CN109336977A

CN109336977A - The binding protein of tumor stem cell marker molecule EpCAM and its application

Info

Publication number: CN109336977A
Application number: CN201811181484.9A
Authority: CN
Inventors: 魏星; 刘志芳; 张伟雄; 戴琳
Original assignee: Jinan University
Current assignee: Jinan University; University of Jinan
Priority date: 2018-10-11
Filing date: 2018-10-11
Publication date: 2019-02-15

Abstract

The invention discloses the binding protein of tumor stem cell marker molecule EpCAM a kind of and its applications.The binding protein is one of entitled NB85 binding protein, NB143 binding protein, NB278 binding protein, NB431 binding protein and NB589 binding protein or the binding protein that at least two combinations are formed.Aforementioned 5 binding proteins are respectively provided with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:4 and SEQ ID NO:5.The invention also discloses the binding protein application in preparations of anti-tumor drugs of above-mentioned tumor stem cell marker molecule EpCAM.Binding protein structural stability of the invention is high, is easy to be transformed, and be humanized, in human body non-immunogenicity, can be preferably applied for the exploitation of anti-tumor drug.

Description

The binding protein of tumor stem cell marker molecule EpCAM and its application

Technical field

The invention belongs to binding protein field, in particular to a kind of binding protein of tumor stem cell marker molecule EpCAM And its application.

Background technique

Studies have shown that there is heterogeneity between tumour cell, tumour cell can form a level knot in tumor entity Structure has in the hierarchical structure a group to have the cell of initial tumor ability, as tumor stem cell.Tumor stem cell theory thinks Tumor stem cell is similar with Common tumors cell, has the characteristics that self-renewing, infinite multiplication, versatility and drug resistance.At present The main path for treating tumour includes operation excision, radiotherapy or chemotherapy, but these methods cannot effectively kill Tumor Stem Cell.Since tumor stem cell has initial tumor ability, using technique for gene engineering, targeting tumor stem cells specificity is designed Biomarker becomes another effective way for the treatment of cancer.

Signal transduction factor (EpCAM) is made of 314 amino acid, includes three structural domains: extracellular fragment structural domain (EpEX), single pass transmembrane structural domain and intracellular domain (EpICD).In some normal tissues and cell, on esophageal squamous Skin, EpCAM protein expression level is lower, and in certain cells with proliferative capacity, such as liver regeneration cell, the table of EpCAM It is relatively high up to level, it expresses in normal gastric mucosa without EpCAM, but is found in 77% patients with gastric cancer gastric mucosa EpCAM is expressed again.In addition, there are also occur more normal group of EpCAM expression when quite a few group is woven in and canceration occurs Knit high situation, such as pancreas, mammary gland, ovary, colon, bladder and prostate.These are studies have shown that EpCAM may organized Certain effect is played during malignant transformation of cells, thereby increases and it is possible to participate in the relevant metabolic process of tumor development.In addition, Also studies have shown that EpCAM albumen extracellular fragment lacks in tumour frequent occurrence, and the grade malignancy phase of such case and tumour It closes.EpCAM albumen participates in the processes such as tumor cell proliferation, formation, invasion, migration, diagnosis, drug resistance and antineoplaston, Therefore, it gathers around and has broad application prospects as the target spot of oncotherapy using EpCAM, there is high medical value.

Summary of the invention

The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of tumor stem cell mark point The binding protein of sub- EpCAM.

Another object of the present invention is to provide the protein-bonded applications of above-mentioned tumor stem cell marker molecule EpCAM.

The purpose of the invention is achieved by the following technical solution: a kind of combination egg of tumor stem cell marker molecule EpCAM It is white, it is that entitled NB85 binding protein, NB143 binding protein, NB278 binding protein, NB431 binding protein and NB589 are combined The binding protein that one of albumen or at least two combinations are formed；

The protein-bonded amino acid sequence of the NB85 is as shown in SEQ ID NO:1；

The protein-bonded amino acid sequence of the NB143 is as shown in SEQ ID NO:2；

The protein-bonded amino acid sequence of the NB278 is as shown in SEQ ID NO:3；

The protein-bonded amino acid sequence of the NB431 is as shown in SEQ ID NO:4；

The protein-bonded amino acid sequence of the NB589 is as shown in SEQ ID NO:5.

The protein-bonded nucleotide sequence for encoding above-mentioned tumor stem cell marker molecule EpCAM is that coding is described NB278 described in the protein-bonded nucleotide sequence of NB143, coding described in the protein-bonded nucleotide sequence of NB85, coding NB589 described in the protein-bonded nucleotide sequence of NB431 described in protein-bonded nucleotide sequence, coding and coding is combined The nucleotide sequence that one of nucleotide sequence of albumen or at least two combinations are formed.

The coding protein-bonded nucleotide sequence of NB85 is preferably as shown in SEQ ID NO:8.

The coding protein-bonded nucleotide sequence of NB143 is preferably as shown in SEQ ID NO:9.

The coding protein-bonded nucleotide sequence of NB278 is preferably as shown in SEQ ID NO:10.

The coding protein-bonded nucleotide sequence of NB431 is preferably as shown in SEQ ID NO:11.

The coding protein-bonded nucleotide sequence of NB589 is preferably as shown in SEQ ID NO:12.

The protein-bonded amino acid sequence of the tumor stem cell marker molecule EpCAM is as follows:

NB85:

MAQVQLLESGGGLVQPGGSLRLSCAASGDRVSYKFMAWVRQAPGKGLEWVSSIRGNDGSTYYADSVKG RFTISRDNSKNTL YLQMNSLRAEDTAVYYCARKGKRHYELPYWGQGTLVTVSSAAA；

NB143:MAQVQLLESGGGLVQPGGSLRLSCAASGVKFSNHDMTWVRQAPGKGLEWVSAINSGGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMGRQARVPHHMRFWGQGTLVTVSSAAA；

NB278:MAQVQLLESGGGLVQPGGSLRLSCAASGVKIIPKSMAWVRQAPGKGLEWVSTIETADGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASVSRSRTNSLKSWGQGTLVTVSSAAA；

NB431:MAQVQLLESGGGLVQPGGSLRLSCAASGFTFSYNNMAWVRQAPGKGLEWVSAIEGKDGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRKGRTEKLSYWGQGTLVTVSSAAA；

NB589:MAQVQLLESGGGLVQPGGSLRLSCAASGVRFSNEVMAWVRQAPGKGLEWVSGIEMKNGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASWGHSRRQQSKFNSWGQGTLVTVSSAAA。

The protein-bonded nucleotide sequence of the tumor stem cell marker molecule EpCAM is as follows:

NB85:ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGT CTCTCCTGTGCAGCCTCCGGAGATAGGGTTAGCTATAAGTTTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTC TAGAGTGGGTATCAAGCATTCGGGGCAATGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCAT CTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTAT TGCGCGAGAAAGGGTAAGAGGCACTACGAGCTGCCGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGG CCGCA；

NB143:ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG TCTCTCCTGTGCAGCCTCCGGAGTTAAGTTTAGCAATCACGATATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGT CTAGAGTGGGTATCAGCCATTAATAGCGGAGGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCA TCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTA TTGCGCGAGAATGGGTCGTCAGGCGCGTGTTCCGCACCACATGCGGTTTTGGGGTCAGGGAACCCTGGTCACCGTC TCGAGCGCGGCCGCA；

NB278:ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG TCTCTCCTGTGCAGCCTCCGGAGTTAAGATTATCCCTAAGTCTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGT CTAGAGTGGGTATCAACCATTGAGACGGCAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCA TCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTA TTGCGCGAGTGTTTCGCGTAGTCGGACCAACTCGCTCAAGTCTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC GCGGCCGCA；

NB431:ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG TCTCTCCTGTGCAGCCTCCGGATTTACGTTTAGCTATAACAATATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGT CTAGAGTGGGTATCAGCCATTGAGGGGAAAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCA TCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTA TTGCGCGAGACGTAAGGGGCGTACGGAGAAGCTGTCGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG GCCGCA；

NB589:ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG TCTCTCCTGTGCAGCCTCCGGAGTGAGGTTCAGCAACGAGGTGATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGT CTAGAGTGGGTAGGCATCGAGAAGATGAACGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCA TCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTA TTGCGCAAGTTGGGGCCACAGCAGGAGGCAGCAGAGCAAGTTCAACAGCTGGGGTCAGGGAACCCTGGTCACCGTC TCGAGCGCGGCCGCA。

From the above, the NB85 binding protein, NB143 binding protein, NB278 binding protein, NB431 is encoded to combine Albumen and the protein-bonded nucleotide sequence of NB589 are respectively by 372,381,375,372 and 381 base compositions, corresponding coding Amino acid be respectively 124,127,125,124 and 127.This 5 binding proteins contain 3 CDR (complementary determining region) areas: CDR1 contains 9 amino acid；CDR2 contains 6 amino acid；CDR3 contains 11-14 amino acid.

The protein-bonded preparation method of the tumor stem cell marker molecule EpCAM, comprising the following steps: pass through base Because of the nucleotide sequence of tumor stem cell marker molecule EpCAM described in composite coding, it is cloned into expression vector, then will be weighed Expression vector after group is transferred to host cell and is expressed, purified, and obtains the knot of the tumor stem cell marker molecule EpCAM Hop protein；Or by albumen synthetic method, obtain the binding protein of the tumor stem cell marker molecule EpCAM.

The tumor stem cell marker molecule EpCAM binding protein application in preparation of anti-tumor drugs.

The tumour be EpCAM high express tumour, including but not limited to cancer of pancreas, breast cancer, bladder cancer, the cancer of the esophagus, Gastric cancer, colon cancer, prostate cancer, lung cancer and ovarioncus.

The present invention has the following advantages and effects with respect to the prior art:

1. the present invention by display technique of bacteriophage, screens extracellular with EpCAM in source of people phagocytosis binding protein precursor library The albumen of part interaction, can obtain the binding protein of human body protein in the case where human body is immunized without using antigen.

2. binding protein provided by the invention has many advantages, such as structural stability height, it is easy to be transformed.

3. binding protein provided by the invention is humanized, in human body non-immunogenicity, can be preferably applied for resisting The exploitation of tumour medicine.

4. binding protein provided by the invention combines highly expressed prokaryotic hosts to carry out protein expression, combination can significantly reduce The production cost of albumen promotes protein-bonded application.

Detailed description of the invention

Fig. 1 is positive colony result figure of the monoclonal phage ELISA screening in conjunction with EpCAM polypeptide.

Fig. 2 is the SDS-PAGE electrophoresis of binding protein expression after purification；Wherein, figure A is the protein-bonded electrophoresis of NB85 Figure, figure B are the protein-bonded electrophoretogram of NB143, and figure C is the protein-bonded electrophoretogram of NB278, and figure D is that NB431 is protein-bonded Electrophoretogram, figure E are the protein-bonded electrophoretogram of NB589；In figure, swimming lane M is albumen Marker, and swimming lane 1 is not induce large intestine bar Bacterium holoprotein sample, swimming lane 2 are the Escherichia coli holoprotein after induction, and swimming lane 3 is broken supernatant, and swimming lane 4 is broken precipitating, swimming Road 5 was column liquid, and swimming lane 6 is to wash miscellaneous liquid, and swimming lane 7-11 is purpose protein eluate 1-5.

Fig. 3 is the combination situation map of the EpCAM binding protein and EpCAM polypeptide using ELISA method detection purifying.

Fig. 4 is the influence knot using mtt assay detection binding protein to tumour cell DU145, MCF-7 and PC-3 proliferative capacity Fruit figure；Wherein, figure A is influence result figure of the binding protein to DU145 proliferative capacity, and figure B is that binding protein is proliferated energy to MCF-7 The influence result figure of power, figure C are influence result figure of the binding protein to PC-3 proliferative capacity；* 0 μ g/mL and * * p of p < 0.05vs < 0.01vs 0 μ g/mL, (n=3).

Fig. 5 is using cell scratch method detection binding protein to tumour cell DU145, MCF-7 and PC-3 transfer ability Result photo figure；Wherein, figure A~C is DU145 cell, MCF-7 cell, PC-3 cell respectively.

Fig. 6 is the data analysis result figure obtained according to Fig. 5；Wherein, figure A~C is DU145 cell, MCF-7 thin respectively Born of the same parents, PC-3 cell；* 0 μ g/mL, * * p < 0.01vs of p < 0.05vs, 0 μ g/mL, (n=3).

Fig. 7 is to be invaded using Transwell invasion method detection binding protein to tumour cell DU145, MCF-7 and PC-3 Influence result photo figure；Wherein, figure A~C is DU145 cell, MCF-7 cell, PC-3 cell respectively.

Fig. 8 is the data analysis result figure obtained according to Fig. 7；Wherein, figure A~C is DU145 cell, MCF-7 thin respectively Born of the same parents, PC-3 cell；* 0 μ g/mL, * * p < 0.01vs of p < 0.05vs, 0 μ g/mL, (n=3).

Fig. 9 is to be migrated using Transwell moving method detection binding protein to tumour cell DU145, MCF-7 and PC-3 Influence result photo figure；Wherein, figure A~C is DU145 cell, MCF-7 cell, PC-3 cell respectively.

Figure 10 is the data analysis result figure obtained according to Fig. 9；Wherein, figure A~C is DU145 cell, MCF-7 thin respectively Born of the same parents, PC-3 cell；* 0 μ g/mL, * * p < 0.01vs of p < 0.05vs, 0 μ g/mL, (n=3).

Figure 11 is thin to tumour using stream type cell analyzer and the bis- transfection reagent box detection binding proteins of Annexin V/PI The influence result photo figure of born of the same parents' DU145, MCF-7 and PC-3 apoptosis；Wherein, figure A~C be respectively DU145 cell, MCF-7 cell, PC-3 cell.

Figure 12 is the data analysis result figure obtained according to Figure 11；Wherein, figure A~C is DU145 cell, MCF-7 thin respectively Born of the same parents, PC-3 cell；* p < 0.05vs is without binding protein control group, and * * p < 0.01vs is without binding protein control group, (n=3).

Figure 13 is to examine binding protein NB143 and NB431 in DU145 prostate gland cancer cell mouse model using zoopery In to tumour increase influence result figure；Figure A is the volume change curve graph of tumour after administration, at the end of figure B is dosage period The tumor size of each group, the tumor weight of each group after scheming C for dosage period；* p < 0.05vs PBS, (n=4/n=3).

Specific embodiment

The present invention is described in further detail with attached drawing combined with specific embodiments below, but embodiments of the present invention It is without being limited thereto.

Embodiment 1 prepares helper phage

(1) the three ride TG1 escherichia coli cloning bacterial strain on the culture dish of a TYE solid medium, pulls line Culture dish is inverted in 37 DEG C of incubator culture about 14h.

(2) TG1 single colonie is seeded in 5mL 2 × TY fluid nutrient medium on picking culture dish, and 37 DEG C, 250rpm cultivated Night.

(3) by bacterium solution that step (2) obtains, 1:100 ratio is forwarded to another 2 × TY of 5mL fluid nutrient medium by volume In, 37 DEG C, 250rpm cultivates to bacterium solution OD₆₀₀About 0.5.

(4) with PBS by KM13 helper phage gradient dilution (10¹²Pfu/mL~10⁴pfu/mL)。

(5) it takes 200 μ L steps (3) to cultivate obtained bacterium solution, the KM13 helper phage that 10 μ L have diluted, mixture is added 37 DEG C of water-bath water-bath 30min are placed in, mixture A is obtained.

(6) low melting-point agarose and 2 × TY fluid nutrient medium are mixed, is heated to melting completely, is then incubated in water 42 DEG C are cooled to, top layer (H-top) culture medium is obtained, wherein the concentration of low melting-point agarose is 1.5%.Take 3mL top layer culture The mixture A that base is obtained with step (5) is mildly mixed, and is then taped against on the TYE solid culture plate of 37 DEG C of preheatings.To top layer culture Base solidifies completely, and culture dish is inverted in 37 DEG C of incubator overnight incubations.

(7) one small plaque of picking is inoculated in (OD in 5mL TG1 bacterium solution₆₀₀=0.5), 37 DEG C, under the conditions of 250rpm Cultivate 2h.

(8) by bacterium solution, 1:100 ratio is forwarded in another 2 × TY of 500mL fluid nutrient medium by volume, 37 DEG C, 250rpm shakes bacterium 2h.

(9) it is 50 μ g/mL, 30 DEG C, 250rpm overnight incubation that kanamycins to its concentration in the medium, which is added,.

(10) bacterium solution is centrifuged and is filtered, obtain supernatant, add the 20%PEG- for being equivalent to 1/5 volume of supernatant NaCl solution is sub-packed in the centrifuge tube of 50mL after mixing and is incubated for 1h on ice, and 4 DEG C, 3200g centrifugation 30min abandon supernatant, then use Precipitating is resuspended in 1mL PBS buffer solution, and 4 DEG C, 3200g centrifugation 5min, supernatant is with 0.45 μM of filter filtration sterilization to get helper phage Body.

(11) sensibility of the helper phage of detection preparation to pancreatin: purified helper phage is taken a small amount of average It is divided into two equal portions, one group adds pancreatin to handle, and one group is not processed.The portion for adding pancreatin to handle is added in the trypsin solution of 1mL 10¹⁰A helper phage is incubated at room temperature 30min.Gradient dilution is carried out (from 10 with PBS buffer solution (pH=7.4,0.01M)¹⁰It arrives 10²A bacteriophage), then take the grade dilution of 10 μ L to infect 200 μ L TG1 bacterium (OD respectively₆₀₀=0.5) it is solid, to be coated on TYE (kanamycins containing final concentration of 50 μ g/mL), 37 DEG C of incubator overnight incubations on body plate.The bacteriophage handled by pancreatin Infecting the clone's number obtained after bacterium should be at least fewer by 10 than clone's number of untreated fish group⁶Times, otherwise abandon the helper phage Prepared product, another plaque of picking are prepared again.

Embodiment 2 prepares phagocytosis binding protein precursor library

(1) thaw on ice containing binding protein plasmid TG1 bacterium (i.e. source of people binding protein phage library, SourceBioscience, London, UK), addition 2 × TY of 500mL fluid nutrient medium (contain 100 μ g/mL ampicillins, 4% (w/v) glucose), 37 DEG C, 250rpm cultivates to OD₆₀₀=0.5.

(2) embodiment 1 is prepared 2 × 10 are added¹²A helper phage, 37 DEG C of water-baths keep the temperature 30min, by 500mL Culture is distributed into the every pipe of 50mL, and 3200g is centrifuged 10min, abandons supernatant, and precipitating is resuspended with 500mL 2 × TY culture medium and (contains 0.1% (w/v) glucose, 100 μ g/mL ampicillins and 50 μ g/mL kanamycins).25 DEG C, 250rpm shake culture 16- 20h is to get phagocytosis binding protein precursor library.

3 purified phage binding protein library of embodiment

(1) 2 preparation-obtained phagocytosis binding protein precursor library of embodiment is distributed into the every pipe of 50mL, totally 10 pipe.

(2) centrifuge tube is centrifuged 20min in 3200g, supernatant is transferred in clean container, addition is equivalent to supernatant The 20%PEG-NaCl solution of 1/5 volume, is sub-packed in the centrifuge tube of 50mL after mixing and is incubated for 1h on ice.

(3) centrifuge tube is centrifuged 30min in 4 DEG C, 3200g, abandons supernatant, is resuspended and is precipitated with 5mL PBS buffer solution, then to The 20%PEG-NaCl solution of 1mL is wherein added, is incubated for 10min on ice.

(4) 4 DEG C, 3200g centrifugation 30min, abandon supernatant, then be resuspended and precipitated with 1mL PBS buffer solution, 4 DEG C, 3200g centrifugation 5min, 0.45 μM of filter filtration sterilization of supernatant.

(5) substantially estimate to prepare resulting phage titre by the absorbance value at measurement 260nm: well by above-mentioned purifying Phage library with PBS dilute 100 times.Phage library titre: bacteriophage number/mL is estimated according to empirical equation below =OD₂₆₀×100×22.14×10¹⁰.Obtained phage library titre is 5.3 × 10¹³PFU/mL.The bacteriophage prepared Library can save 2 weeks at 4 DEG C.

EpCAM binding protein is screened in 4 phagocytosis binding protein precursor library of embodiment

(1) addition 4mL EpCAM extracellular fragment (purchased from biotech firm of upper hypo Thailand) solution in pipe is immunized in NUNC and (uses PBS Buffer solution, the final concentration of 100 μ g/mL of EpCAM extracellular fragment), 4 DEG C of coatings are overnight.

(2) immune pipe is softly rinsed with PBS buffer solution three times, 4mL 2%BSA-PBS solution is added, and room temperature closes 2h.

(3) immune pipe is softly rinsed with PBS buffer solution three times, be added 4mL 2%BSA-PBS solution (wherein comprising 5 × 10¹²The bacteriophage that a embodiment 3 is prepared), it is incubated at room temperature 1h.

(4) immune Guan Shici is softly rinsed with PBST buffer, the trypsin solution that 4mL concentration is 1mg/mL is added, It is incubated at room temperature 1h, bacteriophage is thoroughly eluted.

(5) take three ride of TG1 bacterium in a TYE not with ampicillin from the glycerin storage object of TG1 bacterial strain On solid culture plate, 37 DEG C of incubator culture 14h.Picking TG1 monoclonal strain inoculated is in 5mL 2 × TY fluid nutrient medium, and 37 DEG C, 250rpm shaking table concussion be incubated overnight.Bacterium solution is seeded to another 2 × TY of 5mL Liquid Culture by 1:100 ratio by volume In base, 37 DEG C, 250rpm shaking table shake culture to bacterium solution OD₆₀₀=0.5.

(6) 30mL bacterium solution is taken to be added in step (4) obtained eluent, 37 DEG C of water-bath 1h, 3200g are centrifuged 5min, in abandoning Clearly, precipitating is resuspended with 2 × TY of 1mL fluid nutrient medium.

(7) 6 TYE plates (containing 100 μ g/mL ampicillins, 4% (w/v) glucose) is taken, each plate takes 166 μ L steps (6) coating of gained re-suspension liquid, the plate coated are placed in 37 DEG C of incubator overnight incubations.

(8) 2 × TY of 2mL fluid nutrient medium is added in every block of plate, is scraped bacterium on plate using spreading rod.

(9) 2 × TY of 500mL fluid nutrient medium is added in the bacterium scraped, and (containing 4% (w/v) glucose, 100 μ g/mL ammonia benzyls are green Mycin), 37 DEG C, 250rpm shake culture to bacterium solution OD₆₀₀=0.5, it is thin to add the resulting helper phage infection of embodiment 1 Bacterium, 30 DEG C, 250rpm shake culture stay overnight.

(10) the overnight culture of step (9) be added be equivalent to the 20%PEG-NaCl solution of 1/5 volume of supernatant into Row bacteriophage purifies (mixing of turning upside down, ice bath 4h), then measures phage titre by gradient dilution coated plate.

(11) repeat to combine, elute and infect and etc. (repeat step (1)~(10)) carry out the libraries of subsequent 4 wheel Screening, is successively screened from last round of obtained bacteriophage.

Picking monoclonal carries out ELISA verifying in 5 library of embodiment

(1) after carrying out 5 wheel library elutriations, sterile 96 orifice plate (being named as A plate) is taken, 200 μ L 2 × TY liquid are added in every hole Body culture medium (glucose containing 100 μ g/mL ampicillins, 4% (w/v)), is seeded to 96 with sterile pipette tip picking monoclonal Orifice plate, 37 DEG C, 250rpm shaking table shake culture stay overnight；.

(2) new sterile 96 orifice plate (being named as B plate) is taken, every hole is added 200 μ 2 × TY of L fluid nutrient mediums and (contains 100 μ g/ The glucose of the ampicillin of mL and 4% (w/v)), take the 5 μ L of bacterium solution of step (1) to be seeded to every hole, 37 DEG C, 250rpm shake Swing culture 3h.Glycerol is added in bacterium solution every hole in remaining 96 hole in A plate, and the final concentration of percent by volume 20% of glycerol is made sweet Oil storage object, -80 DEG C of refrigerators freeze.

(3) it includes 4 × 10 that 50 μ L are added into the every hole of B plate⁸2 × TY fluid nutrient medium of a KM13 helper phage is simultaneously light Plate is placed in 37 DEG C of incubation 1h by soft mixing.

(4) the incubation product in 96 orifice plates is gone into 1.5mL EP pipe, 3200g is centrifuged 10min, abandons supernatant, precipitates with 200 μ L 2 × TY fluid nutrient medium, which is resuspended, (contains 100 μ g/mL ampicillins, 50 μ g/mL kanamycins, 0.1% (w/v) grape Sugar), 25 DEG C, 250rpm shake culture stay overnight.

(5) overnight culture in step (4) is gone to new 1.5mL EP to manage, 3200g is centrifuged 10min, obtained supernatant Liquid is transferred to 96 new orifice plates, 4 DEG C of refrigerator storages.

(6) using 0.2 μ g EpCAM extracellular fragment antigen (be purchased from biotech firm of upper hypo Thailand), 96 hole elisa plates of coating, 250 μ L 2%BSA-PBS solution closes elisa plate.Blank control is BSA, and irrelevant antigen control is EGFR polypeptide.

(7) 96 new orifice plates are taken, takes the supernatant in 25 μ L steps (5) to prepare into 75 μ L 2%BSA-PBS solution and bites Mycelium dilution liquid reuses multiple tracks liquid-transfering gun and shifts bacteriophage dilution in the elisa plate into step (6) after closing, room temperature It is incubated for 1h.

(8) PBST board-washing 5 times, 100 μ L are added in every hole, and the diluted HRP of 1:10000 marks anti-M13 conjugate by volume (dilution of 2%BSA-PBS solution), room temperature is shaken slowly is incubated for 1h.

(9) PBST board-washing 5 times, every hole are added 100 μ L TMB (tetramethyl benzidine) chromophoric solutions, become to liquid in hole Indigo plant, every hole are added the 50 μ L 1M concentrated sulfuric acids and terminate reaction.

(10) light absorption value at 450nm is read using spectrophotometer.608 monoclonals of picking are tested, Fig. 1 is wherein 32 The ELISA result of a monoclonal phage.

(11) 5 positive colonies have been finally obtained, have sent Huada gene company to carry out DNA sequencing positive colony.? The DNA result of sequencing is analyzed in DNAMAN, excludes identical nucleotide sequence, 5 sequences are obtained, such as SEQ ID Shown in NO.1~5, it is respectively designated as NB85, NB143, NB278, NB431, NB589.

Embodiment 6 obtains negative control binding protein

The synthesis polypeptide of human epidermal growth factor receptor-2 (Her2) or vascular endothelial growth factor (VEGF) is respectively adopted As antigen, 2 are filtered out from phagocytosis binding protein precursor library not with the monoclonal of EpCAM extracellular fragment antigen binding as yin Property control binding protein.Screening technique step carries out ELISA verifying with embodiment 1 to embodiment 5, and by the monoclonal of picking.

Binding protein NB348 and NB475 in experimental example as negative control are respectively provided with such as SEQ ID NO:6 and SEQ Amino acid sequence shown in ID NO:7.The nucleotide sequence of NB348 and NB475 is encoded respectively such as SEQ ID NO:13 and SEQ ID Shown in NO:14.Wherein, NB348 is obtained by antigen selection of Her2, and NB475 is obtained by antigen selection of VEGF.

The preparation of embodiment 7DH5 α and BL21 (DE3) competent escherichia coli cell

(1) it takes and distinguishes three rides purifying bacillus coli DH 5 alpha and BL21 on two culture dishes containing LB solid medium (DE3), the culture dish for pulling line is placed in 37 DEG C of incubator culture about 14h.

(2) it is inoculated in respectively from the bacillus coli DH 5 alpha and BL21 (DE3) bacterium colony of picking one activation on LB plate respectively In 5mL LB liquid medium, 37 DEG C, 220rpm shake culture overnight (about 12h or so).

(3) ratio of bacteria suspension 1:100 by volume is seeded to respectively in 50mL LB liquid medium, 37 DEG C of vibrations Swing culture 2-3h to bacterium solution OD₆₀₀=0.5 or so.

(4) bacterium solution is transferred to new sterile 50mL centrifuge tube, places 10min on ice.

(5) 4 DEG C, 3000g centrifugation 5min, abandon supernatant.

(6) the 0.1mol/L CaCl being pre-chilled with 10mL₂Solution gently suspends precipitating, on ice static 30min.

(7) 4 DEG C, 3000g centrifugation 5min, abandon supernatant, the 0.1mol/L CaCl of 3mL pre-cooling are added₂Solution is soft to be resuspended Precipitating, completes the preparation of competent cell.

(8) the sterile glycerol aqueous solution of 30% (v/v) of 3mL pre-cooling is added in the competent cell that 3mL is prepared, gently It mixes, then mixed liquor is distributed into the 100 every pipes of μ L, be transferred quickly to -80 DEG C of refrigerator Long-term Cryopreservations.

8 binding protein construction of recombinant vector of embodiment

(1) prepare PCR reaction system: 5 × PrimerStar buffer, 10 μ L, dNTP mixture (every kind of 2.5mM) 4 μ L, NB-F primer (10 μM, 5 '-GATCCATGGCCCAGGTGCAGCTGT-3 ') 1 μ L, NB-R primer (10 μM, 5 '- TCTGCGGCCGCGCTCGAGAC-3 ') 1 μ L, 0.5 μ L of template 10ng, PrimerStarDNA polymerase, aseptic deionized water benefit Enough to 50 μ L.

(2) PCR reacts: 96 DEG C of 10min；95 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec, 30 circulations；72℃5min.

(3) PCR product is purified by PCR product purification kit.

(4) according to following scheme by pET-22b carrier (be purchased from EMD Biosciences (Novagen)) and PCR product into Row double enzyme digestion reaction, digestion system are as follows:

2 μ g of pET-22b vector plasmid, 5 μ L of enzyme cutting buffering liquid, 2 NotI μ L, 2 NcoI μ L, sterile water complement to 50 μ L.

1.4 μ g of PCR product, 6 μ L of enzyme cutting buffering liquid, 2 NotI μ L, 2 NcoI μ L, sterile water complement to 60 μ L.

(5) above-mentioned reaction system is placed in 37 DEG C of heat preservation 15min to get pET-22b double enzyme digestion product and target fragment pair Digestion products.

(6) digestion products are purified by digestion products purification kit.

(7) it is attached reaction, system is as follows: pET-22b double enzyme digestion product 0.03pmol, target fragment double enzyme digestion product 0.3pmol, enzyme connect 2.5 μ L of buffer, 1 μ L of T4DNA ligase, and sterile water complements to 25 μ L.

(8) 4 DEG C of connections overnight, obtain connection product.Connection product is transformed into bacillus coli DH 5 alpha competent cell, is applied It is distributed on the LB plate containing 100 μ g/mL ampicillins, primary dcreening operation is then carried out to bacterium colony by primer NB-F and NB-R, is obtained Positive colony expand culture.Plasmid is extracted, by endonuclease reaction secondary screening, the positive colony that secondary screening obtains is sequenced, and is obtained The recombinant expression plasmid that pET-22b carrier and binding protein recombinate.

The protein-bonded recombinant vector for expressing EpCAM is converted BL21 competent cell by embodiment 9

(1) competent escherichia coli cell that embodiment 7 is prepared is taken out from -80 DEG C of refrigerators, is thawed on ice.

(2) it takes sterile 1.5mL EP pipe to be pre-chilled on ice, 100 μ L competent cells and 10 μ L recombinant expression plasmid carriers is added (embodiment 8 is prepared), it is soft to mix, 30min is placed on ice.

(3) mixture of competence and connection product is kept the temperature into 100s in 42 DEG C of thermostat water baths, then transfer rapidly To cooled on ice 2-3min.

(4) the not antibiotic LB culture medium of 900 μ L, 37 DEG C, 200rpm shake culture 1h, 8000g centrifugation 1min is added Collect bacterial sediment.

(5) 900 μ L supernatants are absorbed, is resuspended and is precipitated with remaining 100 μ L culture solution, bacteria suspension is spread evenly across containing 100 μ On the LB solid medium of g/mL ampicillin and 1% (w/v) glucose.

(6) 37 DEG C of incubator culture 12-16h are to get the BL21 monoclonal bacterial strain containing binding protein expression plasmid.

10 binding protein prokaryotic expression of embodiment and purifying

(1) protein-bonded prokaryotic expression

(1) the BL21 monoclonal strain inoculated in picking embodiment 9 containing binding protein expression plasmid is to 5mL LB liquid (contain 100 μ g/mL ampicillins, 1% (w/v) glucose) in culture medium, 37 DEG C, 220rpm shake culture 12h.

(2) 1mL bacterium solution is taken to be seeded to 100mL LB liquid medium (containing 100 μ g/mL ampicillins), 37 DEG C, 220rpm is cultivated to bacterium solution OD₆₀₀=0.6-1.0.Take 1mL bacterium solution for subsequent destination protein PAGE gel electroresis appraisal (not inducing Escherichia coli holoprotein sample).

(3) IPTG, final concentration of 0.25mM of the IPTG in bacterium solution, 25 DEG C, 220rpm induction table are added in remaining bacterium solution Up to 6h, take the bacterium solution of 1mL for subsequent destination protein PAGE gel electroresis appraisal (the Escherichia coli holoprotein sample after induction Product).

(4) 4 DEG C of bacterium solution after remaining inducing expression, 5000g centrifugation 5min collect bacterial sediment, break bacterium with the 20mL of pre-cooling Bacterial precipitation is resuspended in buffer.

(5) it takes 50mL beaker to be placed in ice chest, bacterial suspension is transferred to beaker, ultrasonication bacterium.Setting ultrasound Pulverizer power is the 40% of 650W, and work 4s, stops 8s, working time 40min.

(6) it is crushed liquid and is transferred to centrifuge tube, 4 DEG C, 15000g centrifugation 30min take 20 μ L supernatants, then take partly precipitated (heavy Form sediment and break the resuspension of bacterium buffer with 20 μ L) it (is broken after induction bacterial cell disruption for subsequent destination protein PAGE gel electroresis appraisal Broken supernatant is crushed deposit sample), remaining supernatant is transferred in 50mL centrifuge tube, and 4 DEG C of preservations waited column purification.

(2) binding protein purifies

(1) Ni-NTA HisBind Resin Purification Resin is taken, balances purifying with the sample-loading buffer of 10 times of column volumes Column.

(2) supernatant loading is taken, 20 μ L is during which taken to cross column liquid for subsequent destination protein PAGE gel electroresis appraisal (mistake Column liquid).

(3) after sample all passes through purification column, column is washed using the sample-loading buffer of 10 column volumes, by purification column side wall On residual sample clean.

(4) foreign protein on purification column is washed away using the miscellaneous buffer of washing of 20 times of column volumes, 20 μ L is during which taken to wash miscellaneous liquid For subsequent destination protein PAGE gel electroresis appraisal (washing miscellaneous liquid), if it find that foreign protein appropriate can increase too much That washes miscellaneous liquid washes miscellaneous volume.

(5) destination protein is eluted using the elution buffer of 5 times of column volumes from purification column, is during which managed with 1.5mL EP Eluent is collected, every pipe collects eluent 1mL, collects 5 pipes altogether.Take 20 μ L for subsequent destination protein from every pipe eluent PAGE gel electroresis appraisal (eluent 1-5).

(6) column is washed with the 6M urea of 5 times of column volumes, then washes column with the distillation of 30 times of column volumes, 5mL is added later 20% ethyl alcohol, closing purifying pipe upper and lower ends, 4 DEG C of preservations.

(3) PAGE gel electrophoresis and coomassie brilliant blue staining

(1) separation gel that 5mL concentration is 12% (w/v) is prepared by formula, being added to encapsulating die, there are also 2- apart from top Then the moulding of 1.5mL dehydrated alcohol is added in the position of 3cm or so, it is solid to be stored at room temperature 30min gelling to be separated.

(2) dehydrated alcohol is completely removed, the concentration glue that 2mL concentration is 5% (w/v) is prepared by formula, is added to adhesive filling mould Tool is inserted into the matched comb of encapsulating die, is stored at room temperature 30min gelling to be concentrated admittedly, comb is pulled out, in preparation to top Sample.

(3) prepared offset plate is put into electrophoresis tank, appropriate electrophoresis liquid is added.

(4) 5 μ L 5 × SDS-PAGE albumen sample-loading buffers, 100 DEG C of heating are added in the protein sample for obtaining purifying 10min。

(5) PAGE gel hole is added in the sample for taking 5 μ L steps (4) to prepare, and 45V voltage is run through after concentration glue, Changing 110V voltage race glue to blue indicates band to glue bottom.

(6) it takes out PAGE gel and is put into dye glue box, appropriate coomassie brilliant blue staining liquid chamber temperature dyeing 30min is added.

(7) dyeing liquor is abandoned, glue is washed for several times with clear water, adds appropriate destainer and decolourize.

(8) decoloration extremely can clearly distinguish purpose band on SDS-PAGE glue, take pictures.As a result as shown in Fig. 2, 5 width SDS- PAGE electrophoretogram is it will be evident that in 15kD or so, and holoprotein is more darker than not inducing holoprotein purpose band after induction, Illustrate this 5 binding proteins by success inducing expression.Last 5 of every width SDS-PAGE electrophoresis is through Ni-NTA HisBind The destination protein eluent electrophoretic band that Resin purification column is collected after purification, it is seen that in the purpose item of 15kD or so It is deeper with single and color, illustrate to have washed off most of foreign protein when crossing column purification, our institutes are mainly contained in eluent The destination protein needed.

The binding protein of embodiment 11ELISA detection purifying and the binding ability of antigen

(1) 96 hole elisa Plates of NUNC are taken, every hole is coated with 100 μ L antigenic solutions respectively, and the solvent of antigenic solution is PBS, resists Former concentration is 2 μ g/mL, antigen (being purchased from biotech firm of upper hypo Thailand) be respectively MBP, VEGF, FGF21, HER2, BMP2, EndoF1, SPB2, EGFR, synthetic antigen segment EpCAM, while 2%BSA-PBS buffer is coated with as blank control, it will wrap 37 DEG C are placed in by good ELISA Plate, stands 2h.

(2) ELISA Plate is taken out, three times with PBS board-washing, excess liq is removed on blotting paper by overturning in plate, and every hole adds Enter 200 μ L 2% (w/v) BSA confining liquids, 37 DEG C of closing 2h.

(3) ELISA Plate is taken out, three times with PBS buffer solution board-washing, excess liq is removed on blotting paper by overturning in plate, 100 μ L binding protein-PBS mixed liquors (binding protein and PBS volume ratio be 1:50) is added in every hole, and ELISA Plate is placed in room temperature and is incubated Educate 60min.Binding protein is respectively NB85 binding protein, NB143 binding protein, NB278 binding protein, NB431 binding protein With NB589 binding protein.

(4) ELISA Plate is taken out, three times with PBST board-washing, 2% (w/v) BSA that 100 μ L contain anti-His-HRP is added in every hole (anti-His-HRP antibody and 2%BSA are diluted with volume ratio 1:5000) is incubated at room temperature 60min.

(5) ELISA Plate is taken out, with PBST board-washing five times, 100 μ L tmb substrate developing solutions are added in every hole, and room temperature is protected from light incubation Light absorption value OD is detected at 10min, microplate reader 450nm, and records data.As a result as shown in figure 3,5 binding proteins can be with EpCAM specific binding, wherein the binding ability of NB143 is most strong.

The binding protein of experimental example 12MTT method detection after purification is to Human Prostate Cancer Cells DU145 and PC-3, human breast carcinoma The influence of the proliferative capacity of cell MCF-7

In (1) 96 porocyte culture plates, every hole inoculation is in 5000, logarithmic growth phase cell, and 100 μ L are added and cultivate entirely Base (the DMEM culture medium of 10% calf serum containing percent by volume, similarly hereinafter), 37 DEG C, 5%CO₂Incubator overnight incubation.

(2) after cell is adherent, every Kong Zhongquan culture medium is removed, it is hungry that DMEM culture medium of the 100 μ L containing 1% serum is added Starve processing 4h.

(3) the DMEM culture medium containing 1% serum is removed.Using 7 binding proteins (NB85, NB143, NB278, NB431, NB589, NB348 and NB475, last 2 are negative control binding protein), binding protein concentration it is different (0,25, 50,100 μ g/mL) 1% serum DMEM culture medium (100 hole μ L/) in culture DU145, MCF-7 and PC-3 (37 DEG C, 5% CO₂72h is cultivated in incubator).

(4) culture medium in tissue culture plate is removed, the DMEM culture medium and 20 μ L MTT of 100 μ L serum-frees of every hole addition are molten Liquid is placed in cell incubator and is incubated for 4h.

(5) culture solution is removed, every hole is added 150 μ L DMSO, tissue culture plate is placed in shaking table and quickly shakes 10min, is made Precipitating sufficiently dissolution.

(6) the light absorption value OD in each hole is measured at microplate reader 570nm.As a result as shown in figure 4,5 EpCAM after purification Binding protein (NB85, NB143, NB278, NB431, NB589) is able to suppress the proliferation of DU145, MCF-7 and PC-3 cell.

The binding protein of 13 cell scarification of experimental example detection after purification is to DU145, MCF-7 and PC-3 cell migration ability Influence

(1) in 12 porocyte culture plates, every hole inoculation is in logarithmic growth phase tumour cell 2 × 10⁵It is a, 500 μ are added The full culture medium of L, 37 DEG C, 5%CO₂Incubator overnight incubation.

(2) after cell is adherent, the full culture medium in tissue culture plate is removed, 500 μ L are added containing 1% serum in every hole DMEM culture medium Nature enemy 4h.

(3) crossed using 200 μ L pipette tips to every hole cell, cleaned 2 times with PBS buffer solution later, will draw fall it is thin Born of the same parents clean, and take pictures and measure the length (L1) of scratch under the microscope.

(4) using 7 binding proteins (NB85, NB143, NB278, NB431, NB589, NB348 and NB475, last 2 For negative control binding protein) binding protein concentration different (0,25,50,100 μ g/mL) 1% serum DMEM culture medium (500 hole μ L/) middle culture tumour cell DU145, MCF-7 and PC-3 (37 DEG C, 5%CO₂It is cultivated for 24 hours in incubator).

(5) length (L2) of scratch is taken pictures and measured after cultivating, and with formula: (L1 ﹣ L2)/L1 × 100% calculating is moved Shifting rate.

As a result as it can be seen in figures 5 and 6, as 5 binding protein concentration increase, the migration of DU145, MCF-7 and PC-3 cell Rate reduces, it was demonstrated that the binding protein of EpCAM after purification is able to suppress the migration of DU145, MCF-7 and PC-3 cell.

Experimental example 14Transwell Matrigel detects binding protein to DU145, MCF-7 and PC-3 cell invasion ability Influence

(1) cell Transwell (8 μm of apertures, 24 orifice plates) are put into 24 porocyte culture plates, small chamber internal surface coating Matrigel matrigel (50 hole μ g/), be placed in a few hours in cell incubator make Matrigel matrigel solidify.

(2) tumour cell of logarithmic growth phase, the Nature enemy 4h in the DMEM culture medium containing 1% serum later will Tumour cell is digested and is collected.

The upper interior of each cell Transwell is separately added into containing 5 × 10 in (3) 24 orifice plates⁴A cell with it is difference dense Degree (0,25,50 and 100 μ g/mL) binding protein (NB85, NB143, NB278, NB431, NB589, NB348 and NB475, last 2 A is negative control binding protein) 1% serum 200 μ L of culture medium, room is added containing 20% (w/v) FBS under every hole DMEM culture medium, 37 DEG C, 5%CO₂Incubator culture is for 24 hours.

(4) cell that do not invade on small interior Matrigel matrigel is wiped with cotton swab, room is added under the every hole of 24 orifice plates 4% (w/v) poly methanol fixes the small ventricular cell 10min of Transwell.

(5) fixer is abandoned, 500 μ L 0.1% (w/v) crystal violet dye liquors are added in lower room, it is small that room temperature dyes Transwell Ventricular cell 30min, distilled water clean cell 3 times, then cell is just being placed on glass slide and is being photographed to record under microscope.

(6) 24 porocyte culture plates are put back into the cell Transwell, 33% (w/v) acetic acid solution, 100 μ L is added in lower room will Crystal violet on cell elutes, eluent in microplate reader at 570nm measure light absorption value OD.As a result as shown in FIG. 7 and 8, Fig. 7 is DU145, MCF-7 and PC-3 tumour cell Transwell invasion figure respectively, it can be seen that invasion cell ratio is without knot in figure Hop protein control is reduced.Fig. 8 is that the crystal violet of DU145, MCF-7 and PC-3 tumour cell after binding protein is invaded is washed respectively The OD value of de- liquid.The results show that the invasion rate of DU145, MCF-7 and PC-3 cell drops as 5 binding protein concentration increase Low, the binding protein of EpCAM after purification is able to suppress the invasion of DU145, MCF-7 and PC-3 cell.

Influence of the experimental example 15Transwell migration experiment detection binding protein to tumor cell migration ability

Method and step is similar with experimental example 13, only omits wherein step (1).As a result as shown in Figures 9 and 10, Fig. 9 is respectively DU145, MCF-7 and PC-3 tumour cell Transwell transition graph, it can be seen that migrating cell ratio is compareed without binding protein in figure It is reduced.Figure 10 is the OD value of the crystal violet eluent of DU145, MCF-7 and PC-3 tumour cell after migrating respectively.As a result it demonstrate,proves Bright, as 5 binding protein concentration increase, the mobility of DU145, MCF-7 and PC-3 cell is reduced, EpCAM after purification Binding protein be able to suppress the migration of DU145, MCF-7 and PC-3 cell.

Effect of 16 binding protein of experimental example to DU145, MCF-7 and PC-3 apoptosis of tumor cells

(1) every hole inoculation is in logarithmic growth phase tumour cell 5 × 10 in 6 porocyte culture plates⁶It is a, it is complete that 1mL is added Culture medium, 37 DEG C, 5%CO₂Incubator overnight incubation.

(2) after cell is adherent, the full culture medium in tissue culture plate is removed, the DMEM that 1mL contains 1% serum is added in every hole Culture medium Nature enemy 4h.

(3) remove tissue culture plate in culture medium, every hole be separately added into containing 50 μ g/mL binding proteins (NB85, NB143, NB278, NB431, NB589, NB348 and NB475, last 2 are negative control) 1% blood serum medium 1mL, while every kind Tumour cell is respectively arranged one without binding protein control group, 37 DEG C, 5%CO₂Incubator culture 48h.

(4) tumour cell is digested, the cell digested is transferred to 1.5mL EP pipe, cleans cell 2 with PBS buffer solution It is secondary.

(5) ddH is used₂4 × combination buffer is diluted to 1 by O ×, take 195 μ L1 × combination buffer to adjust cell density It is 5 × 10⁶A cell/mL.

(6) take 5 μ L fluorescein isothiocynates (Annexin V-FITC) that step (5) are added, room temperature, which is protected from light, is incubated for 10- 15min。

(7) it takes 200 μ 1 × combination buffers of L to be added in step (6) and cleans cell.

(8) 1000g is centrifuged 5min, abandons supernatant, takes 190 μ 1 × combination buffers of L that cell is resuspended, adds 10 μ L iodate Third ingot (PI) dyeing liquor is soft to mix.

(9) apoptosis of tumor cells situation is detected using stream type cell analyzer.As a result as shown in FIG. 11 and 12, Figure 11 difference It is DU145, MCF-7 and PC-3 tumour cell two dimension scatter plot of apoptosis, it can be seen that apoptotic cell ratio is without binding protein in figure Control, which has, increases (right one side of something).Figure 12 is the apoptosis ratio obtained from figure A, C, E respectively.The results show that after purification 5 EpCAM binding protein can promote the apoptosis of A549, MCF-7 and DU145 cell.

Effect of 17 binding protein of embodiment to Prostate Carcinoma of Mice model

(1) according to the external contractile studies of the binding protein of EpCAM, final choice constructs mouse DU145 cell forefront Adenocarcinoma models, and choose external contractile studies best binding protein NB143 and NB431 and carry out zoopery.

(2) secondary culture is carried out to DU145 cell using 10cm Tissue Culture Dish, when cell grows into logarithmic phase, pancreatin Cell is digested and collected, centrifuge 1050g centrifugation 5min is placed in.

(3) supernatant is abandoned, the cell precipitation being collected into is resuspended with PBS, is placed in centrifuge 1050g centrifugation 5min.

(4) supernatant after being centrifuged is abandoned, cell precipitation is resuspended with PBS, and a small amount of cell suspension is taken to be counted by cell counting board Number, remaining suspension are placed in centrifuge 1050g centrifugation 5min.

(5) supernatant after being centrifuged is abandoned, appropriate PBS is added, cell precipitation is resuspended, cell density is adjusted to 5 × 10⁷/mL。

(6) 100 μ L (5 × 10 are drawn using 1mL asepsis injector⁶A DU145 cell) cell suspension in step (5), Suspension is injected in mouse with injected s.c. (4 week old male Balb/C nude mices are purchased from Guangdong medical experiment animal center) Oxter carries out making tumor, and mouse is placed in SPF grades of Animal Lab.s later and is routinely raised.

(7) mouse tumor volume of vernier caliper measurement, gross tumor volume calculation formula: a × b were used every three days²× 0.5。

(8) when 30 mouse tumor volumes rise to average 100mm³It, is divided into 6 groups by left and right at random, and every group 5, Wherein 1 group be blank control group (PBS), 1 group be positive controls (DDP), 2 groups for experiment binding protein group (NB143 and NB431), 2 groups are negative control binding protein group (NB348 and NB475).

(9) it is administered using insulin syringe by tail vein, wherein experiment binding protein group and negative control combination egg White group of every mouse dosage is 10mg/kg, and every mouse dosage of positive controls is 2mg/kg, blank control group every Mouse injects 100 μ L cell PBS buffer solution, and dosage period is 27 days, and administration frequency is 3 days/time, while recording each group mouse Gross tumor volume.

(10) mouse is put to death after completing experiment, strips each group mouse tumor, is measured tumor weight and is taken pictures.As a result such as Figure 13 Shown, the increase that binding protein group gross tumor volume is tested after administration is obviously put compared with blank control group and negative control binding protein group Slow, final volume also may be significantly smaller, it was demonstrated that NB143 and NB431 binding protein after purification is able to suppress the mouse of DU145 induction The tumour of cell model of human prostate carcinoma.

The configuration method of used reagent is as follows in embodiment:

(1) TYE solid medium: agar powder 15g, NaCl 8g, tryptone 10g, yeast extract 5g.

Reagent is dissolved in 800mL deionized water, 121 DEG C of high pressure steam sterilization 30min.Wait for that culture medium is cooling when use To about 60 DEG C or so, it is added 800 μ L ampicillin solution and sterile 20% glucose solution of 40mL, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after mixing, 4 DEG C It saves.

(2) 2 × TY fluid nutrient mediums: NaCl 5g, tryptone 16g, yeast extract 10g.

Reagent is dissolved in 1L deionized water, 121 DEG C of high pressure steam sterilization 20min, room temperature preservation.

(3) 50 × TAE DNA electrophoretic buffers: Tris- alkali 242g, Na₂EDTA·2H₂O 37.2g, acetic acid 57.1mL.

Reagent is dissolved in 800mL deionized water, then is settled to 1L, when use, is diluted to 1 × TAE electrophoretic buffer, Room temperature preservation.

(4) PBS buffer solution (PH=7.4): KH₂PO₄ 0.24g、NaCl 8g、KCl 0.2g、Na₂HPO₄·12H₂O 9.07g。

Reagent is dissolved in 800mL deionized water, adjusting pH value to 7.4, then is settled to 1L, 121 DEG C of high steams go out Bacterium 20min, room temperature preservation.

(5) PBST solution: 1mL Tween-20 is added in 1L PBS buffer solution, it is molten that PBST can be configured to after mixing well Liquid.

(6) 20%PEG-NaCl solution: PEG6000 100g, NaCl 73g.

Reagent is dissolved in 500mL deionized water, after 0.2 μM of filter bacterium, 4 DEG C of preservations.

(7) LB liquid medium: NaCl 2g, tryptone 2g, yeast extract 1g.

Reagent is dissolved in 200mL deionized water, 121 DEG C of high pressure steam sterilization 20min, 4 DEG C of preservations.

(8) LB solid medium: NaCl 2g, tryptone 2g, yeast extract 1g, agar powder 4g.

Reagent is dissolved in 200mL deionized water, 121 DEG C of high pressure steam sterilization 30min.Wait for that culture medium is cooling when use To about 60 DEG C, ampicillin and glucose, the final concentration of 100 μ g/mL of ampicillin, the final concentration of glucose is added It is 1%, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after mixing, 4 DEG C of preservations.

(9) 5 × SDS-PAGE electrophoretic buffers: glycine 47g, Tris- alkali 15.1g, SDS 2.5g.

Reagent is dissolved in 400mL deionized water, then is settled to 500mL, when use is diluted to 1 × SDS-PAGE electrophoresis Buffer, room temperature preservation.

(10) 5 × SDS-PAGE sample-loading buffers: 1M Tris-HCl (pH 6.8) 1.25mL, glycerol 2.5mL, bromophenol blue 25mg、SDS 0.5g。

Reagent is settled to 5mL, using preceding every aliquot is added in 25 μ L beta -mercaptoethanols by aliquot (500 L/ parts of μ) packing In, room temperature preservation.

(11) coomassie brilliant blue R_250 dyeing liquor: coomassie brilliant blue R_250 1g, isopropanol 250mL, acetic acid 100mL, ddH₂O 650mL。

It is sufficiently dissolved after reagent is mixed, room temperature preservation.

(12) coomassie brilliant blue staining destainer: acetic acid 100mL, ethyl alcohol 50mL, ddH₂O 850mL。

It is used after solvent is mixed, room temperature preservation.

(13) bacterium buffer: Tris alkali 2.42g, NaCl 14.6g is broken.

It sufficiently dissolves, is added appropriate 100 × PMSF liquid storage (working concentration is 1 ×) using preceding, -4 after first reagent is mixed DEG C save.

Sample-loading buffer: with broken bacterium buffer.

It washes miscellaneous buffer: measuring 9.9mL sample-loading buffer, 100 μ L of 2M imidazoles is added, mixes well, it is ready-to-use.

Elution buffer: measuring 9mL sample-loading buffer, and 2M imidazoles 1mL is added, mixes well, ready-to-use.

(14) kanamycins solution (50mg/mL): weighing 1g kanamycins powder and be dissolved in 20mL deionized water, uses 0.2 μM of filter filtration sterilization, then it is distributed into the every pipe of 1mL, -20 DEG C of preservations.

(15) it ampicillin solution (100mg/mL): weighs 1g ampicillin powder and is completely dissolved in 10mL deionization In water, using 0.2 μM of filter filtration sterilization, it is distributed into the every pipe of 1mL, -20 DEG C of preservations.

(16) 2%BSA-PBS solution: weighing 1g bovine serum albumin(BSA) powder and be dissolved in 50mL PBS buffer solution, uses 0.2 μM of filter filtration sterilization, ready-to-use or -20 DEG C of preservations.

(17) trypsin solution (1mg/mL): weighing 10mg trypsase powder and be dissolved in 10mL PBS buffer solution ,- 20 DEG C of preservations.

(18) 20% glucose solutions: it weighs 200g glucose powder and is dissolved in 1L deionized water, use 0.2 μM of filter Filtration sterilization, -4 DEG C of preservations.

(19) it 1M sulfuric acid solution: measures the 9.8mL concentrated sulfuric acid and is slowly added in 187mL deionized water, mix well, room temperature is protected It deposits.

(20) 10% ammonium persulfates (APS): it weighs 0.1g ammonium persulfate powder and is dissolved in 1mL deionized water, be distributed into The 200 every pipes of μ L, -20 DEG C of preservations.

(21) it IPTG solution (500mM): weighs IPTG powder 11.915g and is dissolved in 100mL deionized water, use 0.2 μ M filter filtration sterilization is distributed into the every pipe of 1mL, -20 DEG C of preservations.

(22) 30% glycerine water solutions: it measures 15mL glycerol and is added in 35mL deionized water, mix well, use 0.22 μM Filter filtration sterilization, -4 DEG C of preservations.

(23) 100 × PMSF liquid storages: it weighs 1.74g PMSF powder and is dissolved in 100mL isopropanol, -20 DEG C of preservations.

(24) it 2M imidazoles: weighs 1.14g imidazoles powder and is dissolved in the broken bacterium buffer of 10mL, -4 DEG C of preservations.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications done without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Sequence table

<110>Ji'nan University

<120>binding protein of tumor stem cell marker molecule EpCAM and its application

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 124

<212> PRT

<213>people (Homo sapiens)

<400> 1

Met Ala Gly Val Gly Leu Leu Gly Ser Gly Gly Gly Leu Val Gly Pro

1 5 10 15

Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Ala Ala Val Ser

20 25 30

Thr Leu Pro Met Ala Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly

35 40 45

Thr Val Ser Ser Ile Ala Gly Ala Ala Gly Ser Thr Thr Thr Ala Ala

50 55 60

Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr

65 70 75 80

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr

85 90 95

Thr Cys Ala Ala Leu Gly Leu Ala His Thr Gly Leu Pro Thr Thr Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120

<210> 2

<211> 127

<212> PRT

<213>people (Homo sapiens)

<400> 2

Met Ala Gly Val Gly Leu Leu Gly Ser Gly Gly Gly Leu Val Gly Pro

1 5 10 15

Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Val Leu Pro Ser

20 25 30

Ala His Ala Met Thr Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly

35 40 45

Thr Val Ser Ala Ile Ala Ser Gly Gly Gly Ser Thr Thr Thr Ala Ala

50 55 60

Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr

65 70 75 80

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr

85 90 95

Thr Cys Ala Ala Met Gly Ala Gly Ala Ala Val Pro His His Met Ala

100 105 110

Pro Thr Gly Gly Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<210> 3

<211> 125

<212> PRT

<213>people (Homo sapiens)

<400> 3

Met Ala Gly Val Gly Leu Leu Gly Ser Gly Gly Gly Leu Val Gly Pro

1 5 10 15

Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Val Leu Ile Ile

20 25 30

Pro Leu Ser Met Ala Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly

35 40 45

Thr Val Ser Thr Ile Gly Thr Ala Ala Gly Ser Thr Thr Thr Ala Ala

50 55 60

Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr

65 70 75 80

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr

85 90 95

Thr Cys Ala Ser Val Ser Ala Ser Ala Thr Ala Ser Leu Leu Ser Thr

100 105 110

Gly Gly Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<210> 4

<211> 124

<212> PRT

<213>people (Homo sapiens)

<400> 4

Met Ala Gly Val Gly Leu Leu Gly Ser Gly Gly Gly Leu Val Gly Pro

1 5 10 15

Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Pro Thr Pro Ser

20 25 30

Thr Ala Ala Met Ala Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly

35 40 45

Thr Val Ser Ala Ile Gly Gly Leu Ala Gly Ser Thr Thr Thr Ala Ala

50 55 60

Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr

65 70 75 80

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr

85 90 95

Thr Cys Ala Ala Ala Leu Gly Ala Thr Gly Leu Leu Ser Thr Thr Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120

<210> 5

<211> 127

<212> PRT

<213>people (Homo sapiens)

<400> 5

Met Ala Gly Val Gly Leu Leu Gly Ser Gly Gly Gly Leu Val Gly Pro

1 5 10 15

Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Val Ala Pro Ser

20 25 30

Ala Gly Val Met Ala Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly

35 40 45

Thr Val Ser Gly Ile Gly Met Leu Ala Gly Ser Thr Thr Thr Ala Ala

50 55 60

Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr

65 70 75 80

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr

85 90 95

Thr Cys Ala Ser Thr Gly His Ser Ala Ala Gly Gly Ser Leu Pro Ala

100 105 110

Ser Thr Gly Gly Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<210> 6

<211> 129

<212> PRT

<213>people (Homo sapiens)

<400> 6

Met Ala Gly Val Gly Leu Leu Gly Ser Gly Gly Gly Leu Val Gly Pro

1 5 10 15

Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Thr Ser Val Ser

20 25 30

Ser Gly Ala Met Gly Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly

35 40 45

Thr Val Ser Gly Ile Leu Ala Gly Ala Gly Ser Thr Thr Thr Ala Ala

50 55 60

Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr

65 70 75 80

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr

85 90 95

Thr Cys Ala Ala Pro Thr Ser Gly Gly Gly Ser Leu Ala Ser Ala Pro

100 105 110

Ile Ala Ser Thr Gly Gly Gly Thr Leu Val Thr Val Ser Ser Ala Ala

115 120 125

Ala

<210> 7

<211> 128

<212> PRT

<213>people (Homo sapiens)

<400> 7

Met Ala Gly Val Gly Leu Leu Gly Ser Gly Gly Gly Leu Val Gly Pro

1 5 10 15

Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Val Ser Val Ser

20 25 30

Ala Gly Ala Met Gly Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly

35 40 45

Thr Val Ser Ser Ile Thr Ala Gly Ser Gly Ser Thr Thr Thr Ala Ala

50 55 60

Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser Leu Ala Thr

65 70 75 80

Leu Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr

85 90 95

Thr Cys Ala Ala Gly Gly Ala Ala Ala Gly Met His Ser Thr Leu Val

100 105 110

Ser Ser Thr Gly Gly Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<210> 8

<211> 372

<212> DNA

<213>people (Homo sapiens)

<400> 8

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagatagg gttagctata agtttatggc ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaagcattc ggggcaatga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

aagggtaaga ggcactacga gctgccgtat tggggtcagg gaaccctggt caccgtctcg 360

agcgcggccg ca 372

<210> 9

<211> 381

<212> DNA

<213>people (Homo sapiens)

<400> 9

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagttaag tttagcaatc acgatatgac ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcagccatta atagcggagg cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

atgggtcgtc aggcgcgtgt tccgcaccac atgcggtttt ggggtcaggg aaccctggtc 360

accgtctcga gcgcggccgc a 381

<210> 10

<211> 375

<212> DNA

<213>people (Homo sapiens)

<400> 10

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagttaag attatcccta agtctatggc ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaaccattg agacggcaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgagt 300

gtttcgcgta gtcggaccaa ctcgctcaag tcttggggtc agggaaccct ggtcaccgtc 360

tcgagcgcgg ccgca 375

<210> 11

<211> 372

<212> DNA

<213>people (Homo sapiens)

<400> 11

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatttacg tttagctata acaatatggc ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcagccattg aggggaaaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

cgtaaggggc gtacggagaa gctgtcgtat tggggtcagg gaaccctggt caccgtctcg 360

agcgcggccg ca 372

<210> 12

<211> 381

<212> DNA

<213>people (Homo sapiens)

<400> 12

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagtgagg ttcagcaacg aggtgatggc ctgggtccgc 120

caggctccag ggaagggtct agagtgggta ggcatcgaga agatgaacga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcaagt 300

tggggccaca gcaggaggca gcagagcaag ttcaacagct ggggtcaggg aaccctggtc 360

accgtctcga gcgcggccgc a 381

<210> 13

<211> 387

<212> DNA

<213>people (Homo sapiens)

<400> 13

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatatagc gttagctctg agaatatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaggcattt tggcgggaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

tttacgtcgg gtcaggggtc gttgcggtcc gaccccatcc ggtcttgggg tcagggaacc 360

ctggtcaccg tctcgagcgc ggccgca 387

<210> 14

<211> 384

<212> DNA

<213>people (Homo sapiens)

<400> 14

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagttagc gttagcaatg aggctatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaagcatta ctgaccaaag cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

gggcagcgtc gtaggcagat gcattcgtac aaggtcagct cttggggtca gggaaccctg 360

gtcaccgtct cgagcgcggc cgca 384

<210> 15

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gatccatggc ccaggtgcag ctgt 24

<210> 16

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gatccatggc ccaggtgcag ctgt 24

Claims

1. a kind of binding protein of tumor stem cell marker molecule EpCAM, it is characterised in that: the tumor stem cell mark point The binding protein of sub- EpCAM is entitled NB85 binding protein, NB143 binding protein, NB278 binding protein, NB431 combination egg The binding protein that one of white and NB589 binding protein or at least two combinations are formed；

The protein-bonded amino acid sequence of the NB589 is as shown in SEQ ID NO:5.

2. encoding the protein-bonded nucleotide sequence of tumor stem cell marker molecule EpCAM described in claim 1, feature Be: the nucleotide sequence is to encode NB143 described in the protein-bonded nucleotide sequence of NB85, coding to combine The protein-bonded nucleotide sequence of NB278 described in the nucleotide sequence of albumen, coding, NB431 binding protein described in coding Nucleotide sequence, the protein-bonded nucleotide sequence of NB589 described in coding and the coding protein-bonded core of NB589 The nucleotide sequence that one of nucleotide sequence or at least two combinations are formed.

3. the protein-bonded nucleotide sequence of codes for tumor stem-cell marker molecule EpCAM according to claim 2, It is characterized in that: encoding the protein-bonded nucleotide sequence of NB85 as shown in SEQ ID NO:8.

4. the protein-bonded nucleotide sequence of codes for tumor stem-cell marker molecule EpCAM according to claim 2, It is characterized in that: encoding the protein-bonded nucleotide sequence of NB143 as shown in SEQ ID NO:9.

5. the protein-bonded nucleotide sequence of codes for tumor stem-cell marker molecule EpCAM according to claim 2, It is characterized in that: encoding the protein-bonded nucleotide sequence of NB278 as shown in SEQ ID NO:10.

6. the protein-bonded nucleotide sequence of codes for tumor stem-cell marker molecule EpCAM according to claim 2, It is characterized in that: encoding the protein-bonded nucleotide sequence of NB431 as shown in SEQ ID NO:11.

7. the protein-bonded nucleotide sequence of codes for tumor stem-cell marker molecule EpCAM according to claim 2, It is characterized in that: encoding the protein-bonded nucleotide sequence of NB589 as shown in SEQ ID NO:12.

8. the protein-bonded preparation method of tumor stem cell marker molecule EpCAM described in claim 1, it is characterised in that packet It includes following steps: the protein-bonded of tumor stem cell marker molecule EpCAM described in claim 1 is encoded by gene chemical synthesis Nucleotide sequence, is cloned into expression vector, then the expression vector after recombination is transferred to host cell and expressed, purify, Obtain the binding protein of the tumor stem cell marker molecule EpCAM；Or by albumen synthetic method, obtain described The binding protein of tumor stem cell marker molecule EpCAM.

9. tumor stem cell marker molecule EpCAM binding protein application in preparation of anti-tumor drugs described in claim 1.

10. tumor stem cell marker molecule EpCAM binding protein according to claim 9 is in the preparation of antitumor drugs Be that EpCAM high expresses tumour using, it is characterised in that: the tumour, including but not limited to cancer of pancreas, breast cancer, bladder cancer, The cancer of the esophagus, gastric cancer, colon cancer, prostate cancer, lung cancer and ovarioncus.