018-12-21 | 杭州晋建环保科技有限公司 | 1 function region variants JG217 of ray Angiostatin and its application |
| CN105418752B (en) * | 2014-06-06 | 2018-12-25 | 杭州晋建环保科技有限公司 | 1 function region variants JG89 of ray Angiostatin and its application |
| CN105418742B (en) * | 2014-06-06 | 2018-12-25 | 新昌县普达环保科技有限公司 | 1 function region variants JG135 of ray Angiostatin and its application |
| CN105713081B (en) * | 2014-06-06 | 2019-04-12 | 浙江药苑生物科技有限公司 | The aptamer K17 and its screening technique of ray Angiostatin 1 and application |
| CN105481965B (en) * | 2014-06-06 | 2019-04-26 | 浙江药苑生物科技有限公司 | 1 function region variants JG63 of ray Angiostatin and its application |
| CN105481966B (en) * | 2014-06-06 | 2019-05-28 | 广州中昱医学生物科技有限公司 | 1 function region variants JG175 of ray Angiostatin and its application |
| CN105481967B (en) * | 2014-06-06 | 2019-08-13 | 绍兴市高砚智生物科技有限公司 | 1 function region variants JG16 of ray Angiostatin and its application |
| CN105541985B (en) * | 2014-06-06 | 2019-08-16 | 绍兴市高砚智生物科技有限公司 | 1 function region variants JG121 of ray Angiostatin and its application |
| CN105481963B (en) * | 2014-06-06 | 2019-08-16 | 陈斯泽 | 1 function region variants JG50 of ray Angiostatin and its application |
| CN105461792B (en) * | 2014-06-06 | 2019-08-20 | 绍兴市高砚智生物科技有限公司 | 1 function region variants JG164 of ray Angiostatin and its application |
| CN105481962B (en) * | 2014-06-06 | 2019-08-20 | 嵊州市法克机械有限公司 | 1 function region variants JG42 of ray Angiostatin and its application |
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| CN101260151B (en) | 2012-05-09 |
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