CN105085632A - Specific EGFR (epidermal growth factor receptor)-protein-targeted polypeptide and application thereof - Google Patents
Specific EGFR (epidermal growth factor receptor)-protein-targeted polypeptide and application thereof Download PDFInfo
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Abstract
The invention relates to a specific EGFR (epidermal growth factor receptor)-protein-targeted polypeptide and application thereof. The polypeptide is disclosed as the following general formula: X1X2X3X4X5LX6X7X8NPX9X10X11X12X13. The invention also relates to a nucleotide sequence for coding the polypeptide, an expression vector for expressing the polypeptide and a host cell. The invention also relates to the polypeptide, a bivalent or polyvalent formed by the polypeptide, a pharmaceutical composition formed by the polypeptide used as the targeted polypeptide and a preparation or developing preparation capable of killing cancer cells, and application of the polypeptide. The polypeptide can perform targeted functions on EGFR positive tumor cells, and has high selectivity. The peptide provided by the invention can be prepared by a chemical synthesis process, and has the advantages of high purity, low molecular weight, high specificity, no immunogenicity and high safety and reliability.
Description
Technical field
The present invention relates to medicinal chemistry art, be specifically related to a peptide species, particularly relate to a kind of polypeptide and application thereof of special target EGFR albumen.
Background technology
Cancer is the major causes of death in the whole world, data presentation: the newly-increased about 1,410 ten thousand routine cases of cancers in the whole world in 2012, number of cancer deaths reaches 8,200,000, and by comparison, the data of 2008 are respectively 1,270 ten thousand and 7,600,000.The most common cancer of diagnosis is followed successively by lung cancer (1,800,000 in world wide, 13%), mammary cancer (1,700,000,11.9%) and colorectal cancer (1,400,000,9.7%), main lethal cancer is lung cancer (1,600,000,19.4%), liver cancer (800,000,9.1%) and cancer of the stomach (700,000,8.8%).International cancer research institution estimates according to available data, and because population in the world increases and aging, before 2025, the annual newly-increased cancer number of cases in the whole world will up to 1,930 ten thousand examples.2012, cancer new cases over half and the number of cancer deaths of whole world sum occurred in low developed area, are respectively 56.8% and 64.9%.
The methods for the treatment of of current malignant tumour mainly contains operative treatment, chemotherapy, radiotherapy three kinds, and wherein chemotherapy is method with fastest developing speed in oncotherapy in recent years.But chemotherapeutics is while killing tumor cell, also killed and wounded human normal cell, toxic side effect is large.Therefore, anti-tumor drugs targeting and molecular probe research imperative.
EGF-R ELISA EGFR (EpidermalGrowthFactorReceptor) is the acceptor of epidermal growth factor (EGF) cell proliferation and intracellular signaling.EGFR belongs to the one of ErbB receptor family, and this family comprises EGFR (ErbB-1), HER2/c-neu (ErbB-2), HER3 (ErbB-3) and HER4 (ErbB-4).EGFR is also referred to as HER1, ErbB1, and sudden change or process LAN generally can cause tumour.EGFR belongs to Tyrosylprotein kinase receptor, molecular weight 170KDa.EGFR is positioned at surface of cell membrane, by activating with ligand binding.Research shows in many noumenal tumours, there is EGFR high expression level or unconventionality expression.The propagation of EGFR and tumour cell, vasculogenesis, tumor invasion, transfer and apoptotic suppression are relevant.The process LAN of EGFR plays an important role in the evolution of malignant tumour, has the process LAN of EGFR in the tissue such as spongiocyte, kidney, lung cancer, prostate cancer, carcinoma of the pancreas, mammary cancer.
Polypeptide is easy to a large amount of synthesis, and molecular weight is little, and tissue permeability is strong, can prevent the non-specific uptake of reticuloendothelial system simultaneously.In addition, polypeptide can be chemically modified to change its avidity, electric charge, hydrophobicity, stability and solvability, uses in vivo by optimization amendment repeatedly.In cancer target therapy, polypeptide drug and diagnostic probe demonstrate very strong superiority, are the attractive surrogates of one of antibody.In application aspect, polypeptide coordinates with radionuclide, can be used as probe application in molecular imaging; Some naturally occurring polypeptide are used as agent delivery; Peptide and their derivative of reproductive hormone can be used for neoplasm targeted therapy.Therefore high-level efficiency finds that valuable active polypeptide has become cancer diagnosis and treated the vital task faced.
CN102993272A discloses a kind of polypeptide and application thereof of targeting EGFR acceptor, and this polypeptide comes from the little peptide EY of EGFR main autophosphorylation action site
1068iNQ (SEQIDNO:1), DY
1148qQD (SEQIDNO:2), AEY
1173lR (SEQIDNO:3), this polypeptide is used for decorated nanometer lipid carrier.But the avidity of itself and EGFR is high not enough, acts on also more weak.
Summary of the invention
The object of the present invention is to provide a peptide species, the polypeptide of special target EGFR albumen and an application thereof, particularly by this peptide derive and the purposes in targeting EGFR antitumor drug or video picture preparation can prepared with the protein bound product of EGFR and aforementioned polypeptides or its derived product.
For reaching this goal of the invention, the present invention by the following technical solutions:
First aspect, the invention provides a kind of peptide of targeting EGFR albumen, and its aminoacid sequence general formula is: X
1x
2x
3x
4x
5lX
6x
7x
8nPX
9x
10x
11x
12x
13
In above-mentioned peptide chain, the amino-acid residue of one-letter symbol representative is defined as follows: L is leucine, and N is l-asparagine, and P is proline(Pro), X
1-13for variable amino-acid residue.
As optimal technical scheme, X
1be charge residue residue, be preferably Methionin or aspartic acid; X
2be polar amino acid residues, be preferably Methionin, Threonine or tyrosine; X
3be aromatic series or aliphatic amino acid residue, be preferably phenylalanine or Isoleucine; X
4be aromaticity or heterocyclic amino acid residue, be preferably tyrosine or proline(Pro); X
5be aromaticity or heterocyclic amino acid residue, be preferably tyrosine or proline(Pro); X
6be aliphatic amino acid residue, be preferably glycine or L-Ala; X
7be aliphatics or aromatic amino acid residue, be preferably leucine, tyrosine or tryptophane; X
8be aromatic amino acid residue, be preferably tyrosine or tryptophane; X
9be polar amino acid residues, be preferably Threonine or l-asparagine; X
10be polar amino acid residues, be preferably Threonine or L-glutamic acid; X
11be aromatic amino acid residue, be preferably tyrosine or tryptophane; X
12be aromaticity or polar amino acid residues, be preferably phenylalanine, arginine or glutamine; X
13be aromaticity or polar amino acid residues, be preferably tyrosine or glutamine.
In the present invention, described polypeptide source is the polypeptide WP1 (its sequence is: DTCPPLMLYNPTTYQM) based on EGFR and the storage capacity set up is 2 × 10
5polypeptide libraries:
Polypeptide WP1 (sequence: DTCPPLMLYNPTTYQM) is positioned at the extracellular region II structural domain of EGFR, is to form EGFR-EGFR dimer, and the critical sites of EGFR-HER2 heterodimer.Described polypeptide energy specific combination Human epidermal growth factor receptor high expression level positive breast cancer cells, combination rate has remarkable lifting.
As optimal technical scheme, the aminoacid sequence of peptide of the present invention is selected from the aminoacid sequence shown in one of SEQIDNO.1-SEQIDNO.14.
Aminoacid sequence:
| SEQ ID NO.1 | KYFPPLALYNPTEYFY |
| SEQ ID NO.2 | DYFYPLAYYNPTTWQY |
| SEQ ID NO.3 | KTIPYLGLYNPNTWRY |
| SEQ ID NO.4 | KKFYPLGWWNPNTWRY |
| SEQ ID NO.5 | DTFYYLAYWNPNTWRY |
| SEQ ID NO.6 | DYIPYLALWNPNTWFY |
| SEQ ID NO.7 | DKIPYLAWWNPNTWQQ |
| SEQ ID NO.8 | DYIPYLGYYNPTTWQY |
| SEQ ID NO.9 | KTFPPLAYYNPTTWRY |
| SEQ ID NO.10 | DTFPYLGYWNPNTYRY |
| SEQ ID NO.11 | KYIPPLALWNPTEWRQ |
| SEQ ID NO.12 | KKIYPLGYYNPNTYQQ |
| SEQ ID NO.13 | DTIPYLAYWNPNTWRY |
| SEQ ID NO.14 | DYFPPLAWWNPNEYFQ |
Second aspect, present invention also offers a kind of DNA fragmentation, and it comprises the aminoacid sequence of the peptide of coding the invention described above general formula.
As optimal technical scheme, described DNA fragmentation comprises the aminoacid sequence of the peptide of the described the present invention of coding as Suo Shi one of SEQIDNO.1-SEQIDNO.14.
The third aspect, present invention also offers a kind of expression vector, and the encoding amino acid sequence comprising at least one copy is the DNA fragmentation described in the second aspect present invention of peptide shown in general formula.
As optimal technical scheme, expression vector of the present invention, the encoding amino acid sequence comprising at least one copy is the DNA fragmentation described in the second aspect present invention of peptide shown in SEQIDNO.1-SEQIDNO.14.
Fourth aspect, present invention also offers a kind of protokaryon or eukaryotic host cell, and this host cell is containing, for example the expression vector described in third aspect present invention.
5th aspect, present invention also offers a kind of bivalent or multivalent, is assembled by peptide described in peptide described in the general formula 1 of first aspect present invention and SEQIDNO.1-SEQIDNO.14.
Bivalent as above or multivalent have the characteristic of the tumour cell of the targeting EGFR positive and have lethality to tumour cell.
As optimal technical scheme, bivalent of the present invention or multivalent be by connect molecule and/or the covalently bound formation of polymkeric substance or by with is connected molecule and/or mixed with polymers, non-covalent linking formation.
Preferably, described connection molecule and/or polymkeric substance are the combination of any one or at least two kinds in polyoxyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine dendrimer (PAMAM), poly(lactic acid) (PLA) or poly(lactic acid)-thanomin (PLGA).
6th aspect, the present invention still further provides a kind of pharmaceutical composition, the aminoacid sequence comprising first aspect present invention is peptide described in general formula, peptide as described in one of SEQIDNO.1-SEQIDNO.14 or as described in the bivalent of peptide or multivalent as target polypeptide, and the preparation of cancer cells can be killed and wounded.
As optimal technical scheme, peptide of the present invention, bivalent or multivalent, as target polypeptide, are puted together mutually with the preparation that can kill and wound cancer cells or mix.
Preferably, described preparation is any one that can kill and wound the chemicals of cancer cells, bio-pharmaceutical, Nano medication, radiopharmaceuticals, photo-thermal therapy or optical dynamic therapy medicine or wrap up in the carrier of these medicines;
Further preferably, described preparation is any one in alkylating agent, antimetabolite, antitumor natural drug, antitumor antibiotics, hormone and metal complex or tumour radiotherapy target marker.
Further preferably, described carrier is nano material, any one in liposome or oiliness compound, or the mixture be made up of multiple oiliness compound.
The present invention adopt by general formula as described in one of SEQIDNO.1-SEQIDNO.14 peptide and as described in the bivalent of peptide or multivalent and the macromolecular material such as nano material, liposome put together, the peptide that the present invention relates to, bivalent or multivalent can make the compound puting together rear generation more stably be transported to target cell in body.The peptide that the present invention relates to, bivalent or multivalent also can mix with the mixture of oiliness compound or multiple oiliness compound mutually, and the peptide that the present invention relates to also can make obtained mixture more stably be transported to target cell in body.
7th aspect, the present invention still further provides another pharmaceutical composition, the bivalent that the aminoacid sequence that described pharmaceutical composition comprises first aspect present invention is the peptide described in general formula described in peptide, SEQIDNO.1-SEQIDNO.14 or described peptide or multivalent; With video picture preparation.
Preferably, described peptide, bivalent or multivalent are puted together mutually with video picture preparation or are mixed.
Preferably, described video picture preparation is any one in radionuclide, radioisotope labeling thing or molecular image preparation.
Eighth aspect, the bivalent that the aminoacid sequence that present invention also offers first aspect present invention is the peptide described in general formula described in peptide, SEQIDNO.1-SEQIDNO.14 or described peptide or multivalent are for the preparation of the purposes in the medicine for the treatment of, prevention or diagnosing cancer or video picture preparation.
As optimal technical scheme, cancer of the present invention is the cancer of EGFR process LAN.
Preferably, described cancer is lung cancer, mammary cancer, cancer of the stomach, liver cancer, colon and rectum carcinoma, the esophageal carcinoma, leukemia, bladder cancer or cervical cancer.
Peptide of the present invention has the effect of targeting EGFR albumen, can as target head increase medicine or be loaded with medicine carrier as the content in EGFR positive cell such as nano material, liposome, then add pharmaceutically acceptable auxiliary material or novel more effective targeted anticancer medicine made by adjuvant.
Compared with prior art, the present invention has following beneficial effect:
(1) polypeptide of the present invention can play targeting to EGFR positive cell, and selectivity is strong, and polypeptide of the present invention can adopt the method for chemosynthesis to prepare, and purity is high, and molecular weight is little, high specificity, and non-immunogenicity is safe and reliable;
(2) polypeptide of the present invention can in conjunction with Human epidermal growth factor receptor positive breast cancer cells, combination rate polypeptide all more of the prior art has remarkable lifting, very micro-with the keying action of EGFR low expression human embryo kidney (HEK) normal cell 293A cell, show the polypeptide of the present invention as special target EGFR, to the selective keying action of EGFR;
(3) polypeptide of the present invention has the characteristic of targeting EGFR positive tumor cell, also has characteristic tumour cell to lethality, as target polypeptide, can be used for the targeted therapy of tumour or the molecular imaging of target tumor and diagnosis.
Accompanying drawing explanation
Fig. 1 (A) is for flow cyctometry method detection control group 0.01mMPBS is to Human epidermal growth factor receptor positive breast cancer cells (468 clone) actuating signal; Fig. 1 (B) detects fluorescein isothiocyanate (FITC) SEQIDNO.1 that marks to the keying action of Human epidermal growth factor receptor positive breast cancer cells (468 clone) for flow cyctometry method; Fig. 1 (C) detects fluorescein isothiocyanate (FITC) SEQIDNO.2 that marks to the keying action of Human epidermal growth factor receptor positive breast cancer cells (468 clone) for flow cyctometry method; Fig. 1 (D) detects fluorescein isothiocyanate (FITC) SEQIDNO.3 that marks to the keying action of Human epidermal growth factor receptor positive breast cancer cells (468 clone) for flow cyctometry method; Fig. 1 (E) detects fluorescein isothiocyanate (FITC) SEQIDNO.4 that marks to the keying action of Human epidermal growth factor receptor positive breast cancer cells (468 clone) for flow cyctometry method; Fig. 1 (F) is combination rate histogram.
Fig. 2 (A) is for flow cyctometry method detection control group 0.01mMPBS is to negative embryonic kidney normal cell (293A clone) actuating signal of Human epidermal growth factor receptor; Fig. 2 (B) is for the SEQIDNO.1 of flow cyctometry method detection FITC mark is to the keying action of negative embryonic kidney normal cell (293A clone) of Human epidermal growth factor receptor; Fig. 2 (C) is for the SEQIDNO.2 of flow cyctometry method detection FITC mark is to the keying action of negative embryonic kidney normal cell (293A clone) of Human epidermal growth factor receptor; What Fig. 2 (D) flow cyctometry method detected FITC mark is the keying action of SEQIDNO.3 to negative embryonic kidney normal cell (293A clone) of Human epidermal growth factor receptor; Fig. 2 (E) is for the SEQIDNO.4 of flow cyctometry method detection FITC mark is to the keying action of negative embryonic kidney normal cell (293A clone) of Human epidermal growth factor receptor; Fig. 2 (F) is combination rate histogram.
Fig. 3 is that immunofluorescence method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 of FITC mark respectively to the keying action of Human epidermal growth factor receptor positive breast cancer cells (468 clone);
Wherein, nucleus hoechst33342 reagent dyeing; A-C is the cell of SEQIDNO.1 polypeptide and hoechst33342 process, and wherein A is the binding site of SEQIDNO.1 polypeptide, is mainly present in cytolemma, and B is nucleus, and C is the superimposed figure of A, B; D-F is the cell of SEQIDNO.2 polypeptide and hoechst33342 process, and wherein D is the binding site of SEQIDNO.2 polypeptide, is mainly present in cytolemma, and E is nucleus, and F is the superimposed figure of D, E; G-I is the cell of SEQIDNO.3 polypeptide and hoechst33342 process, and wherein G is the binding site of SEQIDNO.3 polypeptide, is mainly present in cytolemma, and H is nucleus, and I is the superimposed figure of G, H; J-L is the cell of SEQIDNO.4 polypeptide and hoechst33342 process, and wherein J is the binding site of SEQIDNO.4 polypeptide, is mainly present in cytolemma, and K is nucleus, and L is the superimposed figure of J, K.
Fig. 4 is that immunofluorescence method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 of FITC mark respectively to the keying action of negative embryonic kidney normal cell (293A clone) of Human epidermal growth factor receptor;
Wherein, nucleus hoechst33342 reagent dyeing; A-C is the cell of SEQIDNO.1 polypeptide and hoechst33342 process, and wherein A is the binding site of SEQIDNO.1 polypeptide, and without binding signal, B is nucleus, and C is the superimposed figure of A, B; D-F is the cell of SEQIDNO.2 polypeptide and hoechst33342 process, and wherein D is the binding site of SEQIDNO.2 polypeptide, and without binding signal, E is nucleus, and F is the superimposed figure of D, E; G-I is the cell of SEQIDNO.3 polypeptide and hoechst33342 process, and wherein G is the binding site of SEQIDNO.3 polypeptide, and without binding signal, H is nucleus, and I is the superimposed figure of G, H; J-L is the cell of SEQIDNO.4 polypeptide and hoechst33342 process, and wherein J is the binding site of SEQIDNO.4 polypeptide, and without binding signal, K is nucleus, and L is the superimposed figure of J, K.
Fig. 5 (A) is for surface plasma resonance (SPR) method detection SEQIDNO.1 is to the keying action of the Human epidermal growth factor receptor albumen of different concns; Fig. 5 (B) is for surface plasma resonance (SPR) method detection SEQIDNO.2 is to the keying action of the Human epidermal growth factor receptor albumen of different concns; Fig. 5 (C) is for surface plasma resonance (SPR) method detection SEQIDNO.3 is to the keying action of the Human epidermal growth factor receptor albumen of different concns; Fig. 5 (D) is for surface plasma resonance (SPR) method detection SEQIDNO.4 is to the keying action of the Human epidermal growth factor receptor albumen of different concns.
Fig. 6 (A) is for surface plasma resonance (SPR) method detection SEQIDNO.1-SEQIDNO.4 and WP1 peptide is to the keying action of the Human epidermal growth factor receptor albumen of same concentration (10 μ g/mL); Fig. 6 (B) is for surface plasma resonance (SPR) method detection SEQIDNO.1-SEQIDNO.4 and WP1 peptide is to the keying action of human epidermal growth factor receptor 2 (HER2) albumen of same concentration (10 μ g/mL); Fig. 6 (C) is for surface plasma resonance (SPR) method detection SEQIDNO.1-SEQIDNO.4 and WP1 peptide is to the keying action of the human serum albumin (HSA) of same concentration (10 μ g/mL).
Embodiment
Further describe the present invention below in conjunction with specific embodiment, it will be understood by those skilled in the art that described embodiment is only that intended as illustrative illustrates the present invention, instead of intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying claim.
The synthesis of embodiment 1 polypeptide of the present invention
1) laboratory apparatus and material
Dimethyl formamide (DMF), piperidines, resin, methylene dichloride (DCM), ninhydrin reaction reagent (triketohydrindene hydrate, Vc, phenol), tetramethyl-urea hexafluorophosphate (HBTU), hexahydropyridine (piperidines), tri isopropyl silane (TIS), dithioglycol (EDT), anhydrous diethyl ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methyl alcohol, each seed amino acid, Solid-phase synthesis peptides pipe.
2) solution preparation
Deprotection liquid---hexahydropyridine: DMF=1:4
Reaction solution---NMM:DMF=1:24
Lysate---TFA (92.5%), TIS (2.5%), EDT (2.5%), H
2o (2.5%)
Triketohydrindene hydrate test fluid---triketohydrindene hydrate: Vc: phenol=1:1:1
3) experimental procedure
Weigh resin and also put into Solid-phase synthesis peptides pipe (hereinafter referred to as reactor), add more than appropriate DMF swelling half an hour.Take out DMF, carry out Fmoc protective reaction with deprotection liquid, be placed in shaking table upper 10 minute.Take out protection liquid, wash 3 times with DMF, DCM, the resin that takes a morsel from reactor (about 5 ~ 10mg) is in test tube, and by washing with alcohol 2 times, ninhydrin method detects and records color, prepares to feed intake, and enters amino acid condensation and reacts.Get corresponding amino acid, HBTU (amino acid: HBTU=1:1) according to the aminoacid sequence order of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide respectively, dissolve with reaction solution, put in reactor, stirring reaction.After 1-2 hour, take a morsel resin from reactor in test tube, by washing with alcohol 2 times, ninhydrin method detects.Take out the liquid in reactor, respectively wash 2 times with DMF, DCM, obtain the peptide resin after first amino acid condensation.Above " Fmoc go protection---amino acid condensation " reactions steps is repeated to gained peptide resin, complete to the reaction of last amino acid, obtain the peptide that sequence number is SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4.After completion of the reaction, DMF, DCM each washing resin 2-3 time, methanol wash column twice, continues to drain 15-20 minute.The partial peptide resin synthesized is taken out, at room temperature in lysate (the first ice bath of lysate 20 minutes) middle cracking two hours in reactor.After resin filter, steaming instrument evaporate to dryness in revolving, washing 3 times with anhydrous diethyl ether (ice bath).Thick peptide uses preparative reverse HPLC-purified, uses HPLC to detect purity >90%.The pure peptide obtained uses mass spectrum (MS, electrospray) qualification.
To last peptide symthesis, take out part peptide resin and add fluorescein isothiocyanate (FITC) fluorescent mark.First be linked on polypeptide by amino acid couplings method by Fmoc-e-Acp-OH, then get appropriate HBTU and FITC and be dissolved in fluorescence coupling solvent, after 3-5 hours, triketohydrindene hydrate surveys solution testing.Marking successfully adopts above-mentioned same method to carry out cracking, purifying and qualification.
The relevant physicochemical character of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3 and SEQIDNO.4 peptide as shown in Table 1 and Table 2.
Table 1
Table 2
Experimental example 1 flow cyctometry method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 respectively to the keying action of Human epidermal growth factor receptor positive breast cancer cells
One, experimental technique
Collector mammary cancer EGFR overexpression cell line 468 cell, be suspended in the RPMI1640 nutrient solution containing 10% heat-inactivated fetal bovine serum, cell density is at 1x10
6about/mL, is sub-packed in the EP pipe of four 1.5mL, and 200 μ L/ manage.Add SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 that fluorescein isothiocyanate (FITC) marks respectively, ultimate density is 50 μMs/L, and the 0.01mMPBS (phosphoric acid buffer pH7.4) of control group with polypeptide equivalent replaces polypeptide.After lucifuge ice bath hatches 30 minutes, 1000g centrifugal 4 minutes collecting cells, add 1mLPBS cleaning, after repeated washing 3 times, add 500 μ LPBS, mixing, use flow cyctometry method fluorescence intensity and combining ratio.
Two, experimental result
As seen from Figure 1, SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide can in conjunction with Human epidermal growth factor receptor high expression level breast cancer cell, combination rate is 97.3%, 96.1%, 93.2%, 81.9% respectively, affinity interaction is remarkable, illustrate that peptide of the present invention is used alone and has affinity interaction to EGFR positive tumor cell, can use as the polypeptide of targeting EGFR.
Experimental example 2 flow cyctometry method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 respectively to the Normocellular keying action of the negative embryonic kidney of Human epidermal growth factor receptor
One, experimental technique
Collector's embryonic kidney EGFR down-regulated express cell strain 293A cell, be suspended in the H-DMEM nutrient solution containing 10% heat-inactivated fetal bovine serum, cell density is at 1x10
6about/mL, is sub-packed in four 1.5mLEP pipes.200 μ L/ manage.Add SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 that fluorescein isothiocyanate (FITC) marks respectively, ultimate density is 50 μMs of ol/L, and the 0.01mMPBS (phosphoric acid buffer pH7.4) of control group with polypeptide equivalent replaces polypeptide.After lucifuge ice bath hatches 30 minutes, 1000g centrifugal 4 minutes collecting cells, add 1mLPBS cleaning, after repeated washing 3 times, add 500 μ LPBS, mixing, use flow cyctometry method fluorescence intensity and combining ratio.
Two, experimental result
As seen from Figure 2, the keying action of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide and EGFR low expression human embryo kidney (HEK) normal cell 293A cell is very micro-, combination rate is all below 3%, illustrate that peptide of the present invention is to the selective keying action of HER2, can use as the polypeptide of special target EGFR.
Experimental example 3 immunofluorescence method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 respectively to the keying action of Human epidermal growth factor receptor positive breast cancer cells
One, experimental technique
Human breast carcinoma EGFR overexpression cell line 468 is suspended in the RPMI1640 nutrient solution containing 10% heat-inactivated fetal bovine serum, is inoculated in three confocol capsules with the density of 3000-5000/ware.Cultivate after 12 hours, substratum in exhaustion capsule, then the substratum 200 μ L of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 polypeptide of the 50 μMs of ol/L marked containing fluorescein isothiocyanate (FITC) is added respectively, control group is with containing the substratum with the PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent, nucleus hoechst33342 reagent dyeing, uses and dilutes according to 1:500.Lucifuge ice bath hatches 30 minutes.PBS cleans, and after repeating to wash 3 times, adds 200 μ LPBS, uses laser confocal microscope observation fluorescent signal.
Two, experimental result
As seen from Figure 3, SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide can act on the surface of Human epidermal growth factor receptor positive breast cancer cells, illustrate that peptide of the present invention is used alone and has keying action to EGFR positive tumor cell, can use as the polypeptide of targeting EGFR.
Experimental example 4 immunofluorescence method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 respectively to the Normocellular keying action of the negative embryonic kidney of people HER2
One, experimental technique
HER2 negative human embryo kidney (HEK) normal cell strain 293A is suspended in the H-DMEM nutrient solution containing 10% heat-inactivated fetal bovine serum, is inoculated in three confocol capsules with the density of 3000-5000/ware.Cultivate after 12 hours, substratum in exhaustion capsule, then the substratum 200 μ L of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 polypeptide of the 50 μMs of ol/L marked containing fluorescein isothiocyanate (FITC) is added respectively, control group is with containing the substratum with the PBS (phosphoric acid buffer pH7.4) of polypeptide equivalent, nucleus hoechst33342 reagent dyeing, uses and dilutes according to 1:500.Lucifuge ice bath hatches 30 minutes.PBS cleans, and after repeating to wash 3 times, adds 200 μ LPBS, uses laser confocal microscope observation fluorescent signal.
Two, experimental result
As seen from Figure 4, SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide is not combined with the negative embryonic kidney normal cell of Human epidermal growth factor receptor, illustrate that peptide of the present invention is selectivity specific binding to EGFR positive tumor cell, can use as the polypeptide of special target EGFR.
Experimental example 5 surface plasma resonance (SPR) method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 respectively to the avidity of Human epidermal growth factor receptor albumen
One, experimental technique
By SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide of 1mg/mL and 0.01mMPBS point on chip, overnight incubation under 4 DEG C of wet condition, then ten minutes are cleaned with 0.1mMPBS, 0.01mMPBS cleans ten minutes, finally use washed with de-ionized water ten minutes, and repeat once, immerse in the 0.01mMPBS containing 5% milk, overnight incubation under 4 DEG C of conditions, then clean ten minutes with 0.1mMPBS, 0.01mMPBS cleans ten minutes, finally use washed with de-ionized water ten minutes, and repeat once, to dry up with nitrogen, upper machine.Moving phase passes through the Human epidermal growth factor receptor albumen of 0.625 μ g/mL, 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL successively, record analysis spr signal.
Two, experimental result
As seen from Figure 5, the spr signal of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide strengthens gradually along with the increase of Human epidermal growth factor receptor protein concentration, calculates the dissociation constant K of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3 and SEQIDNO.4 polypeptide to EGFR albumen according to SPR result
dvalue is respectively: 9.41 × 10
-9m/L, 2.31 × 10
-8m/L, 2.57 × 10
-8m/L, 3.18 × 10
-8m/L, illustrates that polypeptide of the present invention has strong keying action to EGFR, can use as the polypeptide of targeting EGFR.
Experimental example 6 surface plasma resonance (SPR) method detects SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 respectively to the avidity of same concentration Human epidermal growth factor receptor, HER2, HSA albumen
One, experimental technique
By SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide of 1mg/mL and 0.01mMPBS point on chip, overnight incubation under 4 DEG C of wet condition, then ten minutes are cleaned with 0.1mMPBS, 0.01mMPBS cleans ten minutes, finally use washed with de-ionized water ten minutes, and repeat once, immerse in the 0.01mMPBS containing 5% milk, overnight incubation under 4 DEG C of conditions, then clean ten minutes with 0.1mMPBS, 0.01mMPBS cleans ten minutes, finally use washed with de-ionized water ten minutes, and repeat once, to dry up with nitrogen, upper machine.Moving phase passes through Human epidermal growth factor receptor, HER2, HSA tri-kinds of albumen of 10 μ g/mL successively, record analysis spr signal.
Two, experimental result
As seen from Figure 6, SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptide has different spr signals to the Human epidermal growth factor receptor of same concentrations, HER2, HSA tri-kinds of albumen, find out that SEQIDNO.1, SEQIDNO.2, SEQIDNO.3 and SEQIDNO.4 polypeptide has significant binding specificity to EGFR according to SPR result, and SEQIDNO.1 binding signal is the strongest, SEQIDNO.2-SEQIDNO.4 binding signal is close, and result is consistent with Fig. 5 result.SEQIDNO.1-SEQIDNO.4 is faint to HER2 and HSA protein binding signal, illustrates that peptide of the present invention has strong specific binding effect to EGFR, can use as the polypeptide of targeting EGFR.
The result of experimental example 1-6 shows, on a cellular level, it is specially to be combined with the breast cancer cell of the EGFR positive that flow cyctometry and immunofluorescence technique two kinds of experiment tests all show SEQIDNO.1, SEQIDNO.2, SEQIDNO.3 and SEQIDNO.4 polypeptide, and be incorporated into the surface of cell membrane of EGFR protein expression, and be not obviously combined with the embryonic kidney normal cell of EGFR feminine gender; On a molecular scale, surface plasma resonance (SPR) experiment confirms that SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 polypeptide all has strong keying action with EGFR albumen too, and along with the raising of EGFR protein concentration, binding signal is stronger.The Human epidermal growth factor receptor of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 polypeptide and same concentrations, HER2, HSA tri-kinds of albumen have different spr signals in addition, show that SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 polypeptide has significant binding specificity to EGFR.
Can draw from experimental example 1-6, peptide of the present invention has the characteristic of targeting EGFR positive tumor cell, thus in actual applications, can using peptide of the present invention as target polypeptide, put together mutually with the preparation that can kill and wound cancer cells or mix, for the targeted therapy of tumour, or can put together mutually with photographic developer or mix, for molecular imaging and the diagnosis of target tumor.
Applicant states, the present invention illustrates processing method of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned processing step, does not namely mean that the present invention must rely on above-mentioned processing step and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of ancillary component, the concrete way choice etc. of raw material selected by the present invention, all drops within protection scope of the present invention and open scope.
Claims (10)
1. a peptide for special target EGFR albumen, is characterized in that, the general formula of described polypeptide is:
X
1X
2X
3X
4X
5LX
6X
7X
8NPX
9X
10X
11X
12X
13
Wherein, L is leucine, and N is l-asparagine, and P is proline(Pro), X
1-13for variable amino-acid residue;
Preferably, X
1be charge residue residue, be preferably Methionin or aspartic acid; X
2be polar amino acid residues, be preferably Methionin, Threonine or tyrosine; X
3be aromatic series or aliphatic amino acid residue, be preferably phenylalanine or Isoleucine; X
4be aromaticity or heterocyclic amino acid residue, be preferably tyrosine or proline(Pro); X
5be aromaticity or heterocyclic amino acid residue, be preferably tyrosine or proline(Pro); X
6be aliphatic amino acid residue, be preferably glycine or L-Ala; X
7be aliphatics or aromatic amino acid residue, be preferably leucine, tyrosine or tryptophane; X
8be aromatic amino acid residue, be preferably tyrosine or tryptophane; X
9be polar amino acid residues, be preferably Threonine or l-asparagine; X
10be polar amino acid residues, be preferably Threonine or L-glutamic acid; X
11be aromatic amino acid residue, be preferably tyrosine or tryptophane; X
12be aromaticity or polar amino acid residues, be preferably phenylalanine, arginine or glutamine; X
13aromaticity or polar amino acid residues, be preferably tyrosine or glutamine.
2. peptide according to claim 1, is characterized in that, the aminoacid sequence of described peptide is selected from the aminoacid sequence shown in one of SEQIDNO.1-SEQIDNO.14.
3. a DNA fragmentation, is characterized in that, it comprises the nucleotide sequence of peptide described in any one of coding claim 1-2.
4. an expression vector, is characterized in that, described expression vector contains the DNA fragmentation as claimed in claim 3 of at least one copy.
5. a host cell, is characterized in that, described host cell contains expression vector according to claim 4.
6. the bivalent that the peptide according to any one of claim 1-2 is formed or multivalent, it is characterized in that, described bivalent or multivalent have the characteristic of targeting EGFR positive tumor cell and have lethality to tumour cell;
Preferably, described bivalent or multivalent are by connecting molecule and/or the covalently bound formation of polymkeric substance;
Preferably, described bivalent or multivalent be by be connected molecule and/or mixed with polymers, non-covalent linking formed;
Preferably, described connection molecule or polymkeric substance are the combination of any one or at least two kinds in polyoxyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine dendrimer (PAMAM), poly(lactic acid) (PLA) or poly(lactic acid)-thanomin (PLGA).
7. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises the peptide according to any one of claim 1-2 and can kill and wound the preparation of cancer cells;
Preferably, described pharmaceutical composition comprises bivalent according to claim 6 or multivalent and can kill and wound the preparation of cancer cells;
Preferably, described peptide, bivalent or multivalent, as target polypeptide, are puted together mutually with the preparation that can kill and wound cancer cells or mix;
Preferably, described preparation is any one that can kill and wound the chemicals of cancer cells, bio-pharmaceutical, Nano medication, radiopharmaceuticals, photo-thermal therapy or optical dynamic therapy medicine or wrap up in the carrier of these medicines;
Further preferably, described preparation is any one in alkylating agent, antimetabolite, antitumor natural drug, antitumor antibiotics, hormone, metal complex or tumour radiotherapy target marker;
Further preferably, described carrier is nano material, any one in liposome or oiliness compound, or the mixture be made up of multiple oiliness compound.
8. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises peptide described in any one of claim 1-2 and video picture preparation;
Preferably, described pharmaceutical composition comprises bivalent according to claim 6 or multivalent and video picture preparation;
Preferably, described peptide, bivalent or multivalent are puted together mutually with video picture preparation or are mixed;
Preferably, described video picture preparation is any one in radionuclide, radioisotope labeling thing or molecular image preparation.
9. any one of claim 1-2 or peptide according to claim 6, bivalent or multivalent for the preparation of the purposes in treatment, prevention or the medicine of diagnosing cancer or video picture preparation.
10. purposes according to claim 9, is characterized in that, described cancer is the cancer of EGFR process LAN;
Preferably, described cancer is any one in lung cancer, mammary cancer, cancer of the stomach, liver cancer, colon and rectum carcinoma, the esophageal carcinoma, leukemia, bladder cancer or cervical cancer or nasopharyngeal carcinoma.
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| CN106699890A (en) * | 2016-10-31 | 2017-05-24 | 上海大学 | Artificial antibody for targeting EGFR (epidermal growth factor receptor) based on gold nanoparticles and preparation method thereof |
| CN108570108A (en) * | 2017-03-17 | 2018-09-25 | 浙江日升昌药业有限公司 | Cancer target polypeptide, preparation method and applications |
| CN109180787A (en) * | 2018-08-14 | 2019-01-11 | 江苏大学 | Targeting EGFR inhibits the polypeptide of EGF rush tumor cell proliferation |
| CN111393503A (en) * | 2019-01-03 | 2020-07-10 | 北京京东方技术开发有限公司 | Quasi-peptide and preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106699890A (en) * | 2016-10-31 | 2017-05-24 | 上海大学 | Artificial antibody for targeting EGFR (epidermal growth factor receptor) based on gold nanoparticles and preparation method thereof |
| CN106699890B (en) * | 2016-10-31 | 2021-05-07 | 上海大学 | Gold nanoparticle-based artificial antibody targeting Epidermal Growth Factor Receptor (EGFR) and preparation method thereof |
| CN108570108A (en) * | 2017-03-17 | 2018-09-25 | 浙江日升昌药业有限公司 | Cancer target polypeptide, preparation method and applications |
| CN108570108B (en) * | 2017-03-17 | 2021-07-23 | 浙江孚诺医药股份有限公司 | Tumor targeting polypeptide, preparation method and application thereof |
| CN109180787A (en) * | 2018-08-14 | 2019-01-11 | 江苏大学 | Targeting EGFR inhibits the polypeptide of EGF rush tumor cell proliferation |
| CN109180787B (en) * | 2018-08-14 | 2021-08-03 | 江苏大学 | Polypeptide for targeting EGFR to inhibit EGF (epidermal growth factor receptor) and promoting tumor cell proliferation |
| CN111393503A (en) * | 2019-01-03 | 2020-07-10 | 北京京东方技术开发有限公司 | Quasi-peptide and preparation method and application thereof |
| CN111393503B (en) * | 2019-01-03 | 2022-05-13 | 北京京东方技术开发有限公司 | Quasi-peptide and preparation method and application thereof |
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| CN105085632B (en) | 2019-01-25 |
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