CN102924578A - Anti-tumor polypeptide, preparation method and anti-tumor applications thereof - Google Patents
Anti-tumor polypeptide, preparation method and anti-tumor applications thereof Download PDFInfo
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Abstract
The invention relates to an anti-tumor polypeptide, a preparation method and anti-tumor applications thereof. A purpose of the present invention is to provide a polypeptide having an anti-tumor function and a preparation method thereof. A structure of the polypeptide comprises three histidine structures containing an endostatin N-terminal zinc ion binding site, a beta-pleated sheet structure and a serial RGD sequence. According to the invention, a chemical synthesis method for the polypeptide drug is disclosed, and a tumor proliferation inhibition effect of the polypeptide is proved.
Description
Technical field
The present invention relates to the polypeptide drug in biotechnological pharmaceutics field, and preparation method thereof with antitumor application.
Background technology
Tumour is a kind of polygene, multi-factor disease, and traditional oncotherapy comprises operation, chemotherapy, radiotherapy etc., mainly directly take tumour cell as target spot, has Postoperative Residual, useful for drug delivery and penetrance problem, and the shortcoming such as, resistance insensitive to chemotherapy, radiotherapy.And that the growth of tumour is blood vessel is dependent, when diameter of tumor≤2mm, draws nutrition by dispersion between tumour cell, is called " blood vessel early stage "; When tumour further increases, then need new capillary vessel that nutrition is provided, be called " blood vessel phase ".Angiogenesis is closely related to generation, development and the transfer of tumour, therefore, early 1970s Folkman proposes the anti-angiogenic therapy of tumour, and this is envisioned for increasing evidence and supports, and makes this field become the focus of tumor research and the New Policy of oncotherapy.In the middle of multiple angiogenesis inhibitor, Endostatin (endostatin) is the angiogenesis inhibitor that effect is the strongest at present, experiment effect is best.
Endostatin is a kind of Angiostatin, is 20kd molecular weight protein matter, is comprised of 184 amino-acid residues of the C-terminal of collagen XV III.The propagation of the specific inhibition endotheliocyte of energy and the generation of blood vessel suppress formation and the transfer of tumour.By an alkalescence zone that is comprised of 11 arginine, there is stronger avidity in this zone to heparin in its structure, may be that performance is to the inhibiting key structure of vascular endothelial cell zone.It is a highly folding molecular conformation that the molecular structure of Endostatin discloses, and the prompting of the random coil structure of Endostatin N-terminal: its functionally active district may be minimized in these tens amino acid of N end.1,3,11 Histidine and 76 s' aspartic acid has consisted of zine ion in conjunction with the territory, can 1: 1 in conjunction with zine ion, in anti-tumour effect, play a significant role.
The generation of blood vessel relies on original blood vessel tooth expansion, and endotheliocyte must mutually adhere to and adhere to form new born microvessels with extracellular matrix (ECM).In this process, integrin plays an important role, and it participates in the migration of endotheliocyte and the formation of capillary vessel sample pipeline.The RGD sequence is the main aminoacid sequence-Arg-Gly-Asp (arginyl-glycyl-aspartic acid that adheres to identification in the ECM acceptor relevant with the tumour cell adhesion, be called for short RGD), people have synthesized many rgd peptides and derivative, thereby the interaction of competition, interference tumour cell and ECM reaches the purpose that suppresses metastases.Experiment shows and contains RGD tumor-necrosis factor glycoproteins polypeptide, invasion and attack, migration at the obvious check melanin oncocyte of external energy, the in vivo formation of the spontaneity of check melanin knurl and experimental lung metastasis, it is strong to act on more non-repetitive RGD peptide, and this enhancement is relevant with the multiplicity of RGD sequence, multiplicity is more, and inhibiting effect on tumor metastasis is stronger.
Summary of the invention
The purpose of this invention is to provide a kind of polypeptide with anti-tumor function and preparation method thereof, polypeptide of the present invention, its structure comprises 3 Histidines of Endostatin N end zine ion binding site, a β-pleated sheet structure structure, and the RGD sequence of series connection.
Wherein said: 3 Histidine site basic sequences of Endostatin N end zine ion binding site are SEQ ID NO:1 sequence, wherein be 3～8 amino acid between Asp and the Leu, be preferably 4～6, it is characterized in that penultimate is Pro, other are Ala, Phe, Trp, Gly, Ile, Leu, Val, Gln.
Wherein said: β-pleated sheet structure structure master operation is classified SEQ ID NO:2 sequence as, be 5～20 amino acid behind Asn, be preferably 6～10, it is characterized in that second is Pro, and containing a Gly, other are Ala, Ser, Met, Leu, Gly, Asn, Gln, Thr, Tyr.
Wherein said: the RGD structure basic sequence of series connection is that SEQ ID NO:3 sequence serial number is 0～5, is preferably 0～3.
The preferred peptide sequence of the present invention is as follows:
Peptide sequence 1:His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met sees SEQ ID NO:4.
Peptide sequence 2:His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly Asp sees SEQ ID NO:5,
Peptide sequence 3:His Ser His Arg Asp Trp Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly Asp Arg Gly Asp Arg Gly Asp sees SEQ ID NO:6.
Peptide sequence 4:His Ser His Arg Asp Trp Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Trp Met Arg Gly Asp Arg Gly Asp Arg Gly Asp sees SEQ ID NO:7.
Wherein particularly preferred peptide sequence is: His Ser His Arg Asp Trp Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Trp Met Arg Gly Asp Arg Gly Asp Arg Gly Asp, its aminoacid sequence is seen SEQ ID NO:7
The preparation method of polypeptide of the present invention can adopt the polypeptide synthesis method preparation, also can be by the method preparation of gene recombination, and the preferred preparation method of the present invention is as follows:
Anticancer polypeptide of the present invention preferably adopts reaction conditions gentleness, side reaction to reach less the high Fmoc protected amino acid solid-phase synthesis of productive rate.Take Fmoc-Asp (OtBu)-Wang as starting raw material; piperidines with 20% (PIP)/N; dinethylformamide (DMF) solution is sloughed the Fmoc protecting group; condensing agent is N; N-DIC (DIC); access successively protected amino acid (Fmoc-AA) from the sequence C end to the N extreme direction and synthesize to get 32 peptide resins; with lysate [TFA/ water/tri isopropyl silane/1; 2-3-mercaptoethanol=94: 2: 2: 2 (V/V)] cracking; anhydrous diethyl ether is settled out the crude product of anticancer polypeptide, adopts the preparation liquid phase systems, and anti-phase C18 filler is made with extra care purifying; get refined liquid after the desalination; get finished product after freeze-drying, purity is more than 98%.
Carry out the test of vitro inhibition endothelial cell proliferation with anticancer polypeptide of the present invention, estimate the inhibited proliferation to human vascular endothelial ECV304, calculate cell proliferation inhibition rate.The result shows that anticancer polypeptide has certain restraining effect to the ECV-304 Growth of Cells.
Suppress the tumor proliferation test evaluation in the body to murine hepatocarcinoma cell H
22Mice-transplanted tumor and to people's adenocarcinoma of stomach SGC-7901 transplanted tumor in nude mice affects on the growth.The method that adopts in experimentation is: getting inoculation has H
22The nude mice of the mouse of tumour cell and inoculation people gastric cancer SGC-7901 cell line is injected respectively anticancer polypeptide, the rhEndostatin of different concns, and successive administration 15～18d puts to death mouse after the drug withdrawal, observe the tumor growth situation.The result shows that anticancer polypeptide of the present invention has obvious antitumor action.The residual knurl growth model of tumor post-operation has been estimated mouse sarcoma S180cell and the residual knurl affects on the growth of lovo colon cancer cell transplanted tumor postoperative, and test-results shows that anticancer polypeptide has the effect that suppresses the Postoperative Residual tumor growth.Under certain administration concentration, the tumor inhibition effect of anticancer polypeptide of the present invention is better than Endostatin.
The invention has the advantages that: increased series connection RGD sequence quantity, can strengthen the targeting for tumour.In addition, owing to do not contain or contain the Trp of lesser amt in the sequence of the present invention, adopt the synthetic method of polypeptide, compare easier accurate quantification with gene recombination method.
Sequence table explanation of the present invention:
SEQ ID NO:1, the Histidine site sequence,
SEQ ID NO:2, the β-pleated sheet structure structure sequence,
SEQ ID NO:3, the RGD structure sequence,
SEQ ID NO:4, the preferred peptide sequence 1 of the present invention,
SEQ ID NO:5, the preferred peptide sequence 2 of the present invention,
SEQ ID NO:6, the preferred peptide sequence 3 of the present invention,
SEQ ID NO:7, the particularly preferred peptide sequence 4 of the present invention.
Embodiment
Below explanation does not consist of the restriction to this patent.
The present invention describes as an example of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 sequence example.
Embodiment 1
The chemosynthesis of the preferred peptide sequence SEQ of 1 the present invention ID NO:4
(1) with Fmoc-Asp (OtBu)-Wang with DMF swelling 30 minutes, remove and added 20% PIP/DMF solution stirring reaction behind the DMF 5 minutes, with the DMF washing, wash 2 times.
(2) add 20% PIP/DMF solution, stirring reaction with DMF washing 2 times, carried out triketohydrindene hydrate and detects and should be positive after 25 minutes again.
(3) take by weighing Fmoc-Gly-OH (3-5 of the contained amino mole number of resin doubly) and equimolar 1-hydroxyl-benzo-triazole (HOBt) and dissolve ice bath cooling 30min with DMF; Other gets equimolar DIC, again with one times of DCM dilution, in 0 ℃～-10 ℃ water-bath precoolings, the DIC solution of cooling is added in the DMF dissolving of Fmoc-Gly-OH, continue more than the cooling and stirring reaction 60min, join and carry out condensation reaction in the reaction column, reaction end rear (not developing the color with the ninhydrin detection) is finished for 3 times with the DMF washing and is once connect reactive polypeptide.
(4) repeated removal Fmoc protecting group and connect the operation of reactive polypeptide step according to SEQ ID NO:4 sequence, is changed different amino acid and is progressively carried out coupling, until last Fmoc-His (Trt)-the OH coupling is complete.
(5) with lytic reagent [TFA/ water/tri isopropyl silane/1,2-3-mercaptoethanol=94: 2: 2: 2 (V/V)] peptide resin, consumption is 10～13ml/ gram peptide resin, stirs, and adds dry peptide resin again.20～30 ℃ stirring reaction 3-5 hour, filter to collect filtrate, resin washs 2 times with a small amount of TFA again, merging filtrate, 30～35 ℃ are evaporated to 30% of original volume.
(6) concentrated solution is added in the anhydrous diethyl ether of precooling (30ml/ restrains peptide resin), precipitation below-10 ℃ 60 minutes, filters collecting precipitation, use again the anhydrous diethyl ether washed twice of a small amount of precooling.Be transferred to and volatilize ether in the drying tray.
(7) above-mentioned solid is dissolved with 25～45% acetums, membrane filtration stirs the lower saturated I of dropping
2/ acetum to solution is reddish-brown, continues to stir 30 minutes, adds a small amount of Vc aqueous solution to reddish-brown and disappears, and namely gets the oxidation crude product solution, and concentrating under reduced pressure under 30～40 ℃ of conditions gets anticancer polypeptide crude product concentrated solution, and purity is more than 80%.
2 polypeptide separation and purification
Adopt the preparation HPLC separation and purification, flow phase system is the 0.1%TFA-acetonitrile; The preparative liquid chromatography system adopts anti-phase C18 (77mm*250mm) filler of 10 μ m, and UV-detector is set 280nm, flow velocity 90ml/min.
Adopt gradient elution, get crude product solution and be splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collect main peak and with analyzing Liquid Detection purity, merge main peak solution, at 30～40 ℃ of concentrating under reduced pressure, obtain concentrated solution behind the anticancer polypeptide purifying, purity is about 90%, and refining yield is 60%, carries out next step purifying.
Adopt high performance liquid chromatography to carry out desalination, flow phase system is 0.2% acetic acid-acetonitrile.Get the anticancer polypeptide concentrated solution, filter with 0.45 μ m filter membrane, the purifying chromatograph packing material is anti-phase C18 (77mm*250mm) filler of 10 μ m, and the ultraviolet detection wavelength is 280nm, and flow velocity is 90ml/min.
Adopt gradient elution, getting crude product solution is splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed the salt main peak and is used and analyze Liquid Detection purity, merge and to change salt main peak solution, concentrating under reduced pressure under less than 40 ℃ of water bath condition boils off most of acetonitrile with Rotary Evaporators, obtain purity greater than 98% anticancer polypeptide acetate aqueous solution, refining yield is 86%.Freezing preservation.
Embodiment 2
The chemosynthesis of the preferred peptide sequence SEQ of 1 the present invention ID NO:5
(1) with Fmoc-Asp (OtBu)-Wang with DMF swelling 30 minutes, remove and added 20% PIP/DMF solution stirring reaction behind the DMF 5 minutes, with the DMF washing, wash 2 times.
(2) add 20% PIP/DMF solution, stirring reaction with DMF washing 2 times, carried out triketohydrindene hydrate and detects and should be positive after 25 minutes again.
(3) take by weighing Fmoc-Gly-OH (3-5 of the contained amino mole number of resin doubly) and equimolar 1-hydroxyl-benzo-triazole (HOBt) and dissolve ice bath cooling 30min with DMF; Other gets equimolar DIC, again with one times of DCM dilution, in 0 ℃～-10 ℃ water-bath precoolings, the DIC solution of cooling is added in the DMF dissolving of Fmoc-Gly-OH, continue more than the cooling and stirring reaction 60min, join and carry out condensation reaction in the reaction column, reaction end rear (not developing the color with the ninhydrin detection) is finished for 3 times with the DMF washing and is once connect reactive polypeptide.
(4) repeated removal Fmoc protecting group and connect the operation of reactive polypeptide step according to SEQ ID NO:5 sequence, is changed different amino acid and is progressively carried out coupling, until last Fmoc-His (Trt)-the OH coupling is complete.
(5) with lytic reagent [TFA/ water/tri isopropyl silane/1,2-3-mercaptoethanol=94: 2: 2: 2 (V/V)] peptide resin, consumption is 10～13ml/ gram peptide resin, stirs, and adds dry peptide resin again.20～30 ℃ stirring reaction 3-5 hour, filter to collect filtrate, resin washs 2 times with a small amount of TFA again, merging filtrate, 30～35 ℃ are evaporated to 30% of original volume.
(6) concentrated solution is added in the anhydrous diethyl ether of precooling (30ml/ restrains peptide resin), precipitation below-10 ℃ 60 minutes, filters collecting precipitation, use again the anhydrous diethyl ether washed twice of a small amount of precooling.Be transferred to and volatilize ether in the drying tray.
(7) above-mentioned solid is dissolved with 25～45% acetums, membrane filtration stirs the lower saturated I of dropping
2/ acetum to solution is reddish-brown, continues to stir 30 minutes, adds a small amount of Vc aqueous solution to reddish-brown and disappears, and namely gets the oxidation crude product solution, and concentrating under reduced pressure under 30～40 ℃ of conditions gets anticancer polypeptide crude product concentrated solution, and purity is more than 80%.
2 polypeptide separation and purification
Adopt the preparation HPLC separation and purification, flow phase system is the 0.1%TFA-acetonitrile; The preparative liquid chromatography system adopts anti-phase C18 (77mm*250mm) filler of 10 μ m, and UV-detector is set 280nm, flow velocity 90ml/min.
Adopt gradient elution, get crude product solution and be splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collect main peak and with analyzing Liquid Detection purity, merge main peak solution, at 30～40 ℃ of concentrating under reduced pressure, obtain concentrated solution behind the anticancer polypeptide purifying, purity is about 90%, and refining yield is 60%, carries out next step purifying.
Adopt high performance liquid chromatography to carry out desalination, flow phase system is 0.2% acetic acid-acetonitrile.Get the anticancer polypeptide concentrated solution, filter with 0.45 μ m filter membrane, the purifying chromatograph packing material is anti-phase C18 (77mm*250mm) filler of 10 μ m, and the ultraviolet detection wavelength is 280nm, and flow velocity is 90ml/min.
Adopt gradient elution, getting crude product solution is splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed the salt main peak and is used and analyze Liquid Detection purity, merge and to change salt main peak solution, concentrating under reduced pressure under less than 40 ℃ of water bath condition boils off most of acetonitrile with Rotary Evaporators, obtain purity greater than 98% anticancer polypeptide acetate aqueous solution, refining yield is 85%.Freezing preservation.
Embodiment 3
The chemosynthesis of the preferred peptide sequence SEQ of 1 the present invention ID NO:6
(1) with Fmoc-Asp (OtBu)-Wang with DMF swelling 30 minutes, remove and added 20% PIP/DMF solution stirring reaction behind the DMF 5 minutes, with the DMF washing, wash 2 times.
(2) add 20% PIP/DMF solution, stirring reaction with DMF washing 2 times, carried out triketohydrindene hydrate and detects and should be positive after 25 minutes again.
(3) take by weighing Fmoc-Gly-OH (3-5 of the contained amino mole number of resin doubly) and equimolar 1-hydroxyl-benzo-triazole (HOBt) and dissolve ice bath cooling 30min with DMF; Other gets equimolar DIC, again with one times of DCM dilution, in 0 ℃～-10 ℃ water-bath precoolings, the DIC solution of cooling is added in the DMF dissolving of Fmoc-Gly-OH, continue more than the cooling and stirring reaction 60min, join and carry out condensation reaction in the reaction column, reaction end rear (not developing the color with the ninhydrin detection) is finished for 3 times with the DMF washing and is once connect reactive polypeptide.
(4) repeated removal Fmoc protecting group and connect the operation of reactive polypeptide step according to SEQ ID NO:6 sequence, is changed different amino acid and is progressively carried out coupling, until last Fmoc-His (Trt)-the OH coupling is complete.
(5) with lytic reagent [TFA/ water/tri isopropyl silane/1,2-3-mercaptoethanol=94: 2: 2: 2 (V/V)] peptide resin, consumption is 10～13ml/ gram peptide resin, stirs, and adds dry peptide resin again.20～30 ℃ stirring reaction 3-5 hour, filter to collect filtrate, resin washs 2 times with a small amount of TFA again, merging filtrate, 30～35 ℃ are evaporated to 30% of original volume.
(6) concentrated solution is added in the anhydrous diethyl ether of precooling (30ml/ restrains peptide resin), precipitation below-10 ℃ 60 minutes, filters collecting precipitation, use again the anhydrous diethyl ether washed twice of a small amount of precooling.Be transferred to and volatilize ether in the drying tray.
(7) above-mentioned solid is dissolved with 25～45% acetums, membrane filtration stirs the lower saturated I of dropping
2/ acetum to solution is reddish-brown, continues to stir 30 minutes, adds a small amount of Vc aqueous solution to reddish-brown and disappears, and namely gets the oxidation crude product solution, and concentrating under reduced pressure under 30～40 ℃ of conditions gets anticancer polypeptide crude product concentrated solution, and purity is more than 80%.
2 polypeptide separation and purification
Adopt the preparation HPLC separation and purification, flow phase system is the 0.1%TFA-acetonitrile; The preparative liquid chromatography system adopts anti-phase C18 (77mm*250mm) filler of 10 μ m, and UV-detector is set 280nm, flow velocity 90ml/min.
Adopt gradient elution, get crude product solution and be splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collect main peak and with analyzing Liquid Detection purity, merge main peak solution, at 30～40 ℃ of concentrating under reduced pressure, obtain concentrated solution behind the anticancer polypeptide purifying, purity is about 90%, and refining yield is 64%, carries out next step purifying.
Adopt high performance liquid chromatography to carry out desalination, flow phase system is 0.2% acetic acid-acetonitrile.Get the anticancer polypeptide concentrated solution, filter with 0.45 μ m filter membrane, the purifying chromatograph packing material is anti-phase C18 (77mm*250mm) filler of 10 μ m, and the ultraviolet detection wavelength is 280nm, and flow velocity is 90ml/min.
Adopt gradient elution, getting crude product solution is splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed the salt main peak and is used and analyze Liquid Detection purity, merge and to change salt main peak solution, concentrating under reduced pressure under less than 40 ℃ of water bath condition boils off most of acetonitrile with Rotary Evaporators, obtain purity greater than 98.5% anticancer polypeptide acetate aqueous solution, refining yield is 89%.Freezing preservation.
Embodiment 4
The chemosynthesis of the particularly preferred peptide sequence SEQ of 1 the present invention ID NO:7
(1) with Fmoc-Asp (OtBu)-Wang with DMF swelling 30 minutes, remove and added 20% PIP/DMF solution stirring reaction behind the DMF 5 minutes, with the DMF washing, wash 2 times.
(2) add 20% PIP/DMF solution, stirring reaction with DMF washing 2 times, carried out triketohydrindene hydrate and detects and should be positive after 25 minutes again.
(3) take by weighing Fmoc-Gly-OH (3-5 of the contained amino mole number of resin doubly) and equimolar 1-hydroxyl-benzo-triazole (HOBt) and dissolve ice bath cooling 30min with DMF; Other gets equimolar DIC, again with one times of DCM dilution, in 0 ℃～-10 ℃ water-bath precoolings, the DIC solution of cooling is added in the DMF dissolving of Fmoc-Gly-OH, continue more than the cooling and stirring reaction 60min, join and carry out condensation reaction in the reaction column, reaction end rear (not developing the color with the ninhydrin detection) is finished for 3 times with the DMF washing and is once connect reactive polypeptide.
(4) repeated removal Fmoc protecting group and connect the operation of reactive polypeptide step according to SEQ ID NO:7 sequence, is changed different amino acid and is progressively carried out coupling, until last Fmoc-His (Trt)-the OH coupling is complete.
(5) with lytic reagent [TFA/ water/tri isopropyl silane/1,2-3-mercaptoethanol=94: 2: 2: 2 (V/V)] peptide resin, consumption is 10～13ml/ gram peptide resin, stirs, and adds dry peptide resin again.20～30 ℃ stirring reaction 3-5 hour, filter to collect filtrate, resin washs 2 times with a small amount of TFA again, merging filtrate, 30～35 ℃ are evaporated to 30% of original volume.
(6) concentrated solution is added in the anhydrous diethyl ether of precooling (30ml/ restrains peptide resin), precipitation below-10 ℃ 60 minutes, filters collecting precipitation, use again the anhydrous diethyl ether washed twice of a small amount of precooling.Be transferred to and volatilize ether in the drying tray.
(7) above-mentioned solid is dissolved with 25～45% acetums, membrane filtration stirs the lower saturated I of dropping
2/ acetum to solution is reddish-brown, continues to stir 30 minutes, adds a small amount of Vc aqueous solution to reddish-brown and disappears, and namely gets the oxidation crude product solution, and concentrating under reduced pressure under 30～40 ℃ of conditions gets anticancer polypeptide crude product concentrated solution, and purity is more than 80%.
2 polypeptide separation and purification
Adopt the preparation HPLC separation and purification, flow phase system is the 0.1%TFA-acetonitrile; The preparative liquid chromatography system adopts anti-phase C18 (77mm*250mm) filler of 10 μ m, and UV-detector is set 280nm, flow velocity 90ml/min.
Adopt gradient elution, get crude product solution and be splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collect main peak and with analyzing Liquid Detection purity, merge main peak solution, at 30～40 ℃ of concentrating under reduced pressure, obtain concentrated solution behind the anticancer polypeptide purifying, purity is about 90%, and refining yield is 62%, carries out next step purifying.
Adopt high performance liquid chromatography to carry out desalination, flow phase system is 0.2% acetic acid-acetonitrile.Get the anticancer polypeptide concentrated solution, filter with 0.45 μ m filter membrane, the purifying chromatograph packing material is anti-phase C18 (77mm*250mm) filler of 10 μ m, and the ultraviolet detection wavelength is 280nm, and flow velocity is 90ml/min.
Adopt gradient elution, getting crude product solution is splined in the chromatographic column, start the moving phase wash-out, gather collection of illustrative plates, the variation of observation optical density, collection is changed the salt main peak and is used and analyze Liquid Detection purity, merge and to change salt main peak solution, concentrating under reduced pressure under less than 40 ℃ of water bath condition boils off most of acetonitrile with Rotary Evaporators, obtain purity greater than 98% anticancer polypeptide acetate aqueous solution, refining yield is 88%.Freezing preservation.
Embodiment 5
Carried out following antitumor test with the particularly preferred polypeptide of the present invention (embodiment 4):
1 vitro inhibition endothelial cell proliferation activity experiment
The huve cell ECV304 that takes the logarithm vegetative period makes 5 * 10 with the DMEM substratum that contains 10%FBS after the trysinization
4The cell suspension of individual/ml concentration is laid in 96 orifice plates, every hole 100 μ l.The blank group adds the DMEM substratum of 100 μ l, and the administration group adds the anticancer polypeptide of gradient concentration, 3 every group multiple holes, 37 ℃, 5%CO
2Cultivated 3 days, and be changed to the DMEM substratum that contains 10%cck-8,37 ℃, 5%CO
2Use microplate reader OD450nm reading after cultivating 1h, calculate cell proliferation inhibition rate.The result shows that anticancer polypeptide has certain restraining effect to the ECV-304 Growth of Cells, and inhibiting rate is up to 54.1%.
Table 1 embodiment 3 anticancer polypeptides are to the restraining effect of ECV-304 Growth of Cells
2 anti-tumor in vivo activity tests
Get NIH mouse peritoneal injection H
22The knurl strain, 1 week put to death and gets ascites.Get 60 mouse, oxter injection cell concn is 1 * 10
7The ascites 0.2ml of/ml is divided into 6 groups at random with mouse, 10 every group.The inoculation second day begins subcutaneous administration, concentration is respectively 6mg/kg, 2mg/kg, 0.67mg/kg, 0.22mg/kg, the blank group is set to be given with volume physiological saline, positive controls is that rhEndostatin 30mg/kg after continuous 15 days puts to death mouse, peeling off taking-up knurl piece weighs, use the vernier caliper measurement volume, calculate tumor control rate.
People's gastric adenocarcinoma cells SGC-7901 is inoculated in Balb/c nude mice oxter, and every 0.2ml when treating that Subcutaneous tumor grows to diameter 2-3cm, takes out the knurl piece under the aseptic condition, the knurl piece is cut into about 1.5mm * 1.5mm size, in every nude mice right fore oxter inoculation.Long-pending 100～the 300mm that rises to of knurl block to be inoculated
3The time, reject too small or excessive nude mice and the too small nude mice of body weight of gross tumor volume, with qualified nude mice random packet and begin administration, route of administration is tail vein injection, dosage is the same, successive administration is peeled off taking-up knurl piece and is weighed after 18 days, use the vernier caliper measurement volume, calculates tumor control rate.
Table 2 anticancer polypeptide is to H
22The restraining effect of mice-transplanted tumor growth
*p＜0.05
Table 3 anticancer polypeptide is to the restraining effect of SGC-7901 transplanted tumor in nude mice growth
*p＜0.05
The result shows that anticancer polypeptide is to H
22The growth of mice-transplanted tumor and people's gastric adenocarcinoma cells SGC-7901 transplanted tumor in nude mice has obvious restraining effect, and has certain dose-effect relationship, and tumor control rate can reach more than 40%.
The residual knurl growth of the 3 transplanted tumor postoperatives test of pesticide effectiveness
Get the ascites of the tumor-bearing mice of 2 Mice Inoculated S180 sarcoma cells strain under the aseptic condition, be diluted to concentration with stroke-physiological saline solution and be about 1 * 10
7Individual/ml, give right side of mice oxter inoculation 0.2ml.In one week of mouse inoculation tumour cell, gross tumor volume is approximately 350～400mm
3The time, perform an operation to mouse, with 0.5% Sodital sodium solution, according to 0.12ml/20g subcutaneous administration anesthetized animal, remove the knurl piece of half volume under the aseptic condition, the residue gross tumor volume is approximately 60～100mm
3, carry out follow-up test.Begin administration behind the operation 24h, dosage is the same.Administration finished administration after 9 days, and animal is put to death in the neck dislocation, peeled off taking-up knurl piece and weighed, and used the vernier caliper measurement volume, calculated tumor control rate.
Under the aseptic condition, recovery lovo colon cancer cell is used physiological saline with DMEM high glucose medium+10% foetal calf serum cultivation, and is resuspended, counting 2 * 10
7Individual/ml, give oxter, nude mice right side inoculation 0.2ml.
One week of nude inoculation tumour cell, the nearly 350mm of gross tumor volume
3The time, perform an operation to nude mice, with 0.5% Sodital sodium solution, according to 0.12ml/20g subcutaneous administration anesthetized animal, remove the knurl piece of only about half of volume under the aseptic condition, the residue gross tumor volume is approximately 60mm
3, then carry out follow-up test.Administration behind the operation 24h, dosage is the same.Administration finished administration after 9 days, and animal is put to death in the neck dislocation, peeled off taking-up knurl piece and weighed, and estimated curative effect of medication according to knurl piece weight and measurement volumes.
Table 4 anticancer polypeptide is to the residual knurl affects on the growth of kunming mice S180 sarcoma transplanted tumor postoperative
*P＜0.05；
**P＜0.01；
***P＜0.001
Table 5 anticancer polypeptide is to the residual knurl affects on the growth of nude mice lovo colorectal carcinoma transplanted tumor postoperative
*P＜0.05；
**P＜0.01
Each treatment group is showed no acute toxic reaction after administration; Each treated animal activity is normal, and the operative incision healing is good, and the result shows that anticancer polypeptide produces stronger restraining effect to S180 sarcoma and the rear residual knurl growth of lovo colorectal carcinoma transplanted tumor operation, and has statistical significance, significant difference.
Claims (10)
1. tumor protein p53 is characterized in that, contains 3 Histidine sequences of Endostatin N end zine ion binding site, a β-pleated sheet structure structure sequence, and the RGD sequence of series connection.
2. described tumor protein p53 according to claim 1, wherein Endostatin N end zine ion binding site master operation is classified SEQ ID NO:1 sequence as.
3. described tumor protein p53 according to claim 1, wherein β-pleated sheet structure structure master operation is classified SEQ ID NO:2 sequence as.
4. described tumor protein p53 according to claim 1, wherein RGD structure basic sequence is SEQ ID NO:3 sequence.
5. described tumor protein p53 according to claim 2 is 3～8 amino acid between Asp and the Leu in the SEQ ID NO:1 sequence wherein, is preferably 4～6, it is characterized in that penultimate is Pro.
6. described tumor protein p53 according to claim 3 is 5～20 amino acid behind the Asn in SEQ ID NO:2 sequence wherein, is preferably 6～10, it is characterized in that containing at least a Gly.
7. described tumor protein p53 according to claim 4, wherein SEQ ID NO:3 sequence serial number is 0～5, is preferably 0～3.
8. prepare the described tumor protein p53 of claim 1, be selected from:
The preferred peptide sequence 1 of SEQ ID NO:4 the present invention,
The preferred peptide sequence 2 of SEQ ID NO:5 the present invention,
The preferred peptide sequence 3 of SEQ ID NO:6 the present invention,
The preferred peptide sequence 4 of SEQ ID NO:7 the present invention,
SEQ ID NO:7 is the particularly preferred peptide sequence of the present invention.
9. the preparation method of described tumor protein p53 according to claim 8 adopts polypeptide synthesis, comprises liquid phase synthesizing method or solid-phase synthesis.
10. the application of the described tumor protein p53 of claim 1 in the medicine of preparation treatment tumour.
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