CN101143895A - Polypeptide with tumour targeting effects and preparation method thereof - Google Patents

Abstract

The invention relates to a polypeptide with tumor-targeting performance and a preparation method. The structure of the aminophenol sequence of the polypeptide is CASPSGALRSC or CFPVPGHDLVC or CFSVPGHDIVC or CTPMSLSLSEC or CYTYPLGWHIC. The pC89 phage peptide library expressing protein polypeptides with different sequences of 10<SUP>8</SUP> and human breast cancer cell lines MDA-MB-231 are repetitively cocultured for a few times; filtration can penetrate cell membrane to enter into the phages expressing specific polypeptide in cytolymph and / or karyon, and phages are amplified in vitro in order to carry out DNA sequencing and deduce an exogenous amino acid sequence inserted in the phages; the filtered specific polypeptide phages, other human tumor cells and normal cells are cocultured in vitro, and the tumor cell specificity of the polypeptide phages is tested; according to the testing results of polypeptide sequence and cell specificity, the polypeptide with tumor targeting is artifically synthesized. The invention as a specificity carrier of the mammary cancer-targeting genetic therapy has potential clinic application value. The invention also provides a strong technical support for the filtration of affinity specificity polypeptide of other types of malignant tumor cell strains. Moreover, the polypeptide only contains nine to eleven amino acid residues, so the polypeptide can be easily synthesized, the change of spacial position is relatively less, the quality control of the polypeptide is easy, and use is convenient.

Description

A kind of have tumor targeting polypeptide and preparation method thereof

Technical field

The present invention relates to medicine technology field, specifically a kind of have tumor targeting polypeptide and preparation method thereof.

Background technology

Capturing of tumour is the focal issue on biomedical boundary always.Developed in the last hundred years and formed operation, radiotherapy, chemotherapy three big clinical therapy of tumor methods.These treatment meanss have prolonged patient's band knurl lifetime to a certain extent, but they in process of clinical application except excision with destroy the 26S Proteasome Structure and Function that tumour also is damaged to healthy tissues, especially when whole body uses chemotherapeutics, medicine arrives each tissue of human body and normal cell is also brought into play lethal effect by blood circulation, cause systemic adverse reactions, have a strong impact on the final effect of treatment.Therefore, the research and development of novel tumor treatment means seem particularly important.

Along with finishing of the Human Genome Project (HGP) and carrying out in a deep going way of human cancer genome anatomy plan (CGAP), understanding to the tumour molecular level is greatly improved, the formation of tumour mainly is because change has taken place for gene such as P53, the RAS etc. of the sudden change of some oncogenes, cancer suppressor gene or the growth of control cell, if these genes that change can be corrected by us, tumour can fundamentally be suppressed and reverse so.Genetic treatment of tumor is born under this theory just, and it is considered to thoroughly cure the effective ways of tumor disease in the future.Is the precondition that gene therapy plays a role by genophore with the specific position that goal gene or curative drug import tumour cell.But one of main difficult problem of gene therapy for cancer is exactly to develop suitable carriers to carry out whole body therapeutic.Used carrier generally has two classes in the clinical trial at present: virus vector and non-virus carrier, it with the adenovirus carrier carrier that the expression rate of the viral vector of representative is higher than other types, but it is controlled that the potential safety issue that this carrier exists is difficult to, and comprises that producing wild-type virus damage body and viral DNA may be incorporated into and cause heritable variation on the karyomit(e).Another outstanding shortcoming of virus vector is the no target of transduction, can't realize the individuation magnetic target therapy of tumour.Compare with virus vector, the advantage of non-viral vector is more obvious.The non-virus carrier that relates in the therapy of tumor research at present mainly comprises naked DNA, liposome, lipid complex, cationic polymer, receptor-mediated carrier etc.This class kind of carrier is more, and has safety low-poison characteristics, so non-virus carrier is considered to more rising, but they exist the problem of target difference and no target equally.Can see that the research work for the therapy of tumor carrier in the past mainly concentrates on transduction efficiency and the security that how to improve carrier under the external environment, seldom notes its effect of the killing tumor cells of target in vivo.And tumour is the individuation disease, the generation of tumour exists tissue specificity and individual difference, the therapy of tumor final objective is to carry out individualized treatment, therefore, ideal therapy of tumor carrier should possess and can target transports therapeutic substance and enter tumour cell clinically, do not influence Normocellular growth, expression rate height, characteristics such as while safety non-toxic.Do not report this cancer target specific gene treatment carrier up to now both at home and abroad as yet.Do not see that as yet receptor-mediated carrier is used for the affine The specificity of tumour, the affine specific detection of carrying out breast cancer cell with receptor-mediated carrier does not at home and abroad appear in the newspapers as yet.

Summary of the invention

The purpose of this invention is to provide a kind of novel tumor targeting polypeptide that has, it not only possesses high transduction rate, nontoxicity, the more important thing is that it can carry goal gene or antitumor drug target and enter tumour cell it is treated and kills and wounds, simultaneously other normal cell of human body is not had affinity or toxic action.The present invention also provides its preparation method.

Research of the present invention is to be based upon on the existing basis theoretical and a large amount of results of study reliably.

Defective virus HIV-TAT albumen that 1988 famous science magazines " Cell " are reported and the hsv VP22 albumen of reporting subsequently, its transduction efficiency is higher than all gene therapy vectors at present.Further studies show that and to pass cytolemma by albumen such as HIV-TAT, VP22 albumen to enter cell be because comprised in these protein structures by several or tens amino-acid residues and form, and be rich in the peptide chain of arginine (Arginine), be called protein transduction domain (protein-transduction domain, PTD).PTD is that the reaction mechanism by certain receptor-mediated Ag-Ab enters in the cell.Subsequently to studies confirm that of PTD, can also carry the material that originally can not enter cell behind it and various macromole or the biologically active treatment drug coupling and pass cytolemma and in cell, express, bring into play its biological effect.PTD is a class polypeptides matter, it be found to be us and seek specific polypeptide therapy of tumor targeting vector thinking is provided.In addition, road FOR ALL WE KNOW, owing to reasons such as the sudden change of oncogene or cancer suppressor gene or inactivations, the receptive material of different tumor cell surfaces meeting expression specificities, though the understanding to these acceptors is still limited, the existing for us and search out tumour cell targeted polypeptide carrier and lay a good foundation of this phenomenon.If can will separate and analyze its amino acid structure and function relationship with the peptide material of these tumour-specific receptors bind by certain technological method, we just probably find and are satisfactory at the targeting gene therapy carrier of not planting tumour cell so.

Meanwhile, a kind of fast development that can be used for screening the phage peptide library technology (phage display librarytechnique) of specific proteins and polypeptide provides the methodology basis for our realization of above-mentioned research imagination.So-called phage peptide library technology be exactly with the method for molecular cloning with the exogenous nucleic acid fragment with the formal representation of fusion rotein on the outside surface of phage particle.In general, its experimental implementation can be summarized as for two steps: build storehouse and sieve storehouse.At first wait and prepare the nucleic acid fragment that various sequences differ by synthetic, cDNA method or DNase random hydrolysis method, then it is cloned in carrier (phage or phagemid), subsequently by infecting or the superingection intestinal bacteria, making it secrete amalgamation and expression has the segmental phage of external source, is collectively referred to as and builds the storehouse.Go to screen the phage that affinity is arranged with it with immobilized target molecule or target cell afterwards, be referred to as to sieve the storehouse.Display technique of bacteriophage has two tangible advantages, and first phage is easy to amplification, and it two is that the allogenic polypeptide of amalgamation and expression or proteinic aminoacid sequence can be known by inference by the sequence of surveying phage DNA.But phage display amalgamation and expression polypeptide, protein domain and protein.This technology has been widely used in screening acceptor, antibody, lectin etc. and has had the foreign protein of clinical value and novel albumen and peptide molecule.

The case of technology of the present invention is:

Aminoacid sequence with tumor targeting polypeptide of the present invention is: CASPSGALRSC, or CFPVPGHDLVC, or CFSVPGHDIVC, or CTPMSLSLSEC, or the CYTYPLGWHIC preparation method is:

1, in order to find the targeted polypeptide with mammary cancer, uses and express 10 ⁸The pC89 phage peptide library of different sequence protein polypeptides and human breast cancer cell strain MDA-MB-231 cultivate repeatedly repeatedly altogether; Screening can be passed the phage that cytolemma enters the expression specificity polypeptide in cytoplasm and/or the nucleus, and the amplification in vitro phage also carries out sequential analysis, i.e. dna sequencing, and derive its aminoacid sequence;

2, the above-mentioned specific polypeptide phage that filters out and other human tumor cells and normal cell are carried out externally cultivate altogether, detect the cell tumour specificity of polypeptide phage;

3, according to aforementioned polypeptides sequence and cell-specific detected result, and this polypeptide of synthetic (and carry out fluorescent mark at its end, so that detect; To contain all amino acid compositions of this peptide sequence simultaneously but the different homeopeptide of sequence of amino acid carries out the detection of the cell spy property led, the relation that its purpose is to study polypeptide amino acid arrangement position and specificity and wears film);

4, above-mentioned synthetic polypeptide and different people tumour cell and normal cell are cultivated respectively altogether, and detect this peptide sequence whether still have the function of plasma membrane of wearing under no bacteriophage coat protein support, and the eliminating phage is protein mediated wears the film effect;

5, make up specific polypeptide associating expression vector: i.e. pEGFP-polypeptide, pGEX-2T-polypeptide and pGEFP-polypeptide-phallotoxins, survey this specific polypeptide and carry the applicability that the different molecular weight target protein is gone into cell.

6, carry out the spy of animal body inner tissue and lead sexual compatibility (using the isotope labeling polypeptide method), survey the characteristic that its in-vivo tumour specificity guiding is expressed.

The experiment situation:

Through expressing 10 ⁸The pC89 phage peptide library of polypeptide and human breast cancer cell strain MDA-MB-231 carry out five and take turns the phage that common cultivation screening back collection can enter cell, after this part specific phage amplification, select 20 phage clones to carry out dna sequencing at random, derive the aminoacid sequence (seeing Table 1) that these 20 phage clones have comprised 5 fusion polypeptide by sequencing result, wherein the multiplicity with the I sequence is the highest.I polypeptide phage and MDA-MB-231 cell and other human breast cancer cell strain, different tissue sources human tumor cells and human normal cell line are carried out common cultivation, detect I polypeptide phage by immunofluorescence technique and almost only affine specificity (Fig. 1) takes place with the MDA-MB-231 cell.In order to get rid of the influence of striding film effect of bacteriophage coat protein to polypeptide, synthesized the I polypeptide separately, and measure this polypeptide specificity by the confocal fluorescent microscope inspection and enter (Fig. 2) in the MDA-MB-231 cell, and prove that itself and normal cell do not have affinity.In order to confirm that further the I polypeptide has carrying capacity, design and made up GST-I polypeptide amalgamation protein expression vector subsequently, utilize genetic engineering technique that the GST-I polypeptide amalgamation protein of expressing is separated and purifying, this section fusion rotein and MDA-MB-231 cell are carried out the affinity experiment, immunofluorescence detects proof I polypeptide and can carry in the specific MDA-MB-231 of the entering cell of GST-I peptide fusion protein (Fig. 3), and in the cell inner expression regular hour.

The aminoacid sequence structure of five fusion roteins of table 1.

Sequence numbering	The aminoacid sequence structure	The multiplicity of sequence
			I	CASPSGALRSC	6
II	CFPVPGHDLVC	5
			III	CFSVPGHDIVC	4
IV	CTPMSLSLSEC	3
			V	CYTYPLGWHIC	2

I polypeptide phage adds in the MDA-MB-231 cell of vitro culture, behind the effect certain hour, use that the mouse IgG-488 two of anti-M13 phage antibody and green fluorescence mark is anti-to carry out immunofluorescence with the last M13 that enters the phage of cell and develop the color, Phalloidin redyes the result that the back Laser Scanning Confocal Microscope is observed down, (Fig. 1 is the compatibility test result of I polypeptide phage of the present invention and MDA-MB-231 cell) as shown in Figure 1.

The I polypeptide of mark green fluorescence FITC and MDA-MB-231 co-culture of cells, behind the effect certain hour, the polypeptide that does not enter cell that wash-out is unnecessary, the result that the confocal fluorescent microscopically is observed, (Fig. 2 is the compatibility test result of I polypeptide of the present invention and MDA-MB-231 cell) as shown in Figure 2.

The GST-I polypeptide amalgamation protein joins in the MDA-MB-231 cell of vitro culture, the effect certain hour, utilize the anti-mouse HRP-IgG of rabbit of mouse anti blood GST monoclonal antibody and FITC mark and the immune response that GST albumen takes place, detect the GST-I polypeptide amalgamation protein that enters cell, be the observed result of confocal fluorescent microscopically.(Fig. 3 is the compatibility test result of GST-I polypeptide amalgamation protein of the present invention and MDA-MB-231 cell) as shown in Figure 3.

The present invention serves as the research starting point with receptor-mediated carrier, the polypeptide that exploitation has the tumour cell pathoklisis, this section has the affine specific polypeptide of human breast cancer cell and can carry curative drug and gene with different molecular weight size and enter in the specific breast cancer cell, its target in specific breast cancer cell and transduction effect all are higher than other gene therapy vectors at present, have the potential clinical value as the idiosyncratic carrier of mammary cancer targeting gene therapy.Simultaneously, its discovery also provides powerful technical support for the screening of the affine specific polypeptide of the malignant cell strain of other kinds later on.In addition, this polypeptide only contains 9-11 amino-acid residue, and is easily synthetic, and spatial position change is less relatively, is easy to polypeptide is carried out quality control, easy to use.The present invention can improve the target of therapy of tumor greatly, the discovery of this mammary tumor cells specific polypeptide not only can be actively promoted the development of tumour cell targeting gene therapy, and the foundation of this experimental technique also provides experimental basis for the discovery of the targeted polypeptide carrier of other tumour cells, novel tumor related antigen, tumour specific antigen.Positive pushing effect will be played, tumour individuation gene therapy truly can be realized.

Description of drawings:

Fig. 1 be I polypeptide phage of the present invention and MDA-MB-231 cell compatibility test result 1000 *

Fig. 2 be I polypeptide of the present invention and MDA-MB-231 cell compatibility test result 400 *

Fig. 3 be I polypeptide of the present invention and gst fusion protein and MDA-MB-231 cell compatibility test result 1000 *

Embodiment

Embodiment:

Take the logarithm vegetative period MDA-MB-231 cell inoculation in 150mm tissue culture ware, and 37 ℃, 12h changes fresh medium (the L15 nutrient solution that contains 10%FBS), and adds Chloroquine make its final concentration reach 100 μ M, 37 ℃, 30min in nutrient solution; With 3 * 10 ¹¹The pC89 phage peptide library phage of TU adds culture dish, 8h-12h, 5min on ice; 4 ℃ of following 40ml HBSS flushing cells 4 times, HBSS () wash 1 time, and the Subtilisin with 11ml handles cell 1h again, preparation cell suspension, centrifugal removal Subtilisin; HBSS (one) the liquid processing cell 15min that contains 1mM PMSF and 2mM EDTA again with 6ml, washing; Lysing cell, collecting lysate is to contain the phage that can enter the MDA-MB-231 cell that obtains through first round cell screening.

With the above-mentioned cell pyrolysis liquid transformed competence colibacillus intestinal bacteria E.Coli XLI-Blue that collection obtains, transformed bacteria liquid is collected in the dull and stereotyped amplification of LB, adds the M13KO7 helper phage, the preparation phage particle; The phage that obtains after the amplification adds once more and carries out next round screening-amplification in the cell, till phage that adds and the phage that can enter cell are in a basic balance.

This experiment pnagus medius titre is calculated by following formula:

Phage titre (pfu)=blue plaque number * extension rate

As stated above, through 5 take turns screening after, DNA is extracted in 20 phage clones of random choose and separately amplification from transform bacteria LB flat board, with the order-checking of M13 (40) primer (sending biotech firm's order-checking), and derive the peptide sequence that is inserted in these bacteriophage coat proteins.Sequence results shows that detect five different peptide sequences among 20 clones, wherein 6 clones are CASPSGALRSC; 5 clones are CFPVPGHDLVC; 4 clones are CFSVPGHDIVC; 3 clones are CTPMSLSLSEC; 2 clones are CYTYPLGWHIC.These five peptide sequences are numbered I～V number (table 1) one by one.The amino-acid residue of peptide sequence is based on nonpolar hydrophobic amino acid.Comparing not with the international Protein Data Bank of PIR (http://pir.georgetown.edu/), discovery has the consistent functional protein sequence of homology with these five sequences.

Choose the phage that contains the high duplication aminoacid sequence and carry out affine specific detection with the MDA-MB-231 cell.Concrete grammar is: logarithmic phase MDA-MB-231 cell inoculation is in 24 orifice plates, 2 * 10 ⁴/ hole behind 37 ℃ of cultivation 12h, is changed fresh medium and is also added Chloroquine, 30min; Experimental group adds the I that contains the high duplication aminoacid sequence number and the II number little peptide phage that obtains through screening respectively, and control group drips original peptide storehouse phage, RGD-integrinbinding phage respectively, and the phage of adding is dripped poison and is 10 ⁹-10 ¹⁰TU establishes 3 multiple holes for every group; After cultivating 8h-12h again, the PBS flushing with 3.7% formalin and 10%Triton X-100 difference fixed cell 10min, adds and contains I%BSA, 0.025%NaN ₃, 0.1%Saponin damping fluid preact 15min, drip 1: 500 anti-M13 phage antibody effect 1h, the 0.1%Saponin flushing, after the mouse IgG-488 two that added again 1: 800 resists effect 30min, cleaning unnecessary two resists, redye the affine specificity of little peptide phage of fluorescence microscope and MDA-MB-231 cell with Phalloidin.

For whether the further clear and definite above-mentioned polypeptide phage that filters out has cell-specific, with human breast cancer cell strain MDA-MB-435, MCF-7, T47D, different tissue sources tumour cell Hela, HT-1080, A431, SCC-29, Calu3, Calu1, GLC, U251 and normal cell KMB17, L-02 are inoculated in 96 orifice plates respectively.Detect the affinity of I number little peptide phage and these cell strains according to the method described above.

In order to get rid of bacteriophage coat protein the fusion rotein specificity is striden the influence of film function, and further detect the specialized transport ability of fusion polypeptide.Select two peptide sequences that repeatability is the highest among 20 clones (I number and II number) to synthesize (synthetic by biotech firm), and mark the FITC green fluorescence endways, synthetic fluorescent mark polypeptide is called after I peptide, II peptide respectively.The synthetic polypeptide is carried out experiment in vitro with the MDA-MB-231 cell respectively, and concrete operations are as follows: logarithmic phase MDA-MB-231 cell inoculation is in 96 orifice plates, 5 * 10 ³Individual/hole, cultivate 12h for 37 ℃.Be divided into 4 groups according to the difference that adds peptide sequence.Add I number, II peptide respectively according to concentration 200ng/ml, 500ng/ml, 1000ng/ml, establish RGD-integrin protein positive control group simultaneously and do not add the blank group of polypeptide.Every kind of concentration is established 6 multiple holes.Behind effect 12h, 48h, do not enter the polypeptide of cell with the PBS flush away respectively, fluorescent microscope is observation polypeptide and the affine specificity of cell and the best use of time and the activity of polypeptide down.

In order to get rid of synthetic polypeptide and the contingent cross reaction of other cells, again with human breast cancer cell strain MDA-MB-435, MCF-7, T47D, different tissue sources tumour cell Hela, HT-1080, A431, SCC-29, Calu3, Calul, GLC, U251 and normal cell KMB17, L-02 are inoculated in 96 orifice plates respectively, detect the affinity of polypeptide and these cell strains according to preceding method.

The result shows that I peptide and II peptide all can combine and enter in the cell with MDA-MB-231 cell generation specificity, and wherein the affinity with the I peptide is stronger, and affinity is higher than the RGD-integrin albumen with higher cell transduction rate of generally acknowledging at present; Simultaneously, specificity takes place with other tissue-derived tumour cells and combines in these two polypeptide hardly, the more important thing is, these two polypeptide all do not combine with normal cell.The polypeptide that filtered out of proof is that to have a breast cancer cell MDA-MB-231 specific really.

Whether have the exogenous molecule of carrying and enter cell in order to understand the specific polypeptide that filters out, send the cDNA of the composite coding I of company peptide and designed the restriction enzyme site of EcoR I and BamH I at purpose segmental 3 ' and 5 ' end respectively.The synthetic dna sequence dna is inserted among the plasmid pGEX-2T by T4 ligase enzyme system is directed, make up prokaryotic expression carrier.Recombinant plasmid obtains identifying through order-checking.With recombinant plasmid transformed competence colibacillus e. coli bl21 (DE3).Select single colony inoculation in the LB nutrient solution that contains penbritin, 37 ℃ when cultivating the A600nm=0.6 left and right sides, the adding isopropyl-(isopropyl-β-D-thiogalactoside) to final concentration 0.6mmol/L, continuation is with 37 ℃, 250r/min shaking culture 4h, the expression of inducible protein.Bacterium liquid after inducing is centrifugal in 4 ℃ of following 5000r/min, and the thalline PBS of collection is prepared into bacterial suspension, carrying out ultrasonic bacteria breaking after washing 2 times.The centrifuging and taking supernatant liquor, through GST agarose affinity chromatography column purification, the product behind the collection purifying is identified through the 10%SDS-PAGE electrophoresis.The fusion rotein of purifying is carried out the SDS-PAGE electrophoresis according to a conventional method, then albumen is forwarded on the nitrocellulose filter, add 5% skim-milk and put 37 ℃ of sealing 30min, add mouse anti blood again and inhale GST monoclonal antibody (1: 800), 4 ℃ are spent the night.After washing 3 times, add the anti-mouse HRP-IgG of rabbit (1: 1000), put 37 ℃ of reaction 30min.Handle the back with the tetramethyl benzidine 5min that develops the color through the sulfuric acid dextran, termination reaction, the albumen that this detected result demonstration is obtained is the fusion rotein of GST albumen and I polypeptide.In addition, take the logarithm vegetative period MDA-MB-231 cell inoculation in 24 orifice plates, 5 * 10 ⁴Fresh medium behind 37 ℃ of cultivation 12h, is changed in/hole.10 μ mol/L add fusion rotein in the cell culture fluid according to working concentration, and establish and only add the proteic experiment contrast group of GST and do not add any proteic blank group.Continue to cultivate, behind the 12h reactant is washed without in conjunction with the fusion rotein that enters cell with PBS, add mouse anti blood GST monoclonal antibody (1: 800) again in cell, 4 ℃ are spent the night.After washing 3 times, add the anti-mouse HRP-IgG of rabbit (1: 1000) of FITC mark, put 37 ℃ of reaction 30min, through the unconjugated unnecessary antibody of PBS wash-out, fluorescence microscope result.The result shows that this polypeptide can carry GST albumen and enter in the MDA-MB-231 cell.Also fusion rotein and other different types of human breast cancer cell strains are carried out common cultivation according to above-mentioned experimental technique, found that polypeptide can not carry GST albumen and enter in these cells.

Additional instruction:

1. the reagent that relates in the test among the present invention all can be bought in market, or the prescription in the by specification is prepared voluntarily.

2. the cell of using in the process of the test of the present invention is the cell strain that has made up, and can buy.

3. bacterial isolates that relates in the test of the present invention and plasmid all can biotechnological formulation company at home and abroad buy.

The pertinent literature source or the source of the biotechnological formulation of some outbalance in the test below are provided:

The pC89 phage peptide library

Pertinent literature: Vasily V, Ivanenkov, Franco Felici et al.Targeted delivery of multivalent phagedisplay vectors into mammalian cells.Biochimica et Biophysica Acta, 1999 (1448): the 463-472. source: the pC89 phage peptide library among the present invention is so kind as to give by Italian Dr.Alessandra Luzzago.RGD-integrin binding phage, RGD-integrin dietary protein origin: RGD-integrin binding phage among the present invention and RGD-integrin albumen are so kind as to give by the Dr.Stambrook at U.S. University of Cincinnati tumor research center.

Claims (3)

1. one kind has tumor targeting polypeptide, it is characterized in that its aminoacid sequence structure is: CASPSGALRSC; Or CFPVPGHDLVC; Or CFSVPGHDIVC; Or CTPMSLSLSEC; Or CYTYPLGWHIC.

2. the described preparation method with tumor targeting polypeptide of claim 1 is characterized in that carrying out according to the following steps:

1) uses expression 10 ⁸The pC89 phage peptide library of different sequence protein polypeptides and human breast cancer cell strain MDA-MB-231 cultivate repeatedly repeatedly altogether; Screening can be passed the phage that cytolemma enters the expression specificity polypeptide in cytoplasm and/or the nucleus, and the amplification in vitro phage also carries out dna sequencing, and derives the exogenous amino acid sequence of inserting on these phages;

2) the above-mentioned specific polypeptide phage that filters out and other human tumor cells and normal cell are carried out externally cultivate altogether, detect the cell tumour specificity of polypeptide phage;

3) according to aforementioned polypeptides sequence and cell-specific detected result, synthetic has tumor targeting polypeptide.

3. the described detection method with tumor targeting polypeptide of claim 1 is characterized in that carrying out according to the following steps:

1) above-mentioned synthetic polypeptide and different people tumour cell and normal cell are cultivated respectively altogether, and detect this peptide sequence whether still have the function of plasma membrane of wearing under no bacteriophage coat protein support;

2) make up specific polypeptide associating expression vector: i.e. pEGFP-polypeptide, pGEX-2T-polypeptide and pGEFP-polypeptide-phallotoxins, survey this specific polypeptide and carry the applicability that the different molecular weight target protein is gone into cell;

3) carry out the spy of animal body inner tissue with the isotope labeling polypeptide method and lead the sexual compatibility experiment, survey the characteristic that its in-vivo tumour specificity guiding is expressed.

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