CN108570108A - Cancer target polypeptide, preparation method and applications - Google Patents

Cancer target polypeptide, preparation method and applications Download PDF

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Publication number
CN108570108A
CN108570108A CN201710161670.5A CN201710161670A CN108570108A CN 108570108 A CN108570108 A CN 108570108A CN 201710161670 A CN201710161670 A CN 201710161670A CN 108570108 A CN108570108 A CN 108570108A
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cancer
cell
target polypeptide
cancer target
amino acid
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CN108570108B (en
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王富军
赵健
曹雪玮
傅龙云
张涛铸
单含文
杨旭中
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ZHEJIANG REACHALL PHARMACEUTICAL CO Ltd
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ZHEJIANG REACHALL PHARMACEUTICAL CO Ltd
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Priority to CN201710161670.5A priority Critical patent/CN108570108B/en
Priority to CN202110610291.6A priority patent/CN113336830B/en
Priority to CN202110609525.5A priority patent/CN113332448B/en
Priority to CN202110609521.7A priority patent/CN113332447B/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins

Abstract

The present invention relates to biomedicine fields, more particularly to a kind of cancer target polypeptide, preparation method and applications.The invention discloses a kind of cancer target polypeptides.The cancer target polypeptide specificity binding ability of the present invention is more preferable, to mitigate influence of the tumor therapeutic agent to normal cell, reduces the generation of adverse drug reaction, improves therapeutic effect.

Description

Cancer target polypeptide, preparation method and applications
Technical field
The present invention relates to biomedicine fields, more particularly to cancer target polypeptide, preparation method and applications.
Background technology
Tumour is that the world today seriously affects one of human health and three big diseases of life.In many common tumour diseases In disease, as breast cancer, non-small cell lung cancer, colorectal cancer, carcinoma of urinary bladder, oophoroma, gastric cancer, cancer of pancreas, epidermis squamous carcinoma, kidney, It is abnormal often all to there is tumor cell surface EGF-R ELISA (EGFR) in head-neck malignant tumor, glioblastoma etc. The phenomenon that overexpression1, and the high expression of EGFR is usually extremely closely related with cell Proliferation activity.In each normal human The quantity of the EGFR on the surface of cell is usually 4 × 104To 1 × 105Left and right, and the EGFR of each tumor cell surface is then more than 2 ×106, it is 20-50 times of normal cell2.Due to EGFR mediate signal transduction in the proliferation of tumour cell, injury repair, invade It attacks and new vessels is formed etc. plays an important role, therefore EGFR just becomes in tumor diagnosis and therapy and pays special attention to naturally Critical target point, become tumour medicine exploitation and clinical treatment application an important directions3。
The member of Epidermal Growth Factor Receptor Family includes ErbB1/HER1/EGFR, ErbB2/HER2, ErbB3/HER3 and Tetra- kinds of ErbB4/HER4, belongs to tyrosine kinase receptoroid.Human epidermal growth factor receptor albumen is made of 1186 amino acid residues, relatively Molecular weight is 170,000 dalton.Entire receptor protein is made of three parts：(1) extracellular region, it is residual by 621 amino acid of aminoterminal Base is constituted, and there are ligand binding domains；(2) transmembrane region, the hydrophobic plot structure of helical form that is made of 23 amino acid residues it is buried In cell membrane lipid bilayer, receptor protein itself is anchored on cell membrane；(3) intracellular region, by 542 amino acid residue structures At 3 subprovinces can be further subdivided into：Nearly film subprovince, tyrosine kinase subprovince, c-terminus subprovince.
Have now found that there are many epidermal growth factor (EGF) class ligand molecular can and EGFR specificity combination and play life Object effect.These ligand moleculars include epidermal growth factor (EGF), transforming growth factor (TGF- α), amphiregulin (amphireguin, AR), B- cytokines (beta-celluin, BTC), Heparin-binding sample epidermal growth factor (HB-EGF), table The vaccinia growth factor (VGF) etc. of Pi Su (epiregulin, EPR) and vaccinia source.
These ligand moleculars combined with EGFR have very similar and conservative three-dimensional structure, i.e., on space structure Inside ligand molecular by the effect of polypeptide intrachain disulfide bond (Cys6-Cys20, Cys14-Cys31, Cys33-Cys42) shape At typical three cyclic structure4。
The study found that the specific recognition and cohesive process of EGF and EGFR represent such ligand molecular and receptor substantially Interaction process.Three cyclic structures of EGF it is upper and lower, in three positions and EGFR be mutually distinguishable effect, and with Specificity combine, wherein most go deep into the inside of EGFR protein moleculars with the structure of C rings, played in identifying and combining with EGFR Important function5-6。
After EGF class ligand moleculars are combined with EGFR, EGF-R ELISA can be made to form homodimer between each other Or heterodimer, lead to the activation in intracellular tyrosine kinase area, other side's tyrosine residue phosphorylation can be started a system each other Row cascade reaction, signal is passed in nucleus, is finally caused a series of related genes to activate, is led to tumor cell proliferation, withers Inhibition is died, Nasopharyngeal neoplasms are promoted and Radiotherapy chemotherapy is caused to be resistant to, is played an important role during tumor development.Equally, It is synthesized by engineer, the peptide molecule of EGF classes growth factor and its receptor specific recognition and combination is imitated, since it can Similar specific recognition and combination is carried out with EGFR, and the combination of this species specificity of these polypeptides specially designed is not Generate the subsequent signal transmission effect of EGFR molecules7, therefore, the drug targeting that can be used to be directed to tumour cell in vivo is transported It is defeated, carry out the targeted therapy of tumor disease8-9；Either this species specificity polypeptide itself with the tumor growth factor is emulative robs The receptor binding site for taking cell surface by force is in combination, to inhibit the growth of tumour cell, generates the purpose for the treatment of10-11；Or Person is through radioactive label12-13, the polypeptide of fluorochrome label14-15, after being injected into vivo, since it is capable of specificity Assemble in tumor cell surface, so as to the imaging diagnosis, label and treatment for entity tumor16-17.It is this can be in vivo The polypeptide combined outside with tumor cell specific identification is called cancer target peptide for short.
Invention content
The technical problems to be solved by the invention be provide one kind can efficiently specific recognition and have stronger binding force Polypeptide structure, existing EGF-R ELISA specificity can be largely overexpressed with tumor cell surface and is combined, thus It can be used for the markup identifying and diagnosing of tumour cell, or competitiveness is combined with EGFR, or carry tumor therapeutic agent molecule and reach To tumor cell surface, magnetic target therapy effect is played.
For this purpose, one aspect of the present invention discloses, a kind of cancer target polypeptide is shown in YXGXR it includes amino acid sequence First ray and amino acid sequence be another second sequence shown in YXGXR, the First ray and second sequence it Between pass through and connect peptide linkage, wherein Y indicates tyrosine or derivatives thereof；G indicates glycine or derivatives thereof；R indicates arginine Or derivatives thereof；X is indicated containing amino of the side chain with fats group or/and hydroxyl groups or their combinations or derivatives thereof Acid.
In some embodiments, the connection peptide includes the amino acid sequence for forming alpha-helix, the preferably described connection Peptide includes the structure of rigid alpha-helix, and the more preferably described connection peptide includes the sequence that amino acid sequence is HMAATT.
In some embodiments, the connection peptide includes 6 or more amino acid residues；Or the connection peptide includes few In 3 amino acid residues；Or the connection peptide is made of 1 amino acid residue；The connection peptide is by histidine or derivatives thereof Composition.
In some embodiments, it is SEQ ID NO.1-4 or SEQ ID that the cancer target polypeptide, which is amino acid sequence, The polypeptide or its modified derivative of one of NO.8 or SEQ ID NO.9；Preferably as amino acid sequence be SEQ ID NO.1 or The polypeptide of SEQ ID NO.8 or SEQ ID NO.9.
In some embodiments, the cancer target polypeptide can specifically bind EGFR families any member, preferably institute Stating cancer target polypeptide can be combined with EGFR, and Kd is less than 50nM.
On the other hand, the invention also discloses a kind of conjugates, and it includes cancer target polypeptides and one described in claim 1 Kind of active constituent, the two is conjugated by a kind of connector (linker), wherein the active constituent is therapeutic component, diagnosis at Divide, radioactive isotope, radionuclide, toxin or their compositions.
In some embodiments, the connector includes that covalent key connection or non-covalent bond connect, preferably described covalent Key connection includes direct covalent bonds, peptide bond, ester bond, disulfide bond, amido bond, imide bond, phosphodiester bond, urea bond, isocyanic acid Ester bond or combination thereof.
In some embodiments, the therapeutic component includes cell-penetrating peptide (CPP), the preferably described cell-penetrating peptide For polypeptide, the amino acid fragment of the trans-activator TAT from HIV, EC- shown in SEQ ID NO.12 amino acid sequences The amino acid fragment of the heparin binding domain (HBD) in the sources SOD, the amino acid fragment of the heparin binding domain (HBD) in the sources HBEGF, Or their derivative.
In some embodiments, it is the more of one of SEQ ID NO.13-15 that the conjugate, which includes amino acid sequence, Peptide.
In some embodiments, the therapeutic component include radiation treatment ingredient, chemotherapy ingredient, antibody, enzyme, Or combination thereof.
In some embodiments, the radiation treatment ingredient includes radioactive isotope, shown radioactive isotope For iodine -131, lutetium -177, Yttrium-90, samarium -153, phosphorus -32, cesium-131, palladium -103, radium -223, iodine-125, boron -10, actinium -225, Bismuth -213, radium -225, lead -212, thorium -232 or combination thereof.
In some embodiments, the chemotherapy ingredient includes capecitabine (capecitabine), cis-platinum (cisplatin), Herceptin (trastuzumab), fulvestrant (fulvestrant), tamosifen (tamoxifen), Letrozole (letrozole), Exemestane (exemestane), Anastrozole (anastrozole), ammonia Shandong Meter Te (aminoglutethimide), Testolactone (testolactone), Vorozole (vorozole), formestane (formestane), Fadrozole (fadrozole), Letrozole (letrozole), Erlotinib (erlotinib), Afatinib (lafatinib), Dasatinib (dasatinib), Gefitinib (gefitinib), Imatinib (imatinib), Pazopinib, Lapatinib (lapatinib), Sutent (sunitinib), nilotinib (nilotinib), Suo Lafei Buddhist nun (sorafenib), Abraxane (nab-palitaxel) or derivative or their compositions.
In some embodiments, the diagnosis ingredient include radiodiagnosis ingredient, fluorescent component, quantum dot or it Composition, wherein the radiodiagnosis ingredient include Value linear, technetium -99, molybdenum -99, rubidium -82, strontium -82, thallium -201, or Their compositions.
On the other hand, the invention discloses a kind of nucleic acid sequences of coding cancer target polypeptide of the present invention.
On the other hand, the invention discloses a kind of expression vectors including polynucleotide molecule of the present invention, preferably institute Stating expression vector can express in the cell.
On the other hand, the invention discloses a kind of host cell including expression vector of the present invention, the host is thin Born of the same parents are prokaryotic cell or eukaryocyte.
On the other hand, the invention discloses the preparation methods of cancer target polypeptide of the present invention, and it includes cultivate this hair The bright host cell prepares the cancer target polypeptide.
On the other hand, the invention discloses a kind of pharmaceutical compositions, it includes conjugate of the present invention and pharmaceutically may be used The carrier of receiving.
In some embodiments, described includes radiation treatment ingredient, radioactivity nucleic acid, toxin, therapeutic component, or change Learn therapeutic component or combination thereof.
On the other hand, the invention discloses a kind of pharmaceutical compositions, and it includes cancer target polypeptide of the present invention and medicines Acceptable carrier on.
On the other hand, the invention discloses the methods of bion of the treatment with tumour, including giving this bion The conjugate of the present invention of effective dose；The tumour includes to express the cell of at least one EGFR family members.
In some embodiments, the tumour is breast cancer, colorectal cancer, cancer of pancreas, head and neck cancer, melanoma, ovum Nest cancer, prostate cancer, one of non-small cell lung cancer.
In some embodiments, the method also includes while giving the curative drug of effective dose.
In some embodiments, the bion is the mankind.
On the other hand, the invention discloses a kind of solution, described molten it includes the conjugate of the present invention of effective concentration Liquid is the blood plasma of bion.
The present invention to the S3 simulating peptide amino acid sequence structures therein combined with EGFR by carrying out artificial mutation transformation Optimization, obtain the better cancer target peptide of a species specificity binding ability, with molecular imaging reagent (radioactive isotope mark Note, fluorochrome label etc.) it can be used for the diagnostic analysis of tumour after coupling；After being either coupled with tumor therapeutic agent, system At tumour cell target therapeutic agent, it is used for the magnetic target therapy of malignant tumour, to mitigate tumor therapeutic agent to normal thin The influence of born of the same parents reduces the generation of adverse drug reaction, improves therapeutic effect；Or directly utilize itself polypeptide structure energy and target position The characteristic that point (EGFR) height is affine, emulative combination simultaneously shelter the sites EGFR, inhibit to dominance the growth of tumour cell, To play the purpose of tumor disease therapeutic.
Description of the drawings
Fig. 1 .ELBD sequences wear membrane efficiency comparison schematic diagram with natural other sequences.
Fig. 2 .ELBD and mutant fusion protein wear membrane efficiency comparison schematic diagram.
Fig. 3 .EGFP-ELBD-HBD mutant recombinant proteins wear the concentration and time dependence schematic diagram of membrane efficiency.
The broad spectrum activity schematic diagram of Fig. 4 targeting peptides ELBD.
Selective schematic diagram of Fig. 5 .EGFP-ELBD-HBD mutant recombinant proteins to human body cell.
Fig. 6 .EGFP-S3-HBD recombinant proteins and EGFP-ELBD-HBD recombinant proteins are respectively to the knot of HeLa cell surfaces Conjunction ability schematic diagram.
Inhibiting effect comparison schematic diagrams of Fig. 7 recombinant proteins TCS-ELBD-CPP to growth of tumour cell.
Fig. 8 recombinant proteins MAP30-ELBD-CPP is to the relatively more signals of the inhibiting effect of normal cell and growth of tumour cell Figure.
Specific implementation mode
The present invention receives the content of Chinese invention patent CN 201310170530.6 and the bibliography being mentioned above full text Enter herein.
Bibliography：
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6.Nestor JJ Jr,Newman SR,DeLustro B,Todaro GJ,Schreiber AB.A synthetic fragment of rat transforming growth factor alpha with receptor binding and antigenic properties.Biochem Biophys Res Commun.1985；129(1):226- 32.
7.Lin YZ,Ke XH,Tam JP.Growth inhibition by vaccinia virus growth factor.J Biol Chem.1990；265(31):18884-90.
8.Ding Y,Tan W,Hu R,Chen W,Hou Y.Construction of a novel fusion protein harboring mouse interferon gamma and epidermal growth factor receptor binding domain and enhancement of its antitumor activity.Sci China C Life Sci.1997；40(3):293-300.
9.Lelle M,Kaloyanova S,Freide C,Theodoropoulou M,Musheev M,Niehrs C, Stalla G,Peneva K.Octreotide-Mediated Tumor-Targeted Drug Delivery via a Cleavable Doxorubicin-Peptide Conjugate.Mol Pharm.2015；12(12):4290-300.
10.Eppstein DA,Marsh YV,Schryver BB,Bertics PJ.Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide.J Cell Physiol.1989；141(2):420-30.
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18. a kind of tumour cell targeting cell-penetrating peptide, Chinese Patent Application No.：CN 201310170530.6
Herein, term " VGF third ring sample manual simulations peptide " (being also known as herein " S3 simulating peptides ") refers to a kind of small peptide sequence Row have the function of being combined with EGFR specificities, may be empty with or without the function of promoting cell growth and division Between structure match with EGFR film extracellular portion Domain III, can specificity therewith combination, so as to cause or do not cause EGFR's is Dimerized.A kind of structure of representative VGF thirds ring sample manual simulation's peptide is residual with 2 Cys for intramolecule Base, amino acid peptide chain folded inside circlewise structure, space conformation is similar with third ring structure, is matched with the Domain of EGFR It is necessary to play bioactivity for it for III calmodulin binding domain CaMs.The Leu of Tyr, Gly, Arg and C-terminal in this cyclic structure is It is highly conserved, these conserved amino acids it is any missing or C-terminal include Leu big section deletion mutation, can result in receptor In conjunction with active forfeiture.
" cancer target peptide " be refer to a kind of short peptide sequence, be the S3 simulating peptides amino acid sequence structure that is combined to EGFR into Row artificial mutation transformation and optimization obtains the better cancer target peptide structure of a species specificity binding ability.It is thin to can be used for tumour The markup identifying and diagnosing of born of the same parents, or competitiveness are combined with EGFR, or are carried tumor therapeutic agent molecule and reached tumor cell surface, Play magnetic target therapy effect.
Cancer target peptide can quilt after being coupled with molecular imaging reagent (labelled with radioisotope, fluorochrome label etc.) Diagnostic analysis for tumour；After being either coupled with tumor therapeutic agent, tumour cell target therapeutic agent is made, for disliking The magnetic target therapy of property tumour reduces the hair of adverse drug reaction to mitigate influence of the tumor therapeutic agent to normal cell It is raw, improve therapeutic effect；Or directly utilize itself polypeptide structure can with the affine characteristic of target site (EGFR) height, it is competitive Combination and shelter the sites EGFR, inhibit to dominance the growth of tumour cell, to play the purpose of tumor disease therapeutic.
" cell-penetrating peptide " (cell penetrating peptides, CPPs) is also known as protein transduction domain or film transduction Peptide is a kind of by 30 or the polypeptide for capableing of penetration cell film that less amino acid forms, described that there is cell-penetrating peptide to live Property CPPs include but is not limited to sequence as but be not limited to human immunodeficiency virus activating transcription factor TAT, herpe simplex Virus I-type VP22 transcription factors, Penetratin, the Transportan of the homologous Antennapedia of drosophila (Antp), from people's Cell-penetrating peptide such as ARF, BagP, CytC, hCT, hLF, hClock, TCTP, NRTN, it is artificial synthesized by large T antigen nuclear localization sequence From facultative molecule MPG, MAP, the Pep-1 of different hydrophobic peptide fragment compositions, the poly arginine sequence of different length from 4 to 15 Row, SynB1, Polyomavirus Vpl, Bac, NF-KB, SV4OT antigen, HATF3, hCT, pVEC, Integrin, DPV6, S413PV, Poly-P, one kind in heparin binding domain etc., (its amino acid sequence is wherein preferably TAT YGRKKRRQRRR) or EC-SOD carboxyl terminals heparin binding domain (its amino acid sequence is GPGLWERQAREHSERKKRRRESECKAA) or its variant, as Chinese patent CN201210587097.1, CN1049111 are wrapped The cell-penetrating peptide contained；The most preferably heparin binding domain of the HBEGF in the sources HBEGF, amino acid sequence such as SEQ ID Shown in NO.12.
" connection peptide " (linkers) is that each functional domain component is connected in protein so that forms the protein molecule Each functional domain keep its activity conformation, play its respective biological function, be each functional domain biological activity Performance collaboration is provided, is adjusted and destructurization, avoid causing because of factors such as charge, space potential barriers protein molecule biology from living The forfeiture of property.In general, connection peptide is divided into elasticity, and rigidity, three kinds of forms of cuttability.Connection peptide can have a variety of secondary structures State plays its biological function, such as α spirals, β-pleated sheet chain, curling/bending and corner.It is used in naturally occurring connection peptide Rolled form accounts for 59% (Chen etc., Adv Drug Deliv Rev.2012, doi:10.1016/ j.addr.2012.09.039).To connect peptide existing for rolled form, it is elastic with certain space operation so that connected Two structural domains can freely activity relatively.
" tumour cell targeting cell-penetrating peptide " to refer to a kind of short peptide sequence, containing has the active CPPs of cell-penetrating peptide Structural domain and above-mentioned " cancer target peptide ".
Polypeptide described herein, including but not limited to " cancer target peptide ", " cell-penetrating peptide ", " tumour cell targeting is worn Film peptide ", modified derivative can be used prior art conventional method and modify cancer target peptide of the present invention. These method of modifying include that the modification to amino terminus or carboxyl terminal, the replacement of central amino acid residue, side chain are repaiied Decorations etc..For example, thering is the acetylation of N-terminal and the amidation of C-terminal to modify the method for modifying of peptide chain end, to the ammonia to end Base or carboxyl are protected.Peptide chain end can also connect the aliphatic acid of different length.PEG molecules either carry out glycosylation and repair Decorations improve its stability to polypeptide hydrolase to increase the relative molecular weight and steric hindrance of peptide molecule, extend in body The residence time of internal circulation system.It simultaneously can also be by replacing the amino acid residues that easily digest individually, or by L-type ammonia Base acid replaces with D- types amino acid to extend or improve the half-life period of polypeptide drugs.
" conjugate " refers to a kind of short peptide sequence conjugate, and the cancer target polypeptide and active constituent, the two pass through company It is conjugated to connect device (linker), wherein the active constituent includes therapeutic component, or diagnosis ingredient or radioactive isotope, or put Penetrating property nucleic or toxin or their compositions.
The synthesis of polypeptide
Polypeptide of the present invention, including but not limited to " cancer target peptide ", " cell-penetrating peptide ", " tumour cell targeting Cell-penetrating peptide " can carry out synthesis in solid state or liquid phase synthesis according to conventional peptide synthesis technology or liquid-solid phase synthesizes.Such as use polypeptide Synthesizer synthesis in solid state " cancer target peptide " of the present invention, " tumour cell targeting cell-penetrating peptide ", and can further pass through company Device is connect, such as 3- maleimidopropionic acid N-hydroxy-succinamide esters, makes tumour cell targeting cell-penetrating peptide and anticancer drug Including but not limited to adriamycin, Docetaxel, mitomycin, daunorubicin, carboplatin, camptothecine, hydroxycamptothecin, Changchun are new Alkali, bleomycin, 5 FU 5 fluorouracil, ring phosphorus phthalein amine, gemcitabine, the cry of certain animals of first ammonia butterfly, capecitabine, lomustine, rely on pool former times, Capecitabine (capecitabine), cis-platinum (cisplatin), Herceptin (trastuzumab), fulvestrant (fulvestrant), tamosifen (tamoxifen), Letrozole (letrozole), Exemestane (exemestane), Ah that Bent azoles (anastrozole), aminoglutethimide (aminoglutethimide), Testolactone (testolactone), Vorozole (vorozole), formestane (formestane), Fadrozole (fadrozole), Letrozole (letrozole), Erlotinib (erlotinib), Afatinib (lafatinib), Dasatinib (dasatinib), Gefitinib (gefitinib), her horse For Buddhist nun (imatinib), pazopinib, Lapatinib (lapatinib), Sutent (sunitinib), nilotinib (nilotinib), Sorafenib (sorafenib), Abraxane (nab-palitaxel) or derivative or their group It closes object and forms copolymer, you can prepare targeting antitumor drug.
By taking adriamycin as an example, detailed process is：Dimethylformamide dissolves adriamycin and 3- maleimides third respectively Sour n-hydroxysuccinimide is added triethylamine and adjusts pH value, poured into 50ml ether after reaction being stirred at room temperature 2 hours, is precipitated Precipitation washed 2 times with anhydrous ether, centrifuge precipitate simultaneously be dried in vacuo；Take precipitation and tumour cell targeting of the present invention Cell-penetrating peptide is dissolved with dimethylformamide, and triethylamine is added, and is poured into 10ml ether after reaction being stirred at room temperature 2 hours, is precipitated Precipitation washed 2 times with anhydrous ether, centrifuge precipitation, obtain subject copolymers after vacuum drying.
The expression of polypeptide or fusion protein
The present invention includes encoding " cancer target peptide " of the present invention or tumour cell targeting cell-penetrating peptide or melting containing it The DNA of hop protein and the carrier containing these DNA, transformant.
In the present invention, the term " transformant " (transformant) used, the i.e. host with heterologous DNA molecule are thin Born of the same parents.
The invention also includes the cancer target peptides by synthesizing and recombinant technique production is of the invention " or tumour cell targeting The method of cell-penetrating peptide or fusion protein containing it.Can detach and purify by methods known in the art polynucleotides (DNA or RNA), carrier, transformant and organism.
Carrier for the present invention can be such as bacteriophage, plasmid, clay, minichromosome, virus or retrovirus Carrier.The carrier for the polynucleotides that can be used for cloning and/or expressing the present invention can need to replicate and/or express polynucleotides The carrier of polynucleotides is replicated and/or expressed in host cell.It is, in general, that polynucleotides and/or carrier can be used for it is any true Core or prokaryotic cell, including mammalian cell (such as people (such as HeLa), monkey (such as Cos), rabbit (such as rabbit granulophilocyte), rat, Hamster (such as CHO, NSO and baby hamster kidney cell) or mouse cell (such as L cells)), plant cell, yeast cells, insect cell Or bacterial cell (such as Escherichia coli).Example in relation to the suitable carrier suitable for multiple types host cell can be found in for example Ausube etc., Current Protocols in Molecular Biology.Greene Publishing Associates And Wiley-Interscience (1992) and Sambrook et al. (1989).It can use and contain these polynucleotides Host cell carry out the protein that great expression can be used for such as drug, diagnostic reagent, vaccine and therapeutic agent.
A variety of methods have been developed for making polynucleotides via complementary cohesive end and carrier is operable is connected.Example Such as, complementary homopolymer sequence fragment can be added in the DNA section in carrier DNA to be inserted into.Then pass through complementary homopolymeric tail Between hydrogen bond connection carrier and DNA section to form recombinant DNA molecules.
The method that synthetic linker containing one or more restriction sites provides another connection DNA section and carrier. The DNA generated by endonuclease restriction digestion with bacteriophage T4DNA polymerases or e. coli dna polymerase I processing Section, described two kinds of its 3' of polymerase, 5 '-exonucleolytic activities remove γ-single stranded end outstanding, its polymerization is used in combination to live Property filling-in 3 '-female end.Therefore, these it is active combine produce flush end DNA section.Then blunt-ended DNA molecules company can be catalyzed The enzyme connect, as kept the temperature flush end section together with the linkers of big molar excess in the presence of bacteriophage T4DNA ligases.Cause This, reaction product is the DNA section that end carries polylinker sequence.Then these region of DNA are cracked with restriction enzyme appropriate Section, and be connected in the expression vector for having used enzymatic lysis, the enzyme can generate the end compatible with the DNA section.From multiple Businessman can buy the synthetic linker containing multiple restriction endonuclease sites.
Polynucleotides insert should be operably connected to compatible with the host cell of expression polynucleotides appropriate open On mover.Promoter can be strong promoter and/or inducible promoter.The example for some promoters enumerated includes bacteriophage λ PL promoters, Escherichia coli lac, trP, phoA, tac promoter, the early and late promoters of SV40 and retrovirus LTR promoters.Other appropriate promoters are known to the skilled in the art.Expression recombinant vector, which further contains, transcribes Begin, termination site, and contains the ribosome bind site for being useful for translation in transcriptional domain.The coding of the transcript of recombinant vector expression Part may include the translation initiation codon for being located at starting point and the terminator codon for being suitably positioned at the end for being translated polypeptide (UAA, UGA or UAG).
As described above, expression vector may include at least one selected marker.The label includes to eukaryotic cell culture For dihyrofolate reductase, G418, glutamine synthase or neomycin resistance；And it is used for Escherichia coli and other bacteriums Tetracycline, kanamycins or the acillin resistant gene of culture.The representative example of appropriate host includes but not limited to：Carefully Bacterium cell, such as Escherichia coli, streptomycete and salmonella typhimurium cell；Fungal cell, as yeast cells (such as saccharomyces cerevisiae or Pichia pastoris yeast)；Insect cell, such as drosophila S2 and noctuid SF9 cells；Zooblast, such as CHO, COS, NSO, 293 He Bowes melanoma cells；And plant cell.The appropriate culture medium and condition of culture of above-mentioned host cell are known in the art.
In order to efficiently separate purifying or secretion target protein, usually also using convenient for the label protein that isolates and purifies or Tag polypeptide (Tag).There are commonly glutathione-S-transferase (glutathione S-transferase, GST), six poly groups Propylhomoserin peptide (His.Tag), a-protein (protein A) and cellulose binding site (cellulose binding domain) Deng.The form for being made up of fusion protein with target protein particularity albumen or polypeptide, utilizes the label protein after expression Or the special nature of tag polypeptide can be detached and be purified to target protein.Such as His.Tag and Ni-Chelating Sepharose columns are specifically bound.The label protein or tag polypeptide can use site-specific protease after purification Digestion removal fusion sequence, such as available fibrin ferment, enterokinase and Xa factor, to obtain target protein.
The invention also includes containing the present invention nucleotide sequence host cell, the nucleotide sequence through this field Heterologous control regions (such as promoter and/or enhancer) are operable is connected with one or more for the technology known.It can select adjust and insert The expression of the gene order entered, or the host strain with processed gene product can be modified according to required particular form.Certain In the presence of inducer, the expression that certain promoters start can increase；Therefore, the table of the polypeptide through genetic modification can be controlled It reaches.In addition, different hosts cell has characteristic and special translation, post translational processing and modification (such as phosphorylation, cracking) The mechanism of protein.Can select cell line appropriate with ensure to the exogenous proteins of expression carry out desirable modification and Processing.
The transfection of the transfection, cation lipid mediation that are mediated by calcium phosphate transfection, DEAE- glucans, turns electroporation It leads, infect or other methods, you can the nucleic acid of the present invention and nucleic acid recombinant vector are imported into host cell.The method is described in In the laboratory manual of multiple standards, such as Davis, Basic Methods In Molecular Biology (1986).
Encoding the polynucleotides of the fusion protein of the present invention can connect with the carrier containing selected marker in place It is proliferated in master.It is, in general, that can be carried in sediment as imported plasmid in calcium phosphate precipitation object or its compound with charged lipids Body.If carrier is virus, package cell line appropriate to be packed in vitro to it, then can be used transduce to host cell.
It can be identified by the cell of successful conversion by widely-known technique, that is, the DNA recombinations for containing the present invention carry The cell of body.For example, the cell obtained by importing expression recombinant vector can be cultivated to generate required polypeptide.Simultaneously lytic cell is collected, Use such as Southern, J.Mol.Biol.1975,98:503 or Berent etc., Biotech.1985,3:Method described in 208, Detect the presence of DNA in its DNA content.Alternatively, using the presence of protein in antibody test supernatant.
From recombinant cell culture thing recycle and purify by well-known method the fusion protein of the present invention compared with To be advantageous, pressed the method includes sulfuric acid or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, cellulose phosphate Chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, dewatering electric charge effect chromatography and agglutinin chromatograph.At some In embodiment, high performance liquid chroma- tography (HPLC) can be used to be purified.
In some embodiments, above-mentioned one or more chromatography methods can be used to purify the fusion egg of the present invention In vain.In other embodiments, the fusion protein of following one or more column chromatographies present invention can be used, it is described Chromatographic column has：Q Sepharose FF columns, SP Sepharose FF columns, Q Sepharose High Performance columns, Blue Sepharose FF columns, Blue columns, Phenyl Sepharose FF columns, DEAE Sepharose FF, Ni- Chelating Sepharose FF columns or Methyl columns etc..
In addition, the method purifying described in international publication number WO00/44772 (being included in full herein as reference) can be used The fusion protein of the present invention.Those skilled in the art can easily change the wherein described method for the purifying present invention's Merge body protein.Can from including such as bacterium, yeast, higher plant, insect and mammalian cell protokaryon or eukaryon place The fusion protein of the present invention is recycled in the product that main warp recombinant technique generates.
Purposes
Conjugate shown in the present invention can be used to treat the various diseases caused by cell hyperplasia as active constituent, Such as tumour, including but not limited to:Osteocarcinoma class, including:Ewing's sarcoma, osteosarcoma, chondrosarcoma etc.；Brain and cns tumor, including: Acoustic neurinoma, neuroblastoma, neuroglia tumor and other brain tumors, tumor of spinal cord, breast cancer, colorectal cancer, advanced colorectal Intestines rectal adenocarcinoma；Endocrine cancer class, including:Adrenocortical carcinoma, pancreas cancer, pituitary cancer, thyroid cancer, accessory thyroid glands cancer, chest Gland cancer, MEN,muitiple endocrine neoplasms；Human primary gastrointestinal cancers class, including:Gastric cancer, cancer of the esophagus, carcinoma of small intestine, liver cancer, cholangiocarcinoma, stomach and intestine class Cancerous tumour, gallbladder cancer；Apparatus urogenitalis cancer class, including:Emerald green ball cancer, carcinoma of penis, prostate cancer；Gynecologic cancer class, including:Uterus Neck cancer, oophoroma, carcinoma of vagina, uterus/carcinoma of endometrium, private parts cancer, gestational trophoblastic tumor, carcinoma of fallopian tube, sarcoma of uterus； Head and tumor colli class, including:Carcinoma of mouth, lip cancer, glandula cancer, larynx cancer, hypopharyngeal cancer, positive pharynx cancer, rhinocarcinoma, nasal sinus cancer, nasopharynx Cancer；Leukemia class, including:Leukemia of children, acute lymphatic leukemia, acute myelogenous leukemia, chronic lymphatic leukemia, Chronic myelogenous leukemia, hair-like cell leukemia, acute promyelocytic leukemia, plasma cell leukaemia；Marrow Cancer blood disorder, including:The bad syndrome of bone marrow differentiation, myeloproliferative illness, alpastic anemia, Fanconi anemia, Idiopathic macroglobulinemia disease；Lung cancer class, including:Small Cell Lung Cancer, non-small cell lung cancer；Lymph cancer class, including:Huo Qijin Disease, non Hodgkin lymphom, skin-type T- cell lymphomas, peripheral T-cell woods bar tumor, AIDS related lymphomas；Cancer eye Class, including:Retinoblastoma, uveal；Cutaneum carcinoma class, including:Melanoma, non-black melanoma skin Cancer, Merkel cell cancer；Soft tissue sarcoma's class, such as:Children soft tissue sarcoma, adult soft tissue sarcoma, kaposi sarcoma；It secretes Urinary system cancer, including:Kidney wilms' tumor, wing skin cancer, carcinoma of urethra or metastatic cell cancer.
Fusion protein disclosed in this invention, the cancer class treated can be used for first be cervical carcinoma, breast cancer, colorectal cancer, Carcinoma of urinary bladder or lung cancer.
The preferred tumour class that can be treated by the fusion protein of the present invention is solid tumor and Hematological Malignancies.
Term as used herein " tumour " generally refers to the widely disease characterized by the runaway misgrowth of cell Disease.
The effective dose of active constituent used can change with mode of administration and the severity of disease to be treated.To big For the large mammal of part, the accumulated dose for imposing active ingredient daily is about 0.01-1000mg.In general, adult's clinic is given Ranging from 0.01-200mg/ days of dose, preferably 0.05-100mg/ days.
" effective dose " or " therapeutic dose " each means the amount for being enough to generate curative effect.Effective quantity can divide to be administered one or more times.It is logical Often, effective quantity is enough to mitigate, improve, stablize, slow down or postpone the further development of disease.
Composition
Composition for the present invention or containing fusion protein of the present invention.In general, when the present composition is used for When such use, the fusion protein can be mixed with one or more pharmaceutically acceptable carriers or excipient difference to The pharmaceutical dosage form of medicine approach, such as tablet, capsule, powder, granule, syrup, solution, oral solution, spirit, tincture, aerosol Agent, powder spray, injection, injection sterile powder, suppository etc..
" pharmaceutically acceptable " ingredient is suitable for people and/or animal and without excessively bad side reaction (such as toxicity, stimulation And allergy) have rational benefit/risk than substance." pharmaceutically acceptable carrier " is for melting the present invention Fit albumen sends acceptable solvent, suspending agent or the excipient pharmaceutically or on food of animal or people to.Carrier can be Liquid or solid.
The fusion protein of the present invention can pass through oral, intravenous, intramuscular or subdermal routes of administration.
In above-mentioned dosage form can the dosage form of oral administration be：Tablet, capsule, powder, granule, syrup, solution, fine wine Agent.Solid-state carrier includes：It is starch, lactose, calcium monohydrogen phosphate, microcrystalline cellulose, sucrose, white bole, superfine silica gel powder, talcum powder, low Replace hydroxypropyl cellulose, sodium carboxymethyl starch, polyvinylpyrrolidone.And liquid carrier includes：Sterile water, ethyl alcohol, poly- second Glycol, nonionic surface active agent and edible oil (such as corn oil, peanut oil and sesame oil).In the mistake for preparing pharmaceutical composition Usually used adjuvant includes in journey：Flavoring agent, colorant, preservative (such as oxybenzene alkyl butyl ester, sodium benzoate, sorbic acid) and Antioxidant (such as vitamin E, vitamin C, sodium pyrosulfite and dibutyl hydroxy toluene).
The dosage form that can be used for injection administration in above-mentioned dosage form includes：Injection, injection sterile powder, they be by Drug is mixed and made into the form for drug administration by injection with one or more pharmaceutically acceptable excipient.Solvent includes：It is sterile Water, ethyl alcohol, glycerine, propylene glycol, polyethylene glycol.In addition, also need be added bacteriostatic agent (such as benzyl alcohol, butyl hydroxybenzoate, thimerosal), Ooze conditioning agent (such as sodium chloride, glucose), suspending agent (such as sodium carboxymethylcellulose, methylcellulose), solubilizer (Tween-80, Lecithin), antioxidant (such as vitamin E, vitamin C, sodium pyrosulfite) and filler (such as lactose, mannitol).
In terms of easily prepared and administration position, preferred pharmaceutical composition is solid-state composition, especially freeze-dried powder Agent.It is preferred that intravenously administrable.
Below in conjunction with specific embodiment, the present invention is furture elucidated.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.Ratio and percentage are based on weight, unless stated otherwise
Herein, the polypeptide of sequence such as SEQ ID NO.1, is called ELBD or V16 for short, represents same polypeptide.
Embodiment 1：The structure of expression vector
A.EGFP-Tn-CPP series expression vector establishments
(1) structure of EGFP-Tn-HBD series mutations expression vector
In the patent application that Chinese Patent Application No. is CN 201310170530.6, it is thin to we disclose a kind of tumour Born of the same parents' targeting cell-penetrating peptide, it is characterised in that containing with the active CPPs structural domains of cell-penetrating peptide and with targets neoplastic cells VGF third ring sample manual simulation's peptide domains.For seek it is higher wear membrane efficiency and targeting, using this polypeptide structure as base Plinth synthesizes both ends with Bam HI and Sal I restriction enzyme sites according to different mutation demands with the artificial solid phase synthesis process of DNA The mutant nucleotide sequence of serial S3 simulating peptides spline structure, that be expressly set out is polypeptide sequence (SEQ ID as shown in Table 1 NO.1-11)。
The artificial synthesized VGF third ring sequences of table 1 and fractional mutant amino acid sequence
With EGFP-T0-HBD expression plasmid (Chinese Patent Application No.：CN 201310170530.6, in embodiment 1 ESH) set out, while by original EGFP-T0-HBD-pET28a expression vectors Bam HI and Sal I double digestions, and with synthesis Segment utilize T4DNA ligases connection.
Recombinant plasmid dna is recycled in Transformed E .coli DH5 α bacterial strains, culture, and sequence verification, i.e. structure complete series EGFP- Tn-HBD-pET28a expression vectors, n=1-12.Wherein for V16, it is especially named as ELBD, the corresponding expression of structure carries Body is EGFP-ELBD-HBD-pET28a.
Meanwhile building the corresponding expression vector of third ring sequence of other spontaneous growth Factor Sources, EGFP-BTC-HBD- pET28a、EGFP-NGR2-β-HBD-pET28a、EGFP-HGR2-β-HBD-pET28a。
(2) structure of EGFP-Tn-TAT series mutations expression vector
Cell-penetrating peptide TAT nucleosides of the both ends with Sal I and Xho I restriction enzyme sites is synthesized using the artificial solid phase synthesis process of DNA Acid sequence is inserted respectively into the EGFP-Tn-HBD-pET28a series for having used Sal I+Xho I double digestions to recycle in the present embodiment In expression vector, structure carries the EGFP-Tn-TAT-pET28a series expression vectors of TAT cell-penetrating peptides accordingly.
(3) structure of EGFP-ELBD-H2 expression vectors
Using EGFP-ELBD-HBD-pET28a plasmids as carrier, artificial synthesis in solid state both ends carry Sal I and Xho I digestions The DNA sequence dna in site, which passes through E.coli codon optimizations, and encodes H2 cell-penetrating peptides, and wherein H2 is another humanized Cell-penetrating peptide derives from heparin class growth factor.By EGFP-Tn-HBD-pET28a expression vectors Sal I and Xho I double digestions, And it is connect using T4DNA ligases with this segment of synthesis.
Transformed E .coli DH5 α bacterial strains, 37 DEG C are incubated overnight 15h, and Plasmid DNA QIAquick Gel Extraction Kit purifies recombinant plasmid dna, Send DNA sequence dna sequencing company sequence verification.
(4) structure of EGFP-TAT expression vectors
DNA sequence dna of the both ends with Bam HI and Xho I restriction enzyme sites, the sequence are synthesized using the artificial solid phase synthesis process of DNA Row codon optimizes, coding cell-penetrating peptide TAT.This artificial-synthetic DNA's segment is inserted into and has used the bis- enzymes of Bam HI+Xho I In the EGFP-ELBD-HBD-pET28a expression vectors cut, Transformed E .coli DH5 α bacterial strains, 37 DEG C are incubated overnight 15h, plasmid DNA QIAquick Gel Extraction Kits purify recombinant plasmid dna, send DNA sequence dna sequencing company sequence verification.
B. different anti-tumor protein drugs are built with ELBD-CPP recombinant expression carriers
(1) structure of TCS-ELBD-H2 expression vectors
The DNA primer that Bam HI and Xho I restriction enzyme sites are carried using artificial synthesis in solid state, with the plasmid built ELBD-H2 genes in EGFP-ELBD-H2-pET28a are that template carries out PCR amplification, and adds restriction enzyme site Bam HI and Xho I。
Using EGFP-ELBD-H2-pET28a as masterplate, according to ELBD-H2 gene orders, designs following primer and carry out PCR expansions Increase, forward and reverse primer introduces Bam HI and Xho I restriction enzyme sites respectively
Primer 1：5’-CGCGGATCCGGTGGTGGTGGTTCTGGTGGTGGTGGTT-3’
Primer 2：5’-CGCCTCGAGGTCTTTACCTTT-3’
Pcr amplification reaction condition：The reaction condition of PCR amplification is：First 95 DEG C, it is denaturalized 5min, then 95 DEG C, is denaturalized 45s, 58 DEG C of renaturation 30s, 72 DEG C of extension 30s, recycle 33 times, last 72 DEG C, 10min.
Amplified fragments are purified using DNA fragmentation QIAquick Gel Extraction Kit, and with Bam HI and Xho I double digestions, recycle segment. Simultaneously with BamHI and XhoI double digestion TCS-HBD-pET28b vector plasmids, carrier segments are recycled.Be used in combination T4DNA ligases into Row connection.Transformed E .coli DH5 α bacterial strains, 37 DEG C are incubated overnight 15h, and Plasmid DNA QIAquick Gel Extraction Kit purifies recombinant plasmid dna, Send DNA sequence dna sequencing company sequence verification..
(2) structure of MAP30-ELBD-H2 expression vectors
It is similar with the step of described in the structure embodiment of above-mentioned TCS-ELBD-H2 expression vectors.The primer and PCR expand Increasing condition is as described above.
Primer of the synthesis with Bam HI and Xho I restriction enzyme sites, in original plasmid EGFP-ELBD-H2-pET28a ELBD-H2 genes be that template carries out PCR amplification, and add restriction enzyme site Bam HI and Xho I.
Amplified fragments are used into DNA Purification Kits, and with Bam HI and Xho I double digestions, recycle segment.Simultaneously With Bam HI and Xho I double digestion MAP30-HBD-pET28b vector plasmids, carrier segments are recycled.Be used in combination T4DNA ligases into Row connection.Transformed E .coli DH5 α bacterial strains, Plasmid DNA QIAquick Gel Extraction Kit purify recombinant plasmid dna, send DNA sequence dna sequencing company Sequence verification.
Embodiment 2：Recombinant protein expression and purification
A.EGFP series recombinant protein expression and purifications
(1) EGFP-Tn-HBD series recombinant protein expression and purification
1. EGFP-Tn-HBD-pET28a plasmid construction schemes are as described in example 1 above.From the solid LB trainings of preservation strain It supports one conversion of picking on base tablet and there is the single bacterium of EGFP-Tn-HBD-pET28a plasmids to fall within 30ml and contain kanamycins (10- In 37 DEG C of shaken cultivations to OD600 it is about 0.5-1.0 in LB liquid medium 50mg/L).
2. this bacterial culture fluid is taken, with 1% inoculum concentration access certain volume kanamycins containing a certain concentration (10-50mg/ L continue to be enlarged culture in culture medium), until OD600 is about 0.5-1.0.
3. cultivation temperature is adjusted within the scope of 15 DEG C to 20 DEG C, suitable IPTG (0.1-10MM) induction target eggs are added White expression continues to cultivate 10-20h, and 500-1000rpm low-speed centrifugals collect thalline.
4. the thalline collected is suspended again with 20mM Tris-HCl buffer solutions (pH 8.0-8.5), with certain power (10- 100w) carry out ultrasonication.(4-10 DEG C) high speed centrifugation under cryogenic abandons supernatant, retains inclusion body.
5. using the Triton washing inclusion bodys 10-60min of 1.0-0.5%.
6. using the urea-denatured inclusion body 5-30min of 6M-8M.
7. dialysis renaturation, successively reduce dialyzate in urea content until dialyzate urea concentration be 0M.
According to above-mentioned steps, expression and purification EGFP-Tn-HBD recombinant proteins and EGFP-ELBD-TAT recombinate egg respectively In vain.(2) EGFP-ELBD-H2 recombinant proteins expression and purification
1. EGFP-ELBD-H2-pET28a plasmid construction schemes are as described in example 1 above.Then from the solid of preservation strain The conversion of picking one has the single bacterium of EGFP-ELBD-H2-pET28a plasmids to fall within 30ml to contain kanamycins on LB culture medium flat plates In 37 DEG C of shaken cultivations to OD600 it is about 0.5-1.0 in the LB liquid medium of (10-50mg/L).
2. this bacterial culture fluid is taken, with 1% inoculum concentration access certain volume kanamycins containing a certain concentration (10-50mg/ L continue to be enlarged culture in culture medium), until OD600 is about 0.5-1.0.
3. cultivation temperature is adjusted within the scope of 15-20 DEG C, suitable IPTG (0.1-10mM) induction target proteins are added Expression continues to cultivate 10-20h, and 500-1000rpm low-speed centrifugals collect thalline.
4. the thalline collected is suspended again with 20mM Tris-HCl buffer solutions (pH 8.0-8.5), with certain power (10- 100w) carry out ultrasonication.Under the conditions of low temperature (4-10 DEG C), high speed centrifugation (8000-13400rpm), which is collected, contains target egg White supernatant.
5. the supernatant collected carries out affinity protein purification by nickel chelating affinity column.It is chelated with most of nickel affine The method of chromatographic purifying target protein is similar, and gradient elution (20mM imidazoles is carried out using the imidazole buffer with various concentration Elution, the elution of 200mM imidazoles), collect the target protein component under the elution of various concentration imidazoles.
(3) EGFP-TAT recombinant proteins expression and purification
1. EGFP-TAT-pET28a plasmid construction schemes are as described in example 1 above.From the solid LB media of preservation strain The conversion of picking one has the single bacterium of EGFP-TAT-pET28a plasmids to fall within 30ml to contain kanamycins (10-50mg/L) on tablet LB liquid medium in 37 DEG C of shaken cultivations to OD600 be about 0.5-1.0.
2. this bacterial culture fluid is taken, with 1% inoculum concentration access certain volume kanamycins containing a certain concentration (10-50mg/ L continue to be enlarged culture in culture medium), until OD600 is about 0.5-1.0.
3. cultivation temperature is adjusted within the scope of 15-20 DEG C, suitable IPTG (0.1-10mM) induction target proteins are added Expression continues to cultivate 10-20h, and low-speed centrifugal collects thalline.
4. the thalline collected is suspended again with 20mM Tris-HCl buffer solutions (pH 8.0-8.5), with certain power (10- 100w) carry out ultrasonication.High speed centrifugation collects the supernatant containing target protein under cryogenic.
5. the supernatant collected carries out affinity protein purification by nickel chelating affinity column.It is chelated with most of nickel affine The method of chromatographic purifying target protein is similar, and gradient elution (10mM imidazoles is carried out using the imidazole buffer with various concentration Elution, the elution of 200mM imidazoles, the elution of 1M imidazoles), collect the target protein component under the elution of various concentration imidazoles.
(4) EGFP-ELBD-TAT recombinant proteins expression and purification
1. EGFP-ELBD-TAT-pET28a plasmid construction schemes are as described in example 1 above.From the solid LB of preservation strain The conversion of picking one has the single bacterium of EGFP-ELBD-TAT-pET28a plasmids to fall within 30ml to contain kanamycins on culture medium flat plate In 37 DEG C of shaken cultivations to OD600 it is about 0.5-1.0 in the LB liquid medium of (10-50mg/L).
2. this bacterial culture fluid is taken, with 1% inoculum concentration access certain volume kanamycins containing a certain concentration (10-50mg/ L continue to be enlarged culture in culture medium), until OD600 is about 0.5-1.0.
3. cultivation temperature is adjusted within the scope of 15 DEG C to 20 DEG C, suitable IPTG (0.1-10mM) induction target eggs are added White expression continues to cultivate 10-20h, and 500-1000rpm low-speed centrifugals collect thalline.
4. the thalline collected is suspended again with 20mM Tris-HCl buffer solutions (pH 8.0-8.5), with certain power (10- 100w) carry out ultrasonication.High speed centrifugation collects the supernatant containing target protein under 4-10 DEG C of cryogenic conditions.
5. the supernatant collected carries out affinity protein purification by nickel chelating affinity column.It is chelated with most of nickel affine The method of chromatographic purifying target protein is similar, and gradient elution (10mM imidazoles is carried out using the imidazole buffer with various concentration Elution, the elution of 200mM imidazoles, the elution of 1M imidazoles), collect the target protein component under the elution of various concentration imidazoles.
B. different anti-tumor proteins and ELBD-CPP recombinant protein expression and purifications
(1) TCS-ELBD-H2 recombinant proteins expression and purification
1. TCS-ELBD-H2-pET28b plasmid construction schemes are as described in example 1 above.From the solid LB trainings of preservation strain It supports one conversion of picking on base tablet and there is the single bacterium of TCS-ELBD-H2-pET28b plasmids to fall within 30ml and contain kanamycins (10- In 37 DEG C of shaken cultivations to OD600 it is about 0.5-1.0 in LB liquid medium 50mg/L).
2. this bacterial culture fluid is taken, with 1% inoculum concentration access certain volume kanamycins containing a certain concentration (10-50mg/ L continue to be enlarged culture in culture medium), until OD600 is about 0.5-1.0.
3. cultivation temperature is adjusted within the scope of 15-20 DEG C, suitable IPTG (0.1-10mM) induction target proteins are added Expression continues to cultivate 10-20h, and 500-1000rpm low-speed centrifugals collect thalline.
4. the thalline collected is suspended again with 20mM Tris-HCl buffer solutions (pH 8.0-8.5), with certain power (10- 100w) carry out ultrasonication.High speed centrifugation collects the supernatant containing target protein under 4-10 DEG C of cryogenic conditions.
5. the supernatant collected carries out affinity protein purification by nickel chelating affinity column.It is chelated with most of nickel affine The method of chromatographic purifying target protein is similar, and gradient elution (20mM imidazoles is carried out using the imidazole buffer with various concentration Elution, the elution of 50mM imidazoles, the elution of 200mM imidazoles), collect the target protein component under the elution of various concentration imidazoles.
(2) MAP30-ELBD-H2 recombinant proteins expression and purification
1. MAP30-ELBD-H2-pET28b plasmid construction schemes are as described in example 1 above.Then consolidating from preservation strain On body LB culture medium flat plates the conversion of picking one have the single bacterium of MAP30-ELBD-H2-pET28b plasmids fall within 30ml contain card that In 37 DEG C of shaken cultivations to OD600 it is about 0.5-1.0 in the LB liquid medium of mycin (10-50mg/L).
2. this bacterial culture fluid is taken, with 1% inoculum concentration access certain volume kanamycins containing a certain concentration (10-50mg/ L continue to be enlarged culture in culture medium), until OD600 is about 0.5-1.0.
3. cultivation temperature is adjusted within the scope of 15-20 DEG C, suitable IPTG (0.1-10mM) induction target proteins are added Expression continues to cultivate 10-20h, and 500-1000rpm low-speed centrifugals collect thalline.
4. the thalline collected is suspended again with 20mM Tris-HCl buffer solutions (pH 8.0-8.5), with certain power (10- 100w) carry out ultrasonication.The supernatant containing target protein is collected by centrifugation in low-temperature and high-speed.
5. the supernatant collected carries out affinity protein purification by nickel chelating affinity column.It is chelated with most of nickel affine The method of chromatographic purifying target protein is similar, and gradient elution (20mM imidazoles is carried out using the imidazole buffer with various concentration Elution, the elution of 50mM imidazoles, the elution of 200mM imidazoles), collect the target protein component under the elution of various concentration imidazoles.
Embodiment 3：Mutant wears membrane efficiency research
A. mutant wears membrane efficiency comparison：
(1) the membrane penetration effect analysis of the EGFP-Tn recombinant proteins of cell-penetrating peptide HBD is carried
Using 2 μM of recombinant protein sample concentration, the tumour cell of sample and in vitro culture is incubated 12h altogether, with streaming The analysis of cell detection art, the various EGFP-Tn-HBD recombinant proteins of comparative observation are to human breast cancer cell Bcap membrane penetration effect effects. Simultaneously with the third ring sequence recombinant proteins of other spontaneous growth Factor Sources (EGFP-BTC-HBD, EGFP-NRG2- β-HBD, EGFP-HRG- β 2-HBD) compare
With 2 μM of sample concentration, by the human breast cancer cell Bcap cells of a variety of recombinant mutant samples and in vitro culture Be incubated 12h altogether, using Laser Scanning Confocal analysis, observe each mutant wear membrane efficiency.
As a result, it has been found that EGFP-ELBD-HBD to the membrane penetration effect significant effects of Bcap cells is higher than other spontaneous growths The third ring sequence recombinant protein (EGFP-BTC-HBD, EGFP-NGR2- β-HBD, EGFP-HGR2- β-HBD) of Factor Source, figure 1A is laser co-focusing as a result, B is flow cytomery as a result, wherein EGFP-ELBD-HBD is labeled as E-V16-H, EGFP- BTC-HBD is labeled as E-BTC-H, and EGFP-NRG2- β-HBD are labeled as E- labeled as E-NRG2- β-H, EGFP-HRG- β 2-HBD HRG-β2-H。
Wearing membrane efficiency (Fig. 2) and can see in conjunction with obtained by the sequence and laser co-focusing of Table 1, flow cytometry tests Go out, the positions Cys of putative cyclization and quantity have exact influence to wearing film result.Sequence V16-9, V16-3, V16- The film effect of wearing of 2 and V16-1 is below ELBD (V16).Compared to V16-3, V16-2, the effect of V16-1 is worse, with V16- 9 quite.This illustrates that these three Cys have significant impact to the tertiary structure of mutant, may all take part in this structure domain space The annulation process of structure, to generate certain influence to wearing membrane efficiency.From the program using Zhang Lab to the solid of ELBD The result of the computer simulation analysis of structure sees, it is all can in the model configurations of cyclization, only C2-C10 and C2-C26 this Two kinds of possible forms illustrate the process that C2 has the possibility of bigger to take part in cyclization without this forms of C10-C26.And only The third ring structure for having the annulation process that C2 is participated in more similar and natural, contains highly conserved amino acid residue in ring (Y-X-G-X-R)。
V16-6 has lacked this section of sequence of YTGIRCSH compared with ELBD (V16), and it is worn film and is greatly affected, and illustrates N-terminal This section of sequence pair keeps the efficient importance for wearing film concertedness of ELBD (V16).It is also possible to be missing from this section of sequence, result in The missing of second Cys, to affect the space structure cyclization of this structural domain.Because according to computer modeling as a result, A cyclic structure is less likely to form between second Cys and third Cys, the possible reason is between two Cys Against the sequence for thering is one section of putatively to form α-helixstructure, the difficulty of disulfide bond formation between the two is caused.
The insetion sequence that can be seen that intermediate region that may be cyclic from the result of V16-10, V16-4 and V16-5 can be straight Connect influence ring size and shape and important conservative amino acid residues Y-X-G-X-R space orientation, so as to cause film is worn Efficiency it is low.Although all confirming combinations of the Leu47 of the carboxy-terminal end regions EGF to EGF and EGFR in previous Research Literature It plays an important role, but the sequence V16-8 of removal end VVL wears film to mutant in our mutation research result Effect makes a significant impact, and implies in the structure of ELBD-H, theflexible C-terminal tail are not participated in With the cohesive process of receptor, and the Cys having a major impact to domain constructs and the Y-X-G-X-R sequences bound directly with receptor It is only key factor, the alpha-helix insetion sequence among ring may be to play to ensure that the correct of its both sides Y-X-G-X-R sequences takes It is acted on to (optimal orientation), it is made to be conducive to efficiently identify and tie with ErbB receptor bound fractions It closes.Meanwhile from the above, it is believed that the sequence of ELBD can be optimized for RCSHYTGIRCSHGIYTGIRCQH
(2) concentration and time dependence of mutant membrane penetration effect
Albumen concentration gradient and different incubations are carried out using various concentration (0-2 μM) EGFP-ELBD-HBD recombinant proteins The research of time (0-12h).From experimental result it is observed that with the extension of incubation time and carrying for recombinant protein concentration Height, the trend wearing membrane efficiency be significantly improved of the recombinant protein EGFP-ELBD-HBD to human breast cancer cell Bcap cells, it was demonstrated that With concentration and time dependence, (A, B show that concentration dependent, C show Time Dependent to the membrane efficiency of wearing of the recombinant protein in Fig. 3 Property)
B. the wide spectrum Journal of Sex Research of targeting peptides ELBD：
In order to investigate its broad spectrum activity as the universality of cancer target peptide and with cell-penetrating peptide use in conjunction, we will ELBD sequences and HBD (a kind of Membrane-permeable Peptide from Human from people's EC-SOD heparin binding domains, Chinese patent ZL200810044084.3), TAT (cell-penetrating peptide in the sources HIV), HBP (the heparin binding domain sequence in heparin class growth factor, A kind of Membrane-permeable Peptide from Human) etc. a variety of cell-penetrating peptides (abbreviation CPP) merged, the recombinant protein sample of acquisition, with 30 μM EGFP-TAT, 2 μM of E-ELBD-TAT, 30 μM of EGFP-HBD, 2 μM of EGFP-ELBD-HBD recombinant proteins are being incubated 12h With flow cytomery, it wears membrane efficiency to Bcap cells afterwards.
As a result：Confirm that ELBD sequences can not only greatly improve the membrane efficiency of wearing of HBD as targeting peptides, while also to classics Cell-penetrating peptide TAT is promoted, and is also similar situation on HBP.Illustrate that ELBD has collaboration to the CPP in different type and source Improve targeting membrane penetration effect, it was demonstrated that ELBD sequences can largely promote various sources and the film of wearing of sequence cell-penetrating peptide is imitated Rate.(Fig. 4)
Selectivity of the C.EGFP-ELBD-HBD recombinant proteins to cell
11 kinds of cells are selected to study the tumor-targeting feature of EGFP-ELBD-HBD recombinant proteins.Including 9 kinds of people Body tumour cell：HeLa (human cervical carcinoma cell), Bcap (human breast cancer cell), A549 (human lung carcinoma cell), (people is pernicious by A357 Melanoma), T24 (human bladder cancer cell), MGC-803 (gastric carcinoma cells), 95D (human cytomegalovirus lung carcinoma cell), BxPC-3 (people Pancreatic cancer cell), 5637 (human bladder cancer cells) and 2 kinds of normal human cells：MRC-5 (human embryonic lung fibroblasts), 293T (people's renal epithelial cell).
Select recombinant protein a concentration of 1 μM is incubated 12h.The results show that EGFP-ELBD-HBD recombinant proteins are to MRC-5 With two kinds of normal cells of 293T almost without obviously wearing film；And to HeLa, Bcap, A549, the cancer cells such as T24, A357 have not Film (Fig. 5) is worn with degree.The generation of this phenomenon may be to be led since the receptor targeted of different cancer cell surfaces is distributed difference It causes recombinant protein EGFP-ELBD-HBD to show different cancer cells and different wears film ability.But for normal cell, Its membrane receptor, such as EGFR, EGF-R ELISA is far below cancer cell, and this iuntercellular characteristic becomes ELBD targeting peptides Key with tumor-selective.
Embodiment 4：Mutant and the ELISA method of tumor cell surface binding ability are analyzed
It is determined using ELISA method and wears the relatively high EGFP-S3-HBD of membrane efficiency (Chinese Patent Application No. CN 201310170530.6) with the bond strength of EGFP-ELBD-HBD two kinds of recombinant proteins and HeLa Cells surface.
By human cervical carcinoma cell with 1x103-1x105The density of a cells/well is inoculated in 96 porocyte culture plates (with for 24 hours It is practical operation concentration to cover with 96 orifice plates), 37 DEG C of cultures are washed 3 times with PBS afterwards for 24 hours, each 15min.The 0.1- of 4 DEG C of precoolings is added 0.25% glutaraldehyde, 50 holes μ l/, in 4 DEG C of fixed cell 10-45min；The cell fixed is washed 3 times, each 15min with PBS, It is stayed overnight in 4 DEG C of closings with 200 holes μ l/ with 1%BSA/PBS solution；(contain 0.05% Tween- in PBS with PBST buffer solutions 20) it washes 3 times, each 15min；Recombination fusion protein is added to according to certain doubling dilution in 96 orifice plates, 50 holes μ l/, each Concentration sets 3 parallel holes, 37 DEG C of incubation 2h；After washing 3 times with PBST, the anti-His-tag monoclonal antibodies (l of mouse is added:1000-l: 3000 dilutions), 50 holes μ l/, 37 DEG C of incubation 2h；With PBST board-washings 3 times, the sheep anti-mouse igg of horseradish peroxidase-labeled is added Secondary antibody (1:800-1:2500 dilutions), 50 holes μ l/, 37 DEG C of incubation 2h；With PBST board-washings 5 times, tmb substrate developing solution is added, The holes 100-200 μ l/, room temperature are protected from light 10-30min.Reaction is terminated with 50 holes μ l/ of sulfuric acid of 2mol/L, immediately in microplate reader The upper light absorption value measured at 450nm.
From fig. 6 it can be seen that ELBD can be combined under lower concentration with HeLa cell surface receptors, show ELBD Sequence be easier is combined with cancer cell surfaces, and more easily combination cell surface further promote recombinant protein wear film imitate Rate.
Embodiment 5：ELBD-CPP targets improvement of the cell-penetrating peptide to protein drug pharmacological action：
TCS is a kind of pharmaceutical protein with anti-tumor activity including building stem tuber from plant, is lost with ribosomes The activity of living protein (RIP), can inhibit protein to synthesize in cell free system in vitro.
After the gene order of TCS and ELBD mutant nucleotide sequence and H2 cell-penetrating peptide sequence amalgamation and expressions, TCS-ELBD-H2 significantly improves (Fig. 7) inhibiting rate of B16 (mouse melanoma cells).
A kind of MAP30 also protein drugs with anti-tumor activity come from seed of bitter gourd, and a kind of RIP albumen.By it with after ELBD-H2 amalgamation and expressions, MAP30-ELBD-H2 is aobvious to the inhibiting rate of tumour cell HeLa, B16 cell It writes and improves, however the inhibiting rate of normal cell MRC-5 is not significantly improved (Fig. 8).
The scope of the present invention is not limited by the specific embodiments described, and the embodiment is only intended to as illustrating the present invention The single example of various aspects further includes function equivalent method and component in the scope of the invention.In fact, in addition to described herein Content outside, those skilled in the art with reference to described above and attached drawing can easily grasp to the present invention a variety of improvement. The improvement is also fallen within the scope of the appended claims.Every bibliography mentioned above is all included in full to be made herein For reference.
SEQUENCE LISTING
<110>Zhejiang Reachall Pharmaceutical Co., Ltd.
<120>Cancer target polypeptide, preparation method and applications
<130>Specification sequence table
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<170> PatentIn version 3.3
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Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Met Ala Ala Thr
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Claims (38)

1. a kind of cancer target polypeptide is that First ray and amino acid sequence shown in YXGXR are it includes amino acid sequence Another second sequence shown in YXGXR, by connecting peptide linkage, wherein Y tables between the First ray and second sequence Show tyrosine or derivatives thereof；G indicates glycine or derivatives thereof；R indicates arginine or derivatives thereof；X indicates band containing side chain Fats group or/and hydroxyl groups or they combine or derivatives thereof amino acid.
2. cancer target polypeptide as described in claim 1, it is characterised in that the connection peptide includes the amino for forming alpha-helix Acid sequence.
3. cancer target polypeptide as described in claim 1, it is characterised in that the connection peptide includes the structure of rigid alpha-helix.
4. cancer target polypeptide as described in claim 1, it is characterised in that the connection peptide is comprising amino acid sequence The sequence of HMAATT.
5. cancer target polypeptide as described in claim 1, it is characterised in that the connection peptide includes that 6 or more amino acid is residual Base.
6. cancer target polypeptide as described in claim 1, it is characterised in that the connection peptide includes residual less than 3 amino acid Base.
7. cancer target polypeptide as described in claim 1, it is characterised in that the connection peptide is made of 1 amino acid residue.
8. cancer target polypeptide as claimed in claim 7, it is characterised in that the connection peptide is by histidine or derivatives thereof group At.
9. cancer target polypeptide as described in claim 1, it is characterised in that the cancer target polypeptide is that amino acid sequence is The polypeptide or its modified derivative of one of SEQ ID NO.1-4 or SEQ ID NO.8 or SEQ ID NO.9.
10. cancer target polypeptide as described in claim 1, it is characterised in that the cancer target polypeptide can be specifically bound EGFR families any member.
11. cancer target polypeptide as claimed in claim 10, it is characterised in that the cancer target polypeptide can be combined with EGFR, Its Kd is less than 50nM.
12. a kind of conjugate, it is characterised in that comprising cancer target polypeptide described in any one of claim 1 to 11 claim and A kind of active constituent, the two are conjugated by a kind of connector (linker)；And the wherein described active constituent is therapeutic component, is examined It is broken into point, radioactive isotope, radionuclide, toxin or their compositions.
13. conjugate as claimed in claim 12, the connector includes covalently key connection or non-covalent bond connection.
14. conjugate as claimed in claim 13, the covalent key connection includes direct covalent bonds, peptide bond, ester bond, two sulphur Key, amido bond, imide bond, phosphodiester bond, urea bond, isocyanic acid ester bond or combination thereof.
15. conjugate as claimed in claim 12, it is characterised in that the therapeutic component includes cell-penetrating peptide (CPP).
16. conjugate as claimed in claim 15, it is characterised in that the cell-penetrating peptide is SEQ ID NO.12 amino acid Polypeptide shown in sequence, the amino acid fragment of the trans-activator TAT from HIV, the heparin binding domain in the sources EC-SOD (HBD) amino acid fragment, the amino acid fragment or their derivative of the heparin binding domain (HBD) in the sources HBEGF.
17. conjugate as claimed in claim 15, it is characterised in that the conjugate includes that amino acid sequence is SEQ ID The polypeptide of one of NO.13-15.
18. conjugate as claimed in claim 12, it is characterised in that the therapeutic component includes radiation treatment ingredient, chemistry Therapeutic component, antibody, enzyme or combination thereof.
19. conjugate as claimed in claim 18, it is characterised in that the radiation treatment ingredient includes radioactive isotope, Shown radioactive isotope is iodine -131, and lutetium -177, Yttrium-90, samarium -153, phosphorus -32, cesium-131, palladium -103, radium -223 is iodo- 125, boron -10, actinium -225, bismuth -213, radium -225, lead -212, thorium -232 or combination thereof.
20. conjugate as claimed in claim 18, it is characterised in that the chemotherapy ingredient includes capecitabine (capecitabine), cis-platinum (cisplatin), Herceptin (trastuzumab), fulvestrant (fulvestrant), Tamosifen (tamoxifen), Letrozole (letrozole), Exemestane (exemestane), Anastrozole (anastrozole), aminoglutethimide (aminoglutethimide), Testolactone (testolactone), Vorozole (vorozole), formestane (formestane), Fadrozole (fadrozole), Letrozole (letrozole), Erlotinib (erlotinib), Afatinib (lafatinib), Dasatinib (dasatinib), Gefitinib (gefitinib), her horse For Buddhist nun (imatinib), pazopinib, Lapatinib (lapatinib), Sutent (sunitinib), nilotinib (nilotinib), Sorafenib (sorafenib), Abraxane (nab-palitaxel) or derivative or their group Close object.
21. conjugate as claimed in claim 12, it is characterised in that the diagnosis ingredient includes radiodiagnosis ingredient, fluorescence Ingredient, quantum dot or their compositions.
22. conjugate as claimed in claim 21, it is characterised in that the radiodiagnosis ingredient include Value linear, technetium -99, Molybdenum -99, rubidium -82, strontium -82, thallium -201 or their compositions.
23. a kind of coding any one of claim 1 to 11 claim the cancer target polypeptide nucleic acid sequence.
24. a kind of expression vector including nucleic acid sequence described in claim 23.
25. expression vector described in claim 24, it is characterised in that the expression vector can express in the cell.
26. a kind of host cell including nucleic acid sequence described in claim 23.
27. a kind of host cell including expression vector described in claim 24.
28. host cell described in claim 26, it is characterised in that the host cell is prokaryotic cell or eukaryocyte.
29. a kind of preparation method of the cancer target polypeptide, it includes one of the culture claim 26-27 hosts are thin Born of the same parents prepare the cancer target polypeptide.
30. a kind of pharmaceutical composition, it includes conjugate described in claim 12 and pharmaceutically acceptable carriers.
31. the pharmaceutical composition described in claim 30, it is characterised in that it also includes radiation treatment ingredient, radioactive nucleus Element, toxin, therapeutic component, chemotherapy ingredient or combination thereof.
32. a kind of pharmaceutical composition, it includes any one of claim 1 to the 11 claim institute cancer target polypeptide and medicines Acceptable carrier on.
33. a kind of method of bion of the treatment with tumour, including giving the claim of this bion effective dose 12 conjugates.
34. the method for claim 33, it is characterised in that the tumour includes to express at least one EGFR family members' Cell.
35. the method for claim 33, it is characterised in that the tumour is breast cancer, colorectal cancer, cancer of pancreas, neck Cancer, melanoma, oophoroma, prostate cancer, one of non-small cell lung cancer.
36. the method for claim 33, also includes while giving the curative drug of effective dose.
37. the method for claim 33, it is characterised in that the bion is human individual.
38. a kind of solution, it includes conjugates described in the claim 12 of effective concentration, it is characterised in that the solution is biology The blood plasma of individual.
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