CN108640970A - Target polypeptide and its application of dystopy ATP5B accesses - Google Patents
Target polypeptide and its application of dystopy ATP5B accesses Download PDFInfo
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Abstract
It is valine V or methionine M the present invention provides a kind of polypeptide of targeting dystopy ATP5B accesses and its application, motif NPLKXDWG, wherein X, the N-terminal and/or C-terminal of motif are connected with 0 or 2 amino acid；Sequentially connected two amino acid of N-terminal is proline P and valine V, and sequentially connected two amino acid of C-terminal is leucine L and proline P.The present invention also provides the polypeptides in the application for preparing tumour diagnostic reagent and tumor-targeting drug.Polypeptide provided by the invention can influence the aggregation capability of blood platelet, and reduce the formation of membrane glycoprotein GPIIb/IIIa compounds.The peptide fragment has combination for Partial tumors cell, and polypeptide connection toxic sequences compound can inhibit to express the proliferation of the tumour cell of dystopy ATP5B under a certain concentration.Therefore, which is early diagnosis of tumor, targeted therapy provides important theory and practice basis, is had broad application prospects.
Description
Technical field
The present invention relates to a kind of target polypeptides, and in particular to it is a kind of targeting dystopy ATP5B accesses polypeptide and its application.
Background technology
Malignant tumour（Including leukaemia）The important disease for endangering human health at present, although after operative treatment,
After radiotherapy, chemotherapy, in recent years in biological therapy（Including cell therapy, immunization therapy etc.）Even targeted therapy
Aspect obtains many progress, but still has quite a few patient to fail in various treatments.It is various just control in effective case,
How to take appropriate measures prevents from needing the important topic solved in transfer or recurrence and oncotherapy.Research and development are more efficient
Or cheaper antitumor drug, it is always field scientist and the target that medical workers are pursued.
Before at least 50 years, scholars just recognize that blood platelet plays a significant role in Malignant tumor of bonal metastasis：Entity tumor
After cell enters blood vessel, with the platelet-shaped in blood at aggregation（Platelet- tumor aggregates, PTA）；PTA
Formation be conducive to metastases, and antiplatelet measure can inhibit metastases.We study PTA formed mechanism when,
It finds for the first time and proves the ATP5B in Partial tumors cell membrane surface ectopic expression（ATP synthase β subunit, ATP
Synthase beta-subunit is distributed only in the case of " normal " on the inside of mitochondrial inner membrane）, the glycoprotein IIB with platelet surface（GPIIb）
Interaction participates in the formation for mediating PTA；Further, a sequence peptide fragment from GPIIb can be combined efficiently in tumour cell
On the ATP5B on surface；Using the polypeptide as targeting peptides, known toxic polypeptide can be directed to the tumour of expression dystopy ATP5B
On cell, to realize the target killing to tumour cell.Meanwhile often occurring with new vessels inside entity tumor, it is existing
Data shows that neovascular endothelium cell also expresses dystopy ATP5B, and the Surface of Vascular Endothelial Cells of normal steady state is not expressed then
Dystopy ATP5B, therefore the target killing to new vessels also can be achieved at the same time in the target polypeptide.
It domestic and international Brief Review on Research Progress and is analyzed as follows：
1, about neoplasm targeted therapy
The concept of " targeted therapy " and trial most occur earlier than the 1980's with monoclonal antibody technique, and with monoclonal antibody medicine
Object is popularized and is more taken seriously.But the data of accumulation is found, only special in certain specific molecular of tumor cells expression, that is, tumour
Hapten（Tumor-Specific Antigen）And be not present, and most tumors related antigen（Tumor-Associated
Antigen）Also it is expressed in normal cell, therefore " targets " hazard boundary of the antibody of these molecules far beyond tumour cell, than
If currently used Rituximab can eliminate the high lymphoma cell for expressing CD20 simultaneously, same expression CD20 is also eliminated
The normal development stage bone-marrow-derived lymphocyte, to cause the immunosupress of complete set.In recent years the Chimeric antigen receptor T cell occurred
（CAR-T）Therapy（Such as it is directed to the various CAR-19 of CD19）There is also similar limitations.Therefore at this stage, still need to combine tumour
The characteristics of cell itself, combines mature biotechnology, finds new neoplasm targeted therapy scheme or strategy.
Targeted therapy is the essential characteristic of modern medical treatment, especially in all tumours including neoplastic hematologic disorder
In treatment, targeted therapy is the major fields of medicament research and development.In conjunction with applied in being treated in hematologic malignancies chemical drugs, life
The action target spot of object medicine is classified（There is intersection each other）, application or present Research and development trend to targeted therapy etc. make one point
Analysis：
2, the major target class of neoplasm targeted therapy
Clinic no matter is applied at present, has researched and developed or the target of neoplasm targeted therapy still in phase of basic research
Point, is divided into three categories substantially：（1）Tumor cell surface antigen：Drug or drug candidate for this kind of target spot are mainly single
Anti- class preparation or derivatives thereof（Including Fab segments, humanization improver, with the other compositions person of coupling）, directly to carrying spy
The tumour cell for determining antigen is attacked, is killed.Exemplary includes：The natural monoclonal antibody or humanization monoclonal antibody of anti-CD20
The antibody drug Brentuximab vedotin that Ofatumumab, CD30 monoclonal antibody are constituted with vedotin, and be directed to
The bispecific antibody of double target spots.The latter can simultaneously tumor cell surface antigen and CTL surface antigens, by tumour cell with
Killing cell connects together and activates the latter, to improve killing-efficiency or overcome immunosurveillance escape mechanism.Anti- CD19/
CD3 double antibodies, anti-CD 33/CD3 double antibodies, anti-CLL1/CD3 double antibodies, AntiCD3 McAb 0/CDl6a double antibody etc. be also respectively at
Different conceptual phases.（2）The closely related signal path molecule of tumour：This kind of drug is mainly by rightCellVarious letters
Number access is interfered, to realize the treatment to tumour cell, such as Btk inhibitor Ibrutinib, PKCl3 inhibitor
Enzastaurin, PI3K inhibitor Iderlalisib or micromolecular inhibitor AMG319, Bcl.2 inhibitor ABT0199/
Inhibitor Everolimus, PI3K isomers and mTORCl and TORC2 inhibitor SAR2454096 of ABT-199, mTORCl etc.,
MDM2 antagonists RG7112 etc..（3）The inhereditary material of tumour cell：Inhereditary material changes（Including series jump or apparent modification）
It is one of tumorigenic material base.Although theoretically tumour cell can be kept extensive by correcting the inhereditary material being mutated
It is multiple normal, but due to technology restriction, it is clinically and infeasible；And epigenetic can then be realized by drug and adjusted, such as
Using general deacetylase inhibitor Panobinostat and other chemotherapeutics（Ifosfamide+carboplatin+Etoposide）It closes
Refractory Hodgkin lymphoma is recurred with treatment, obtains first-stage success.
In addition to this, antitumor immunity of organism cell or the targeted therapies for tumour cell microenvironment are also directed to, such as
The antitumor activity etc. that can restore T cell with antibody or other modes inhibition T cell PD-L1/PD-1 reciprocations, because indirect
Tumour cell is acted on, is omited herein.
3, blood platelet-possible application of the tumor accumulation mechanism target spot in treatment and prevention of tumour
Effect of the 3.1 blood platelets-tumor accumulation in metastases：Hematogenous metastasis is the main path of tumour amphi position transfer.It is early
People just recognize the presence of blood platelet-tumor accumulation phenomenon before a century[1], until nineteen sixty generation recognize that the mechanism exists
Importance in metastases simultaneously begins through and interferes the mechanism and prevent metastases[2, 3].Traditionally it is thin to be incorporated in tumour
The blood platelet of cellular surface has facilitation to transfer, clinically antiplatelet strategy is applied to prevent metastases.Blood platelet promotees
Mechanism into metastases includes：Direct " umbrella " effect to tumour cell；Increase the viscosity of tumour cell or cell mass
And it is easy to vascular wall side collection；Mediate with endothelial cell in conjunction with, discharge protease and degradation of esoderma intercellular matrix or substrate
Film is to make tumour cell group be easy to move out.It is singly wherein " umbrella " mechanism, also there are at least four aspect factors：It is hindered by physics
Make monitoring and attack of the tumour cell from immunocyte every mechanism, to be conducive to survival of the tumour cell in blood phase and turn
It moves[4, 5]；Interfere the lethal effect of certain cytokine on tumor cells；Blood platelet shifts the MHC-I class molecules of own face
To tumor cell surface, make its " camouflage " at normal cell, to escape the identification and killing of immunocyte[6]；Blood platelet is logical
It crosses the tumour cell that directly effect induction combines and epithelial-mesenchymal conversion occurs, to promote to shift, and leading structure is suitable for transfer
The microenvironment " tabernacle " that stove is planted and expanded[7, 8].Although most evidences think that blood platelet plays " accomplice " work in metastases
With, but there are also evidence, to show that the blood platelet for being incorporated into tumour cell may have the survival of tumour cell or proliferation unfavorable
On one side.For example, no matter to entering the solid tumor cell of blood or originating from the leukaemia cell of blood, blood platelet is possible to induce
Apoptosis or direct killing tumour cell[9, 10]；But various cells are inconsistent to the sensibility of blood platelet lethal effect, Sagawa etc.
Report blood platelet can direct killing K562, KU812, LU99A, KGl tumour cell, but for U937, MIA, PaCa-2,
MOLT-4 tumour cells are invalid[11].The work of our early stages is it has also been found that the blood platelet in physiological range quantity can be different
Inhibit to degree the proliferation of various tumour cells[12], it is white then to prove that the blood platelet for being incorporated into leukaemia cell can change recently
Sensibility of the blood disease cell to chemotherapeutics[13].Therefore " net " effect for the tumour cell that blood platelet touches it depends on
Two kinds effect balances, such as nearest German scholar blood platelet combination B16 tumour cells are proved in mouse tumor model after promote
It is trapped within lung into tumour, but also inhibits the proliferation of tumour cell simultaneously[14]。
3.2 blood platelets-tumor accumulation mechanism：The molecule for participating in PTA formation is currently known there are about 10 pairs, wherein main
Be various glycoprotein on film, the same molecule on sometimes two kinds of cell membranes（Such as GPIIbIIIa）Match by another
Body（Such as fibrin）Connection.Although the molecular species of platelet surface is relatively fixed, tumour cell film surface participates in PTA shapes
At molecular spectra then may with tissue-derived, differentiation state, even individual difference and it is different, wherein existing may generally express
Molecule such as GPIIbIIa, it is also possible to have the molecule only expressed in Partial tumors.It is formed if certain molecule participates in PTA and belongs to swollen
The related even tumour specific antigen of tumor（It is not expressed or low expression in normal structure）, it is likely that become by interfering PTA machines
Make and prevent the target molecule of metastases.The dystopy ATP5B of this project group selection is such.
Ectopic expressions of 3.3 ATP5B on tumor cell membrane：" current money " of cellular energy metabolism is ATP, catalysis
The Major Enzymes of synthesis ATP are the F1F0-ATP synthase positioned at mitochondrial inner membrane.The enzyme is made of totally 24 kinds of albumen, and ATP5B is
Three β subunits of the parts F1.Under normal conditions, which is distributed only over mitochondrial inner membrane.But Das reported K562 for the first time in 1994
There is also ATP5B albumen with A439 cell surfaces, and the albumen is identified by lymphocyte NK or LAK cell[15].Then successively
Detect the presence of ATP5B on the cell membrane of other tumours, for example, Dowling compare it is maternal from MDA-MB-435S but
The memebrane protein of two different clones of invasiveness finds that 16 kinds of albumen make every effort to overcome grand kind of up-regulated expression in height invasion, wherein just including
ATP5B[16]；One thunder seminar of Jilin University Lee then finds in high metastatic prostate cancer cell strain PC-3M compared with female parent strain PC-3
The dystopy ATP5B expression quantity highers of cell[17]；The bright seminar of Wei Yuquan, Yang Jin proves in non-small cell lung cancer and cancer beside organism
The expression ratio of dystopy ATP5B is 72% respectively（23/32）With 26%（16/62）, and the ratio in squamous carcinoma and Small Cell Lung Cancer
Example is respectively then 67%（22/33）With 0%（0/10）[18]；Gao Feng seminars then prove the ATP5B of tumour cell film surface by line
Mitochondrial membrane is transported so far, but may be unrelated with the grade malignancy of tumour[19]（The universality of the conclusion is worth discussion, because should
Tumor cell line quantity of the article for comparing grade malignancy is very little and source is different, such as wherein two plants of " high malignancy " cells
System is respectively lung cancer 95-D and liver cancer HepG2, with the comparativity of the lung cancer A549 and hepatic cell line L-02 of " low potential malignancy " all compared with
Difference[19]）；Yang Zhulin seminars then find the loss or low expression of dystopy ATP5B expression totally in clinical 126 gallbladder cancer cases
It is big gross tumor volume, high TNM classifications, Lymph Node Metastasis, the risk factor of other position invasion[20]。
Other than tumour cell, it is now known that if other cells, which may also express dystopy ATP5B-, will use tumour table
For the dystopy ATP5B in face as therapy target, then the cell of other expression dystopy ATP5B is by as the target spot of side effect, therefore outstanding
It is needed to pay attention to.It is consistent with cancer target screening, although being currently known vascular endothelial cell expression dystopy ATP5B,
But current evidence is all in original cuiture or in vitro culture and the blood vessel endothelium in exponential phase is thin rather than original in body
Position vascular endothelial cell；Prove that dystopy ATP5B can be used as angiostatin on vascular endothelial cell at present[21]、kringle 1–
5 （K1–5）[22], pigment epitheli μM-derived factor, that is, PEDF[23]Receptor play a role, these ligands
Endothelial cell proliferation is caused to inhibit even apoptosis after being combined with ATP5B.If dystopy ATP5B is only in the vascular endothelial cell of proliferation
Upper expression and expressed in non-proliferative vascular endothelial cell, then the ATP5B of new vessels will can be used as oncotherapy in tumour
In adjection target spot without causing the side effect to normal blood vessels -- as the anti-new vessels in previous oncotherapy
Strategy.Data show that being directed to the PEDF of neovascular endothelium cell known to originally, the different of tumor cell surface also can be combined in
Position ATP5B is to inhibit the apoptosis of tumour cell[24], prompt ectopic expression can after the ATP5B of different cells is by ligand binding
Identical access can be mediated, cause similar effect.
Invention content
The purpose of the present invention is to provide a kind of polypeptides of targeting dystopy ATP5B accesses and the polypeptide to be examined in preparation tumour
Application in disconnected reagent and tumor-targeting drug.
The polypeptide of targeting dystopy ATP5B accesses of the present invention, motif NPLKXDWG, wherein X for valine V or
Methionine M, the N-terminal and/or C-terminal of motif are connected with 0 or 2 amino acid.
Further, sequentially connected two amino acid of the N-terminal is that proline P and valine V, the C-terminal connect successively
Two amino acid connect are leucine L and proline P.
The amino acid sequence of the polypeptide is amino acid sequence shown in SEQ ID NO.1 ~ SEQ ID NO.8, specifically such as
Shown in lower：
N2G：NPLKVDWG,
N2Gm：NPLKMDWG,
N2G-LP：NPLKVDWG-LP,
PV-N2G：PV-NPLKVDWG,
PV-N2G-LP：PVNPLKVDWGLP,
N2Gm-LP：NPLKMDWG-LP,
PV-N2Gm：PV-NPLKMDWG,
PV-N2Gm-LP：PVNPLKMDWGLP.
The polypeptide of targeting dystopy ATP5B accesses of the present invention is in the application for preparing tumour diagnostic reagent.
The polypeptide of targeting dystopy ATP5B accesses of the present invention is in the application for preparing tumor-targeting drug.
A kind of neoplasm targeted therapy reagent includes the polypeptide of targeting dystopy ATP5B accesses of the present invention.
Preferably, the polypeptide of the targeting dystopy ATP5B accesses is coupled on bioactive molecule, and the bioactive molecule includes tool
There is the toxicity molecule for the treatment of function of tumor.
Preferably, the polypeptide of the targeting dystopy ATP5B accesses, which is coupled at, passs on drug carrier, and the drug carrier of passing is nanometer
Grain or microballoon.
Preferably, the polypeptide expression of the targeting dystopy ATP5B accesses is on recombinant toxin molecule, the recombinant toxin point
Son passes through genetic recombination by the corresponding DNA sequence dna of polypeptide of targeting dystopy ATP5B accesses and the DNA sequence dna of protein toxin molecule
Recombinant gene expression afterwards forms.
Advantageous effect：The present invention provides a kind of polypeptides of targeting dystopy ATP5B accesses and its application, the present invention to utilize mouse
The monoclonal antibody of anti human platelet membrane glycoprotein GPIIb（SZ-22）Three-wheel is carried out to random 12 peptide library of phage display
Elutriation screening that biology is affine, after clone's large amplification of gained, carries out extraction and the sequencing of DNA, then to measurement
Bacteriophage monoclonal sequence is compared to obtain peptide section sequence N2G：NPLKVDWG passes through fluidic cell skill after artificial synthesized
Art and Western blot technologies prove that peptide fragment N2G can block the monoclonal of mouse anti human platelet membrane glycoprotein GPIIb anti-
Body（SZ-22）And the combination of Normal Human Platelets.Meanwhile the peptide fragment can influence the aggregation capability of blood platelet, and reduce film sugar
The formation of Protein G PIIb/IIIa compounds.The peptide fragment has combination for Partial tumors cell, and using N2G as targeting peptides
The complex polypeptide constituted with known toxicity peptide fragment KLAKLAKKLAKLAK（NPLKVDWG-LP-KLAKLAKKLAKLAK）Certain
The proliferation that can inhibit tumour cell under concentration embodies the N2G polypeptides and is preparing tumour diagnostic reagent and tumor-targeting drug
Aspect is with a wide range of applications.
Description of the drawings
Fig. 1 is the sequence analysis for 11 polypeptides that PhD is filtered out and the comparison chart with CD41 gene orders（Used
PhD12 carriers on, be GGG immediately in subsequent three amino acid of 12 peptides, so being mended behind tri- polypeptides of H10, A11, B10
Position g）.
Fig. 2 is that flow cytometer detection polypeptide is small to the monoclonal antibody SZ-22 and blood of mouse anti human platelet membrane glycoprotein GPIIb
The blocking effect figure of hardened conjunction, wherein A, B scheme a concentration of 0.1 μ g of corresponding SZ-22 antibody；C, D schemes corresponding SZ-22 antibody
A concentration of 1 μ g.
Fig. 3 is the monoclonal antibody that Western blot methods detect polypeptide to mouse anti human platelet membrane glycoprotein GPIIb
The blocking effect figure that SZ-22 is combined with blood platelet total protein, wherein A, B scheme a concentration of 0.1 μ g of corresponding SZ-22 antibody；C、D
Scheme a concentration of 1 μ g of corresponding SZ-22 antibody.
Fig. 4 N2G polypeptides（Peptide 8）To the histamine result figure of the platelet aggregation of ADP inductions（With platelet aggregation
Figure calculates aggregation rate）.
Fig. 5 N2G polypeptides（Peptide 8）To the histamine result figure of the activation of ADP inductions（It is dyed with PAC-1 and shows that blood is small
Plate activates）.
Fig. 6 is flow cytomery FITC-N2G polypeptides and the intensity map of Peptide S and four kinds of cell combinations.
Fig. 7 is the fluorescence microscopy figure that four kinds of cells are combined with FITC-N2G polypeptides.
Fig. 8 is the maximum suppression concentration comparison diagram of Peptide 1 and Peptide 5.
Fig. 9 is influence diagrams of the Peptide 1 and Peptide 5 to seven plants of tumor cell proliferations.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, but examples are merely exemplary, not to this hair
Bright range constitutes any restrictions.It will be understood by those skilled in the art that without departing from the spirit and scope of the invention
Can the details and form of technical solution of the present invention be modified or be replaced, but these modifications and replacement each fall within the present invention's
In protection domain.
Embodiment 1：Target the acquisition of the polypeptide sequence of dystopy ATP5B accesses
By the monoclonal antibody of purified mouse anti human platelet membrane glycoprotein GPIIb（SZ-22）With 1010The bacteriophage of pfu
12 peptide libraries of displaying（New England Biolabs Products, embedded 12 amino in the pIII albumen of M13 bacteriophages
The long random peptide of acid）It is incubated at room temperature 10 ~ 60min, TBST washes away unbonded bacteriophage, and lower knot is eluted by glycine elution liquid
The bacteriophage for closing SZ-22 antibody carries out same 2 wheel elutriation and screening after amplification titrates, then selects 12 bacteriophage Dan Ke
The grand extraction for carrying out DNA and sequencing, translate into polypeptide, to more corresponding to phage clone by the nucleotide sequence of acquisition
Peptide sequence carries out homology analysis using ncbi database and Clustal softwares, it is found that wherein 11 peptide fragments are high each other
Spend it is homologous, and with people's CD41 albumen（GPIIb）Homologous, aligned sequences are：NPLKVDWG is named as N2G polypeptides.（See Fig. 1）.Root
According to the sequence of its corresponding GPIIb, respectively forwardly and/or two amino acid that extend back, or by V therein variations it is M, then divides
Following polypeptide sequence is not constituted：
N2G：NPLKVDWG；
N2Gm：NPLKMDWG；
N2G-LP：NPLKVDWG-LP；
PV-N2G：PV-NPLKVDWG；
PV-N2G-LP：PVNPLKVDWGLP；
N2Gm-LP：NPLKMDWG-LP；
PV-N2Gm：PV-NPLKMDWG；
PV-N2Gm-LP：PVNPLKMDWGLP.
Embodiment 2：The combination of synthesis polypeptide segment and blood platelet
According to N2G sequences, through chemically synthesized polypeptide.Blood platelet is prepared from normal human peripheral blood and is adjusted to suitable concentration, respectively
By various concentration（100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM and 0 μM）N2G polypeptides（Peptide 8）With 0.1 μ g/1 μ g
SZ-22 antibody is incubated at room temperature 30-60min；MTB is added after blood platelet incubation at room temperature 30min is added（Modified Tyrode’s
buffer）Buffer solution is washed, and blood platelet is resuspended in MTB buffer solutions, the sheep anti-mouse igg antibody of PE labels is added as secondary antibody,
Room temperature, which is protected from light, is incubated 30min, and MTB buffer solutions are added and are washed, and is detected with MTB buffer solutions resuspension blood platelet and upper machine more
The blocking degree that peptide combines SZ-22 antibody with blood platelet, rondom polypeptide Peptide S groups and the blank group conduct for being not added with polypeptide
Negative control（See Fig. 2）, the results showed that, compared with the blank group and rondom polypeptide Peptide S groups that are not added with N2G polypeptides, with
The increase of N2G peptide concentrations, the blocking effect combined with blood platelet to SZ-22 antibody is more apparent, and to low concentration but satisfies
In the SZ-22 antibody of platelet surface, the effect that polypeptide blocks it is more notable for the combination of sum.
It extracts Normal Human Platelets total protein and carries out protein SDS-PAGE electrophoresis, respectively by various concentration（100 μM, 10 μM,
1 μM, 0.1 μM, 0.01 μM and 0 μM）Polypeptide Peptide 8 and 0.1 μ g/1 μ gSZ-22 antibody, 4 DEG C incubation 30-60min, it is more
Peptide is incubated with SZ-22 antibody mixtures as primary antibody and blood platelet albumen band, then anti-with the sheep anti-mouse igg of HRP labels
Body is incubated therewith as secondary antibody, and for β-Tubulin albumen as internal reference, exposure imaging observes polypeptide to SZ-22 antibody and blood platelet
In conjunction with blocking degree, rondom polypeptide Peptide S groups and be not added with the blank group of polypeptide as negative control.As a result（See Fig. 3）
It has been shown that, compared with the blank group and rondom polypeptide Peptide S groups that are not added with polypeptide, with the increase of peptide concentration, to SZ-22
The blocking effect that antibody is combined with blood platelet is also more apparent, dose-dependent effect is presented, existing for the SZ-22 antibody of low concentration
In the case of, the effect that polypeptide blocks it will be apparently higher than in the presence of high concentration SZ-22 antibody.
Embodiment 3：Effect of the N2G polypeptides to platelet activation
1）Influence of the polypeptide to platelet aggregation
Platelet aggregation instrument detects：Extraction Normal Human Platelets are simultaneously adjusted to suitable concentration, blood platelet and 10 μM of Peptide 8
In incubation at room temperature 30min, platelet aggregation instrument returns to zero polypeptide with MTB buffer solutions, do not add peptide blank group blood platelet and
The blood platelet of rondom polypeptide Peptide S groups incubation is added as negative control, 10 μM of ADP are in 37 DEG C of aggregation instrument moderate stimulation blood
Platelet is assembled, and platelet aggregation instrument observes the degree of platelet aggregation.As a result（See Fig. 4）The sky for showing and being not added with polypeptide
White group and rondom polypeptide Peptide S groups are compared, and 10 μM of polypeptides can reduce the aggtegation caused by ADP stimulating platelets.
2）The influence that polypeptide forms platelet membrane glycoprotein GPIIb/IIIa compounds
Flow Cytometry detects：Extraction Normal Human Platelets are simultaneously adjusted to suitable concentration, blood platelet and 10 μM of Peptide 8
ADP stimulations group is not added and 10 μM of ADP stimulations groups are added in 37 DEG C of incubation 30min, adds in incubation at room temperature 30min for polypeptide
After PAC-1 antibody carries out dyeing 30min, the formation of upper machine testing membrane glycoprotein GPIIb/IIIa compounds.As a result（See Fig. 5）Table
Bright, compared with being not added with the blank group of polypeptide, being added without ADP stimulations group and rondom polypeptide Peptide S groups, 10 μM of polypeptides can subtract
The formation of membrane glycoprotein GPIIb/IIIa compounds caused by few ADP stimulating platelets.
Embodiment 4：The interaction of N2G polypeptides and tumour cell
N2G polypeptides of the present invention are because of itself and platelet membrane glycoprotein GPIIb（CD41 albumen）With high homology,
Therefore the interaction of mediating platelet and tumour cell is gone back, and there is combination, and complex polypeptide for Partial tumors cell
（NPLKVDWG-LP-KLAKLAKKLAKLAK）It can inhibit the proliferation of tumour cell under a certain concentration.
1）The binding ability of N2G polypeptides and tumour cell
Tumour cell（K562、NB4）And normal Skin Cell（HACAT）And skin cancer cell（A431）With 10 μM of artificial conjunctions
At 8 polypeptides of Peptide with FITC labels be incubated 30min in 4 DEG C, PBS washes away unbonded peptide molecule, and streaming is thin
Born of the same parents' technology detects the average fluorescent strength of N2G polypeptides and various cell combinations.Tumour cell（K562、NB4）And normal skin
Skin cell（HACAT）And skin cancer cell（A431）With 10 μM of artificial synthesized 8 polypeptides of Peptide with FITC labels in
It is incubated at room temperature 30min, PBS washes away unbonded peptide molecule, and immunofluorescence technique detects N2G polypeptides and various cell combinations are strong
Degree.As a result（See Fig. 6,7）Show and control cell（HACAT, A431）It compares, the mean fluorecence of N2G polypeptides and tumor combination is strong
Degree is increased（Rondom polypeptide Peptide S groups and 8 groups of indifferences of target polypeptide Peptide are shown in Fig. 6, because rondom polypeptide with
Target polypeptide aminoacid ingredient is identical, only amino acid formed peptide fragment position be varied from, this may prompt tumour cell for
The requirement of the amino acid tandem of polypeptide is not very high）.
2）The half-inhibition concentration of complex polypeptide
Artificial synthesized complex polypeptide Peptide 1 is carried out on the basis of the polypeptide that phage display filters out（NPLKVDWG-
LP-KLAKLAKKLAKLAK）With control group polypeptide Peptide 5（KLAKLAKKLAKLAK）, carry out half-inhibition concentration
(IC50) measurement detects various concentration polypeptide using the method for above-mentioned MTT（200 μM, 160 μM, 120 μM, 80 μM,
40 μM, 10 μM, 5 μM, 1 μM, 0.1 μM, 0.01 μM, 0 μM）The inhibition of acute promyelocytic leukemia cell NB4 is made
With addition polypeptide group is as a control group.As a result（See Fig. 8）Show that the IC50 of Petide 1 is 60.34 μM.Peptide's 5
IC50 is 159.6 μM, and complex polypeptide shows the inhibiting effect of NB4 cells certain dose dependent, with peptide concentration
Raising, inhibiting rate increase.And complex polypeptide half-inhibition concentration will be significantly lower than control group polypeptide.
3）Influence of the complex polypeptide to blood bome tumor cell Proliferation
Artificial synthesized complex polypeptide is carried out on the basis of the polypeptide that phage display filters out, by tumour cell（K562、NB4）
And normal Skin Cell（HACAT）And skin cancer cell（A431）With various concentration（10 μM, 1 μM, 0.1 μM）It is artificial synthesized
Complex polypeptide Peptide 1 and polypeptide Peptide 5 be incubated respectively in 37 DEG C for 24 hours, 48h, using mtt assay detect complex polypeptide
In 48h to the influence of tumor cell proliferation, polypeptide group is added and is not added with the blank group of polypeptide as negative control.As a result
（See Fig. 9）It has been shown that, with 5 groups of polypeptide Peptide being added and compared with being not added with the blank group of polypeptide, complex polypeptide is dense at 10 μM
Tumour cell is significantly inhibited under degree（K562、NB4）Proliferative capacity；But to normal Skin Cell（HACAT）And skin
Cancer cell（A431）, 1 unrestraints of complex polypeptide Peptide effect.
Bibliography
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227.。
Sequence table
<110>First Affiliated Hospital of Soochow University,Suzhou
<120>Target polypeptide and its application of dystopy ATP5B accesses
<141> 2018-03-29
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 1
Asn Pro Leu Lys Val Asp Trp Gly
1 5
<210> 2
<211> 8
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 2
Asn Pro Leu Lys Met Asp Trp Gly
1 5
<210> 3
<211> 10
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 3
Asn Pro Leu Lys Val Asp Trp Gly Leu Pro
1 5 10
<210> 4
<211> 10
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 4
Pro Val Asn Pro Leu Lys Val Asp Trp Gly
1 5 10
<210> 5
<211> 12
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 5
Pro Val Asn Pro Leu Lys Val Asp Trp Gly Leu Pro
1 5 10
<210> 6
<211> 10
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 6
Asn Pro Leu Lys Met Asp Trp Gly Leu Pro
1 5 10
<210> 7
<211> 10
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 7
Pro Val Asn Pro Leu Lys Met Asp Trp Gly
1 5 10
<210> 8
<211> 12
<212> PRT
<213>Chemical synthesis (artificial sequence)
<400> 8
Pro Val Asn Pro Leu Lys Met Asp Trp Gly Leu Pro
1 5 10
Claims (8)
1. a kind of polypeptide of targeting dystopy ATP5B accesses, which is characterized in that its motif is NPLKXDWG, and wherein X is valine V
Or methionine M, the N-terminal and/or C-terminal of motif are connected with 0 or 2 amino acid.
2. a kind of polypeptide of targeting dystopy ATP5B accesses according to claim 1, which is characterized in that the N-terminal connects successively
Two amino acid connect are proline P and valine V, and sequentially connected two amino acid of C-terminal is leucine L and proline
The amino acid sequence of P, the polypeptide are amino acid sequence shown in SEQ ID NO.1 ~ SEQ ID NO.8.
3. the polypeptide of the targeting dystopy ATP5B accesses described in claim 1-2 any one is preparing answering for tumour diagnostic reagent
With.
4. the polypeptide of the targeting dystopy ATP5B accesses described in claim 1-2 any one is preparing answering for tumor-targeting drug
With.
5. a kind of neoplasm targeted therapy reagent, which is characterized in that including the targeting dystopy ATP5B accesses described in claim 1-2
Polypeptide.
6. a kind of neoplasm targeted therapy reagent according to claim 5, which is characterized in that the targeting dystopy ATP5B is logical
The polypeptide on road is coupled on bioactive molecule, and the bioactive molecule includes the toxicity molecule with treatment function of tumor.
7. a kind of neoplasm targeted therapy reagent according to claim 5, which is characterized in that the targeting dystopy ATP5B is logical
The polypeptide on road, which is coupled at, to be passed on drug carrier, and the drug carrier of passing is nanoparticle or microballoon.
8. a kind of neoplasm targeted therapy reagent according to claim 5, which is characterized in that the targeting dystopy ATP5B is logical
For the polypeptide expression on road on recombinant toxin molecule, the recombinant toxin molecule is corresponding by the polypeptide of targeting dystopy ATP5B accesses
The DNA sequence dna of DNA sequence dna and protein toxin molecule is formed by the recombinant gene expression after genetic recombination.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006117250A2 (en) * | 2005-05-03 | 2006-11-09 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Importation of mitochondrial protein by an enhanced allotopic approach |
| CN105237630A (en) * | 2015-11-02 | 2016-01-13 | 青岛农业大学 | Pesticin and phage lysozyme fusion protein and encoding gene and application thereof |
| CN105859866A (en) * | 2016-05-27 | 2016-08-17 | 郑州大学 | FAP source anti-tumor CTL epitope peptide P265 and application thereof |
| CN107056888A (en) * | 2017-03-01 | 2017-08-18 | 中山大学肿瘤防治中心 | ATP1A1 target polypeptides and application |
-
2018
- 2018-03-29 CN CN201810270410.6A patent/CN108640970B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006117250A2 (en) * | 2005-05-03 | 2006-11-09 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Importation of mitochondrial protein by an enhanced allotopic approach |
| CN105237630A (en) * | 2015-11-02 | 2016-01-13 | 青岛农业大学 | Pesticin and phage lysozyme fusion protein and encoding gene and application thereof |
| CN105859866A (en) * | 2016-05-27 | 2016-08-17 | 郑州大学 | FAP source anti-tumor CTL epitope peptide P265 and application thereof |
| CN107056888A (en) * | 2017-03-01 | 2017-08-18 | 中山大学肿瘤防治中心 | ATP1A1 target polypeptides and application |
Non-Patent Citations (1)
| Title |
|---|
| T WANG等: "《Ectopic ATP synthase β subunit proteins on human leukemia cell surface interact with platelets by binding glycoprotein IIb》", 《HAEMATOLOGICA》 * |
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