CN103387603B - Polypeptide related to RTN4B, and preparation and application thereof - Google Patents
Polypeptide related to RTN4B, and preparation and application thereof Download PDFInfo
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- CN103387603B CN103387603B CN201310136244.8A CN201310136244A CN103387603B CN 103387603 B CN103387603 B CN 103387603B CN 201310136244 A CN201310136244 A CN 201310136244A CN 103387603 B CN103387603 B CN 103387603B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to a polypeptide related to RTN4B and preparation and application thereof. The polypeptide related to RTN4B has an amino acid sequence as represented by SEQ ID No. 1. The invention further discloses a preparation method for the polypeptide related to RTN4B based on chemical synthesis and application of the polypeptide related to RTN4B. The polypeptide related to RTN4B provided by the invention can inhibit biological activity of RTN4B in vitro and inhibit growth of tumor cells in vivo.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of RTN4B related polypeptide and preparation and application thereof.
Background technology
RTN4B belongs to reticolon family (RTNs), because gaining the name containing special endoplasmic reticulum location, has conservative RHD structural frames (reticulon-homology domain).Four genes are contained in this family, RTN1, RTN2, RTN3 and RTN4, and the research report wherein about RTN4 is more.RTN4(has another name called Nogo/ASY/RTN-X) gene have multiple alternative splicing this; main code RTN4A(GenBank accession number AF148537); RTN4B(GenBank accession number AF148538) and RTN4C(GenBank accession number AF087901) three kinds of albumen; this many [Oertle T, Schwab ME.Nogo and its paRTNers.Trends Cell Biol.2003Apr specific expressed in testis of some other montage; 13 (4): 187-94.].
RTN4B wide spectrum in all healthy tissuess of other except liver is expressed, and the research work prompting RTN4B of forefathers participates in regulating cell apoptosis and tumor growth.The people such as Tsujimoto find RTN4B can and Bcl-2; Bcl-xL interacts and recruits in endoplasmic reticulum by Bcl-XL and Bcl-2; reverse Apoptosis inhibitor effect [the Tagami S of Bcl-XL and Bcl-2; Eguchi Y; Kinoshita M; Takeda M; Tsujimoto Y.A novel protein; RTN-XS, interacts with bothBcl-XL and Bcl-2on endoplasmic reticulum and reduces their anti-apoptotic activity.Oncogene.2000Nov23; 19 (50): 5736-46.].The people such as Yutsudo find that RTN4B occurs Transcription inhibition in the sample of small cell lung cancer; and overexpression RTN4B significantly can promote apoptosis [the Li Q of tumour cell; Qi B, Oka K, Shimakage M; Yoshioka N; Inoue H, Hakura A, Kodama K; Stanbridge EJ, Yutsudo M.Link of a new type ofapoptosis-inducing gene ASY/Nogo-B to human cancer.Oncogene.2001Jul5; 20 (30): 3929-36.].But; Schwab group points out there is support in the est database that the expression of RTN4B is originated at a lot of tumor tissues; and RTN4B does not promote apoptotic sign [Oertle T in SaOS-2 or Chinese hamster ovary celI; Merkler D, SchwabME.Do cancer cells die because of Nogo-B Oncogene.2003Mar6; 22 (9): 1390-9.].Therefore, whether the mechanism of RTN4B modulate tumor growth also there is no final conclusion with apoptosis-related.But research in recent years finds that RTN4B is probably relevant with reconstructing blood vessel, even angiogenesis further.2004, the people such as Acevedo found that RTN4B has expression in mammalian Vascular Endothelial cell and smooth muscle cell.RTN4B recombinant protein can promote the migration of vascular endothelial cell and suppress the migration of smooth muscle cell.The vessel wall of abnormal thickening has been there is in RTN4A/B knock out mice after blood vessel injury; intravascular space reduces; by adenovirus carrier, RTN4B is imported in Mice Body again; the vessel wall that this exception thickens can recover again normal form [Acevedo L; Yu J; Erdjument-Bromage H; Miao RQ; Kim JE; Fulton D; Tempst P, Strittmatter SM, Sessa WC.A new role for Nogo as a regulator of vascularremodeling.Nat Med.2004Apr; 10 (4): 382-8.].Subsequently; the people such as Miao have screened acceptor molecule NgBR [the Miao RQ of RTN4B at Surface of Vascular Endothelial Cells; Gao Y; Harrison KD, Prendergast J, Acevedo LM; Yu J; Hu F, Strittmatter SM, Sessa WC.Identification of a receptor necessary for Nogo-B stimulatedchemotaxis and morphogenesis of endothelial cells.Proc Natl Acad Sci U S A.2006 Jul18; 103 (29): 10997-1002.].Striking the interaction subtracted between experiment proof RTN4B and NgBR is that VEGF angiogenic molecule induction of vascular new life is necessary.Find in zebra fish model animals; the interaction of RTN4B-NgBR can by Akt path modulating vascular new life [the Zhao B in activating cells; Chun C; Liu Z; Horswill MA, Pramanik K, Wilkinson GA; Ramchandran R, Miao RQ.Nogo-B receptor is essential for angiogenesis inzebrafish via Akt pathway.Blood.2010Dec9; 116 (24): 5423-33.].
Applicant of the present invention has cloned people RTN4 gene the earliest, and in the research work in early stage, deep functional analysis has been carried out to the RTN4B of tumor cells expression, apply for a patent " a kind of people's albumen and encoding sequence and method for making and purposes " thereof [Authorization Notice No.: CN 1137383C], wherein relate to the synthesis of RTN4B albumen and the application in diagnosing cancer of liver test kit thereof.
The dependency of RTN4B and tumour is reported in studying at many sections, and is confirmed by the previous research work of the present inventor.RTN4B expression change not only closely related with tumor development, and RTN4B can participate in regulate tumor growth: overexpression RTN4B can promote that tumour cell grows in vivo, and cut down RTN4B can inhibition tumor cell growth in vivo.
But how to regulate tumor growth about RTN4B, the effect utilizing RNT4B how to realize Tumor growth inhibition as molecular target is still problem in science urgently to be resolved hurrily.This area, in the urgent need to regulating at in-depth explanation RTN4B on the basis of the mechanism of tumor growth, is molecular target with RTN4B, explores novel effective antitumour medicine.
Prior art has reported the several functions of RTN4B, as to apoptosis, reconstructing blood vessel, the angiogenesis of tumour cell, stick, migration etc. is relevant.RTN4B related polypeptide of the present invention can suppress the biologic activity of RTN4B, means its potential pharmaceutical use.
Fifth aspect present invention, discloses the purposes of described RTN4B related polypeptide in the medicine preparing treatment or prophylaxis of tumours.
In one embodiment of the invention, specifically list RTN4B related polypeptide of the present invention to the restraining effect of liver cancer, this means that RTN4B related polypeptide of the present invention is expected to become cancer therapy drug.The method of embodiment is by abdominal injection polypeptide, suppresses the growth of swelling of the axilla oncocyte transplanted tumor.It comprises step: (a) by tumor cell transplantation to nude mice oxter; B polypeptide injects in nude mouse by the mode of intraperitoneal administration by (); C the growth velocity of () record transplanted tumor in nude mice is also by the Growth of Cells in immunohistochemical analysis knurl body and angiogenic growth situation [Technical Reference: Yamada S; Bu XY; Khankaldyyan V; Gonzales-Gomez I; McComb JG, Laug WE.Effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth in nude mice.Neurosurgery.2006Dec; 59 (6): 1304-12].Result confirms that RTN4B related polypeptide of the present invention can realize Tumor growth inhibition by suppressing the ability that vascular endothelial cell sticks.
Fifth aspect present invention, also discloses a kind of method for the treatment of or prophylaxis of tumours, for using described RNT4B related polypeptide to object.
Described tumour is selected from its growth or breeds and the expression of RNT4B albumen or active relevant tumour.
Further, described tumour is liver cancer.
Sixth aspect present invention, discloses described RTN4B related polypeptide and is preparing the application in the medicine or preparation suppressing vascular endothelial cell to stick.
In one embodiment of the invention, the method is that inhibition tumor cell is at the Adhering capacity on RTN4B surface, and it comprises step: vascular endothelial cell and polypeptide are hatched by (a) by after polypeptide and tumour cell being hatched; B () uses RTN4B albumen coated cell culture dish; C vascular endothelial cell is inoculated in culture dish by (); D () to detect in 1 hour vascular endothelial cell at the ratio [Technical Reference: Gonzalez AM of RTN4B surface adhesion; Gonzales M; Herron GS; Nagavarapu U; Hopkinson SB; Tsuruta D, Jones JC.Complex interactions between the laminin alpha4subunitand integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo.Proc Natl AcadSci U S A.2002Dec10; 99 (25): 16075-80.].
Seventh aspect present invention, discloses a kind of medicine or preparation, and its activeconstituents contains described RTN4B related polypeptide.
Described medicine can be medicine or the preparation of the biologic activity effect for suppressing RTN4B; Or be used for the treatment of or the medicine of prophylaxis of tumours; Or be, the medicine sticked for suppressing vascular endothelial cell or preparation.
In described medicine or preparation, also pharmaceutical excipient can be comprised.
Described pharmaceutical excipient is for solving the active or biological effectiveness of the formability of preparation, validity, stability, security, raising, when oral, produces the preparation associated problem such as acceptable mouthfeel or smell and the medicinal material that adds in prescription except main ingredient.
Pharmaceutical excipient can be various carrier, thinner, weighting agent, bonding agent and other vehicle, and this depends on administering mode and designed dosage form.Various sanitas, lubricant, dispersion agent, correctives, wetting Agent for Printing Inks, antioxidant, sweeting agent, tinting material, stablizer also can add wherein.Concrete, described pharmaceutical excipient can include but not limited to carbohydrate (as: lactose, glucose, sucrose), starch (as W-Gum and potato form sediment), cellulose and its derivates is (as crafty sodium carboxymethylcellulose pyce, ethyl cellulose, methylcellulose gum, Microcrystalline Cellulose), tragakanta powder, Fructus Hordei Germinatus, gelatin, talcum, solid lubricant (as stearic acid and Magnesium Stearate), calcium sulfate, vegetables oil is (as peanut oil, Oleum Gossypii semen, sesame oil, sweet oil, Semen Maydis oil and theobroma oil), wax, fat, ethanol, polyvalent alcohol is (as propylene glycol, glycerine, Sorbitol Powder, mannitol and polyoxyethylene glycol), polyvinylpyrrolidone, Lalgine, emulsifying agent (as Tween), wetting agent (as Sodium Lauryl Sulphate BP/USP), tinting material, seasonings (as sweeting agent), tablet agent, stablizer, antioxidant, sanitas (as methyl hydroxybenzoate and propyl ester), apirogen water, isotonic salts solution, one or more combination in damping fluid (as phosphate buffered saline buffer).
When selecting pharmaceutical excipient, should select compatible with RTN4B related polypeptide of the present invention, can be blended with it and significantly can not reduce the auxiliary material of the effect of pharmaceutical composition under normal conditions.
Medicine of the present invention or preparation can be tablet, pill, pulvis, solution, syrup, sterilizing injecting solution etc.Pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.The pharmaceutical composition of such as Tablet and Capsula and so on, is prepared by ordinary method.Medicine should aseptically manufacture.
The medicine prepared or preparation can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.When using medicine, be that the fusion rotein of the present invention of safe and effective amount or its antibody are applied to object.Such as every day about 50 micrograms/kg body weight-Yue 500 mg/kg body weight.In addition, polypeptide of the present invention also can use together with other treatment agent.
The present invention devises the short peptide molecules of target RTN4B according to the biologic activity of RTN4B and action pathway.Prove through test, RTN4B related polypeptide of the present invention can suppress the biologic activity of RTN4B in vitro, suppress vascular endothelial cell to stick, the growth of inhibition tumor cell in vivo, thus realize the control to tumor growth, there are the potentiality being developed to cancer therapy drug.
Summary of the invention
The object of the invention is open a kind of RTN4B related polypeptide and preparation and application thereof.
First aspect present invention, discloses a kind of RTN4B related polypeptide, and it contains the aminoacid sequence shown in SEQ ID NO.1 or its conservative variation's polypeptide.
SEQ ID NO:1:RRGSS。
RTN4B related polypeptide of the present invention can be improvement on synthesis or recombinant polypeptide.
RTN4B related polypeptide of the present invention can be modified or not modified polypeptide.
Described modification refers to and transforms protein on a molecular scale, does not usually change primary structure.Namely in vitro the side-chain radical of protein is passed through manual method, the macromole that group that is natural with some or that synthesize particularly has biocompatibility carries out covalently bound, thus the character of change protein is as improve its anti-proteolysis performance, optimizing solubility property, increasing the transformation period or be easier to purifying etc.Concrete, these modifications can be glycosylation, PEGization, tag antibody etc.
Described tag antibody refers to the Special Areas or group that determine antigen-specific in antigen molecule, is the structure or sequence that are combined with antibodies specific.Can be used for DNA recombinant technology and build the fusion rotein comprising goal gene and Epitope tag, and then by special signature's antibody to its qualification and purifying, to reach the demand of research.Conventional tag antibody includes but not limited to: myc, HA, Flag, His, GST etc.
Confirm through test, the biologic activity of RTN4B related polypeptide energy vitro inhibition RTN4B of the present invention.
Preferably, the aminoacid sequence of described RTN4B related polypeptide is SEQ ID NO.1.
RTN4B related polypeptide of the present invention can be linear polypeptide or cyclic peptide.
Second aspect present invention, discloses the preparation method of described RTN4B related polypeptide, comprises the linear polypeptide adopting chemical synthesis to obtain described RTN4B related polypeptide.
After obtaining linear polypeptide, also further the cyclisation of described linear polypeptide head and the tail can be obtained cyclic peptide.
In a case study on implementation of the present invention, be that utilize the condensation reaction artificial synthetic polypeptide chain of peptide bond, it comprises step with solid-phase resin carrier, amino acid for material:
A first the carboxy terminal amino acid of peptide chain is covalently attached on solid phase carrier by ();
B (), through deaminizating protecting group, is reacted with the next amino acid condensation of excessive activated carboxyl, thus extend peptide chain;
C the amino acid of () circulation step b successively in catenation sequence is until extend to peptide chain N-terminal;
D () is cut peptide chain from solid phase carrier and is sloughed Side chain protective group, purified acquisition linear polypeptide.
The condition that above steps adopts and reagent are the routine in Peptide systhesis.
Further, when preparing cyclic peptide, described steps d is: cut peptide chain from solid phase carrier, sloughs Side chain protective group after cyclisation, purified acquisition cyclic peptide.
RTN4B related polypeptide of the present invention also can adopt recombinant technology to produce from protokaryon or eucaryon host.
Third aspect present invention, discloses a kind of polynucleotide of separation, and it comprises a nucleotide sequence, and this nucleotide sequence is selected from lower group: (a) encodes the polynucleotide of described RTN4B related polypeptide; B polynucleotide that () is complementary with polynucleotide (a).
Polynucleotide of the present invention can be DNA form or rna form.DNA can be strand or double-strand.
Polynucleotide of the present invention can be used for the recombinant expression vector of construction expression RNT4B related polypeptide of the present invention.Recombinant expression vector refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral as adenovirus, retrovirus or other carriers.The carrier be suitable in the present invention includes but not limited to: the expression vector based on T7 (Rosenberg, et al.Gene, 1987,56.125) of expressing in bacterium; The pMSXND expression vector (Lee and Nathans, J Bio Chem.263:3521,1988) of expressing in mammalian cell and the carrier deriving from baculovirus in expressed in insect cells.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is usually containing replication orgin, promotor, marker gene and translation controlling elements.The conveniently purifying of recombinant expression vector, further when building recombinant expression vector, can also increase the encoding sequence of tag antibody to facilitate purifying in one end of RTN4B related polypeptide encoding gene.
Fourth aspect present invention, discloses the purposes of described RTN4B related polypeptide in the medicine or preparation of the biologic activity of preparation suppression RTN4B.
Accompanying drawing explanation
Fig. 1 polypeptide regulates the restraining effect test of cell adhesion ability to RTN4B.
Ordinate zou: versus cell adherence rate
X-coordinate:
NT:BSA control wells
P4+PBS: the hole of adding RTN4B-P4 and contrast PBS
P4+138: the hole of adding RTN4B-P4 and polypeptide of the present invention
Fig. 2 polypeptide is tested growth of tumour cell restraining effect.
X-coordinate:
CRDfV: contrast cyclic peptide cRDfV group
CRGDfV: positive control cRGDfV group
CRRGSS: cyclic peptide group of the present invention
Embodiment
When the present invention obtains described small peptide by the method for solid phase synthesis, utilize HPLC technology to carry out purifying, and utilize its purity of mass spectroscopy, obtain highly purified small peptide.
The present invention in vitro vascular endothelial cell is sticked in experiment and has been carried out small peptide application, has found that this small peptide can suppress sticking of vascular endothelial cell well.The research report of RTN4B and our work all find that RTN4B can promote sticking of vascular endothelial cell (as HUVEC), and the activity of this event conditioning tumor microenvironment Endothelial Cell is sticked likely via mediation, promote neonate tumour blood vessel (Fig. 1).After utilizing small peptide of the present invention and vascular endothelial cell to hatch, RTN4B is significantly suppressed (Fig. 2) to the regulating effect of the Adhering capacity of vascular endothelial cell.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.In test, unaccounted per-cent is all weight percentage.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; Theseries METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
The solid phase synthesis of embodiment 1 polypeptide of the present invention
Synthesis material and related reagent:
(1) (fluorenylmethyloxycarbonyl-S-trityl-L-cysteine) (Suzhou heavenly steed Pharmaceutical Group fine chemicals company limited) such as protected amino acid raw material: Fmoc-L-Cys (Trt)-OH;
(2) condensation reagent and fluorescence, vitamin H raw material: HBTU(benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate) (Suzhou heavenly steed Pharmaceutical Group fine chemicals company limited), HOBT(1-hydroxy benzo triazole) (Suzhou heavenly steed Pharmaceutical Group fine chemicals company limited), DIEA(N, N-diisopropylethylamine) (Solution on Chemical Reagents in Shanghai company of traditional Chinese medicines group), FITC(fluorescein isothiocyanate) (SIGMA), D-BIOTIN(D-vitamin H) (SIGMA);
(3) solvent: DMF (dimethyl formamide) (Dikma), DCM (methylene dichloride) (Dikma), acetonitrile (Fisher);
(4) resin: 2-chlorotrityl chloride resin (2-chlorine trityl chloride resin) (Tianjin Southern opens Compositech Inc.)
(5) deprotecting regent: piperidines (Solution on Chemical Reagents in Shanghai company of traditional Chinese medicines group);
(6) cutting reagent: TFA(trifluoroacetic acid) (J.T.Baker), TIS (tri isopropyl silane) (ALDRICH), EDT (1，2-ethandithiol);
(7) nitrogen (Shanghai is than Europe gas industry company);
(8) anhydrous diethyl ether (a chemical reagent company limited is tried in Shanghai); (9) precision electronic balance (Beijing Sai Duolisi balance company limited).
Plant and instrument:
(1) SYMPHONY type 12 channel polypeptide synthesizer (model: SYMPHONY, software: Version.201 producer: Protein Technologies.Inc);
(2) SHIMADZU high performance liquid chromatograph (model: preparative, analysis mode, software: Class-VP.Sevial System, manufacturer: SHIMADZU);
(3) LABCONCO Freeze Drying Equipment (model: Freezone.Plus.6, manufacturer: LABCONCO;
(4) whizzer (Anting Scientific Instrument Factory, Shanghai's model: TDL-40B).
Building-up process:
The preparation of linear polypeptide:
(1) resin swelling: 2-chlorotrityl chloride resin is put into reaction tubes, adds DMF (15ml/g), vibration 30min.
(2) connect first amino acid: fall solvent by husky core suction filtration, add Fmoc-L-Cys (the Trt)-OH amino acid of 3 times of molar excess, then add the DIEA of 10 times of molar excess, finally add DMF and dissolve, vibration 30min.
(3) deprotection: remove DMF, adds 20% Piperidine/DMF solution (15ml/g), 5min, removes and adds 20% Piperidine/DMF solution (15ml/g) again, 15min.
(4) detect: take out piperidine solution, get tens grainy resins, wash three times, add triketohydrindene hydrate, KCN with ethanol, each one of phenol solution, 105 DEG C-110 DEG C heating 5min, deepening blue is positive reaction.
(5) wash: DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, DMF (10ml/g) twice.
(6) condensation: protected amino acid (Fmoc-L-Cys (Trt)-OH) three times is excessive, and HBTU tri-times is excessive, all dissolves with as far as possible less DMF, adds reaction tubes, add NMM(N-methylmorpholine at once) ten times excessive, reaction 30min.
(7) washing: DMF (10ml/g) once, methyl alcohol (10ml/g) twice, DMF (10ml/g) twice.
(8) operation of 2-7 step is repeated, the amino acid from right to left successively in catenation sequence.
(9) drain, wash resin according to following method.DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, DMF (10ml/g) twice, DCM (10ml/g) twice, drains 10min.
(10) cut from resin and slough Side chain protective group: preparation cutting liquid (10mL/g) TFA94.5%; Water 2.5%; EDT2.5%; TIS1%, clipping time 120min.
(11) dry up washing: dried up by lysate nitrogen, wash six times with ether, then normal temperature volatilizes as far as possible.
(12) use HPLC purified polypeptide: by thick peptide pure water or add a small amount of acetonitrile dissolve, according to following condition purifying: pump A:0.1% trifluoroacetic acid pure water solution; The pure acetonitrile solution of pump B:0.1% trifluoroacetic acid; Total flux: 1.0ml/min; Volume: 30ul; Wavelength: 220nm;
Gradient:: Time(unit: minute) A B
05.00 90% 10%
30.00 20% 80%
30.10 stop
< detector A>
Post: Venusi MRC-ODS C18 30x250mm
(13) finally by the solution freeze-drying after purifying, the linear polypeptide finished product that sequence is RRGSS had both been obtained.
The preparation of cyclic peptide:
Step (1)-(9), (11)-(14) and linear polypeptide are prepared and are unanimously obtained the cyclic peptide finished product that sequence is RRGSS, and wherein step 10 adopts following three steps to substitute:
(101) polypeptide is cut from resin: preparation cutting liquid: TFA 1%; DCM99%.Cutting liquid consumption is the cutting liquid that every 1 gram of resin peptide adds 10ml, and the reaction times is 2h, and Room-temperature seal cuts.
(102) cyclisation: cutting liquid nitrogen dries up, it is excessive to add HBTU tri-times, with as far as possible less DMF dissolve, add reaction tubes, it is excessive to add NMM ten times at once, reaction 30min.
(103) Side chain protective group is sloughed: preparation cutting liquid (10mL/g) TFA94.5%; Water 2.5%; EDT2.5%; TIS1%, clipping time 120min.
Qualification: the linear polypeptide taken a morsel respectively and cyclic peptide finished product, the molecular weight identification being MS meets expection, order-checking qualification, and sequence meets expection, and HPLC purity assay is that 95%, MS method qualification structure meets expection.
The application of embodiment 2 polypeptide of the present invention in vitro in cell adhesion
Raw material and related reagent: phosphoric acid buffer, pancreatin (invitrogen company), M200 substratum and LSGS serum (cascade biologics company), bovine serum albumin (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), MTS/PMS test kit (Promega company), people HUVEC cell (ScienCell company).
Plant and instrument: cell culture incubator (Heraeus company), inverted microscope (Leica company), horizontal centrifuge (Eppendorf whizzer), microplate reader (Bio-Rad company).
Experimentation:
(1) orifice plate prepares: PBS dilutes RTN4B-P4 albumen (the part extracellular region section of RTN4B albumen, has the activity promoting cell adhesion disclosed in ZL00111791.2, can through Peptide systhesis), wraps by 96 orifice plates, every hole 100 μ L.4 DEG C are spent the night.After next day, PBS washed one time, add 1%BSA and close, every hole 150 μ L.Place 1h for 37 DEG C, then after washing one time with the serum free medium of preheating, can be used for cell inoculation.
RTN4B-P4 protein amino acid sequence (SEQ ID NO:2):
SPQAEPVWTPPAPAPAAPPSTPAAPKRRGSSGSVVVDLLYWRDIKKTGVV。
RTN4B-P4 albumen adopts the method for Peptide systhesis to prepare.
(2) cell inoculation: when cell breed in culturing bottle to 90% degree of converging time be replaced with serum free medium and continue to cultivate and can carry out digestion after 16h and gather in the crops.Discard the former substratum of cell, first with several milliliters of 1 × PBS washed cells one time, then add 1-2mL tryptic digestive juice, rock evenly and discard Digestive system, only stay about 0.5mL in bottle, culturing bottle is put in 37 DEG C of incubators and digest about 1-2min, treat cell rounding, when can depart from from bottle wall, the substratum added containing serum stops trysinization, dispel cell, proceed in sterile centrifugation tube, the centrifugal 5min of 1000rpm.After discarding centrifugal supernatant, with the serum free medium containing 0.1%BSA, cell is adjusted to 5 × 10
5the density of/mL.
(3) to blockade combination: linear polypeptide prepared by the cell suspension after dilution and embodiment 1 is hatched 30min at 37 DEG C, and working concentration is 20mM, then by cell suspension inoculation in 96 orifice plates, every hole 100 μ L.Control group adds the PBS of same volume.
(4) plate and colour developing is washed: 1h after cell inoculation, sucks substratum.Retain three parallel holes as full cell hole.The serum-free culture agent of all the other each hole preheatings or PBS washing, repeated washing three times is until non-adherent cell is washed away completely.Remaining cell MTS test kit colour developing calculates cell count.The method of calculation of versus cell adherence rate are that the OD value of experimental port is divided by the business after the OD value of full cell hole.
Test-results: as shown in Figure 1, compared with contrast NT group, RTN4B-P4(P4+PBS group) can cell adhesion be promoted.But after the polypeptide prepared as endotheliocyte and the present invention is hatched, endotheliocyte significantly declines (P4+138 group) at the Adhering capacity on P4 surface.
Result shows, and polypeptide can suppress RTN4B to regulate cell adhesion ability.Because linear loop peptide can suppress RTN4B to regulate cell adhesion ability, cyclic peptide with it sequence is consistent, therefore thinks also to have and suppresses RTN4B to regulate cell adhesion ability.
The application of embodiment 3 polypeptide of the present invention in vivo in Growth of Cells
Raw material and related reagent: phosphoric acid buffer, pancreatin (invitrogen company), DMEM substratum and foetal calf serum (Gibco company), 3-4 week age nude mice (this Leco Corp. of Shanghai), tumor cell line SMMC-7721 etc.
Plant and instrument: cell culture incubator (Heraeus company), inverted microscope (Leica company), horizontal centrifuge (Eppendorf whizzer), SPF animal housing.
Experimentation:
1. diluted by cyclic peptide PBS, be configured to the mother liquor of 5mg/mL, pH value is adjusted to 7.4.
2. raise nude mice (3-4 age in week).
3. cultivate SMMC7721 liver cancer cell, collecting cell, resuspended to 2*10 with serum free medium
7/ mL.
4. by cell suspension inoculation to nude mice by subcutaneous, every mouse 200uL, totally 40 mouse.
5. cell infusion is after 4 days, and 40 mouse are divided into 4 groups, carries out polypeptide injection in knurl, every mouse 200uL.Continuous injection 13 days.
6. within after drug withdrawal the 3rd day, get knurl.
7. carry out tumor weight analysis, often group removes maximum and minimum value.
Blank: isopyknic PBS.
Contrast cyclic peptide group: cRDfV, adopts Peptide systhesis and the cyclization method of reference example 1 prepares.
Positive controls: cRGDfV(has entered the RGD cyclic peptide of clinical III phase, adopts Peptide systhesis the cyclization method of reference example 1 to prepare.
Test cyclic peptide group: adopt cyclic peptide prepared by embodiment 1.
(4) knurl body record: measure a tumorous size in every two days, and according to a × b
2calculate tumorous size.
(5) last injection gets knurl in latter two days, and knurl body is weighed and taken pictures.
Result: as shown in Figure 2, compare with contrast cyclic peptide, cRRGSS(tests cyclic peptide group) and cRGDfV(positive controls) there is the effect of suppression knurl bulk-growth, result shows, cyclic peptide of the present invention can the growth of inhibition tumor cell.Due to linear peptides of the present invention, sequence is consistent with it, therefore thinks that linear peptides also has the effect of the growth of inhibition tumor cell.
Claims (12)
1. a RTN4B related polypeptide, its aminoacid sequence is as shown in SEQ ID NO.1, and described RTN4B related polypeptide is cyclic peptide.
2. RTN4B related polypeptide as claimed in claim 1, is characterized in that, the biologic activity of described RTN4B related polypeptide energy vitro inhibition RTN4B.
3. the preparation method of RTN4B related polypeptide as claimed in claim 1 or 2, comprises the linear polypeptide adopting chemical synthesis to obtain described RTN4B related polypeptide, and the cyclisation of described linear polypeptide head and the tail is obtained cyclic peptide.
4. the preparation method of RTN4B related polypeptide as claimed in claim 3, is characterized in that, comprise the following steps:
A first the carboxy terminal amino acid of peptide chain is covalently attached on solid phase carrier by ();
B (), through deaminizating protecting group, is reacted with the next amino acid condensation of excessive activated carboxyl, thus extend peptide chain;
C the amino acid of () circulation step b successively in catenation sequence is until extend to peptide chain N-terminal;
D () cuts peptide chain from solid phase carrier, slough Side chain protective group after cyclisation, purified acquisition cyclic peptide.
The purposes of 5.RTN4B related polypeptide in the medicine or preparation of the biologic activity of preparation suppression RTN4B, the aminoacid sequence of described RTN4B related polypeptide is as shown in SEQ ID NO.1.
6. purposes as claimed in claim 5, it is characterized in that, described RTN4B related polypeptide is cyclic peptide or line style polypeptide.
The purposes of 7.RTN4B related polypeptide in the medicine preparing treatment or prophylaxis of tumours, the aminoacid sequence of described RTN4B related polypeptide is as shown in SEQ ID NO.1, and described RTN4B related polypeptide is cyclic peptide.
8. the purposes of RTN4B related polypeptide as claimed in claim 7, is characterized in that, described tumour is selected from the expression of its growth or propagation and RNT4B albumen or the relevant tumour of activity.
9. the purposes of RTN4B related polypeptide as claimed in claim 8, it is characterized in that, described tumour is liver cancer.
10.RTN4B related polypeptide is preparing the application in the medicine or preparation suppressing vascular endothelial cell to stick, and the aminoacid sequence of described RTN4B related polypeptide is as shown in SEQ ID NO.1.
11. purposes as claimed in claim 10, is characterized in that, described RTN4B related polypeptide is cyclic peptide or line style polypeptide.
12. 1 kinds of medicines or preparation, its activeconstituents contains RTN4B related polypeptide described in claim 1 or 2.
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| CN201310136244.8A CN103387603B (en) | 2012-05-08 | 2013-04-18 | Polypeptide related to RTN4B, and preparation and application thereof |
| PCT/CN2013/075186 WO2013166944A1 (en) | 2012-05-08 | 2013-05-06 | Rtn4b-related polypeptide, and, preparation and applications thereof |
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| CN201210141116.8 | 2012-05-08 | ||
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1311439A (en) * | 2000-03-02 | 2001-09-05 | 复旦大学 | Human RTN 4B protein and code sequence, and its preparation and use |
| CN1745094A (en) * | 2002-12-06 | 2006-03-08 | 新加坡综合医院有限公司 | Peptides, antibodies thereto, and their use in the treatment of central nervous system damage |
| CN101015683A (en) * | 2007-01-18 | 2007-08-15 | 复旦大学 | Application of human RTN4B protein in preparation of medicine for healing wound |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1311439A (en) * | 2000-03-02 | 2001-09-05 | 复旦大学 | Human RTN 4B protein and code sequence, and its preparation and use |
| CN1745094A (en) * | 2002-12-06 | 2006-03-08 | 新加坡综合医院有限公司 | Peptides, antibodies thereto, and their use in the treatment of central nervous system damage |
| CN101015683A (en) * | 2007-01-18 | 2007-08-15 | 复旦大学 | Application of human RTN4B protein in preparation of medicine for healing wound |
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| Title |
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| Comparative proteomic analysis of early salt stress-responsive proteins in roots of SnRK2 transgenic rice;NAM,M.H. et al;《Proteome Science》;20120331;第10卷;第2段 * |
| Link of a new type of apoptosis-inducing gene ASY/Nogo-B to human cancer;LI,Q. et al;《Oncogene》;20010705;第20卷;第3929页摘要，第3930页图1，第3935页左栏 * |
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