CN110407924B

CN110407924B - Binding proteins targeting CD133 and uses thereof

Info

Publication number: CN110407924B
Application number: CN201910672222.0A
Authority: CN
Inventors: 魏星; 陈柔
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2019-07-24
Filing date: 2019-07-24
Publication date: 2021-12-21
Anticipated expiration: 2039-07-24
Also published as: CN113912696B; CN113912695A; CN113912694B; CN113912697B; CN113912696A; CN113912695B; CN113912694A; CN110407924A; CN113912697A

Abstract

The invention discloses a binding protein targeting CD133 and application thereof. The invention screens proteins combined with CD133 extracellular domain by utilizing phage display technology from a human protein library, and finally screens 7 combined proteins; the 7 binding protein genes are amplified, sequenced and cloned to an expression vector for expression and purification of a binding protein prokaryotic system, and then ELISA detection, MTT, apoptosis and Transwell invasion experiments are carried out, and the results show that the 7 binding proteins have strong binding capacity with antigens, can effectively inhibit the proliferation and invasion of tumor cells and can induce the apoptosis of the tumor cells. The protein provided by the invention is a fully humanized binding protein, does not generate immunogenicity when applied to a human body, and has the effect of inhibiting tumor cells in vitro. The 7 binding proteins can lay the foundation for the later development of tumor protein drugs.

Description

Binding proteins targeting CD133 and uses thereof

Technical Field

The invention relates to the field of binding protein, in particular to binding protein targeting CD133 and application thereof.

Background

The characteristics of immobility, migration and loss of contact inhibition of tumor cells make the tumor one of the diseases which are extremely difficult to cure for human. At present, the tumor treatment means mainly comprises surgical resection, radiotherapy or chemotherapy, but the existence of a group of tumor stem cells with similar stem cell characteristics in the tumor leads to extremely high tumor recurrence rate after treatment. In 2006, the american cancer research institute defined tumor stem cells as: cells in tumors that have the ability to self-renew and can produce heterogeneous tumor cells. The cell can be in a dormant state in vivo for a long time, has various drug-resistant molecules and is insensitive to external physicochemical factors for killing tumor cells, so that the tumor recurs again after most common tumor cells are killed by a conventional tumor treatment method. Based on the characteristics of the tumor stem cells, the protein targeting the specific marker molecules on the surface of the tumor stem cells is developed through a genetic engineering technology and is likely to become new eosin for treating cancers.

As one of the surface-specific marker proteins for stem cells and tumor stem cells, CD133 was first isolated from CD34 hematopoietic stem cells by artificial AC133 monoclonal antibody, such as Yin. CDl33 belongs to one of the Prominin family members, is located on human chromosome 4, is approximately 152kb in size, and contains at least 37 exons. The CD133 protein consists of 865 amino acids, has a molecular weight of about 120kDa, and comprises: extracellular NH₂-terminal, 5-transmembrane domain, 2 extracellular loops, 2 cysteine-rich intracellular loops, intracellular-COOH structure. CD133 has been shown to be a surface marker of hematopoietic stem cells and neural stem cells; CD133 is expressed in tumor stem cells of tumors such as brain glioma, colon carcinoma, malignant melanoma, etc., and CD133 is expressed in brain tumor, breast carcinoma and colon carcinoma⁺Cell ratio CD133^-The cell has strong tumorigenicity; through gene chip and clinical analysis, CDl33 was found⁺Tumor cells exhibit greater proliferative capacity and poorer prognosis. The results of the above studies all show that the expression of CDl33 in tumor stem cells has important significance for the development of cancer. Therefore, CD133 can be used as a new target for developing antitumor drugs.

Compared with the traditional monoclonal antibody, the binding protein consisting of only one heavy chain variable region has the advantages of small molecular weight, easy blood brain barrier crossing, low immunogenicity, strong specificity and the like. Relevant research results show that the binding protein can effectively target antigens and has anti-tumor effect in vivo and in vitro. At present, no study has been made on a protein binding to the tumor stem cell marker protein CD 133.

Disclosure of Invention

The invention mainly aims to overcome the defects of the prior art and provide a binding protein of a targeting tumor stem cell marker molecule CD 133.

Another object of the present invention is to provide a method for preparing the above binding protein targeting CD 133.

It is still another object of the present invention to provide the use of the above binding protein targeting CD 133.

The purpose of the invention is realized by the following technical scheme:

a binding protein targeting CD133 is a binding protein formed by one or the combination of at least two of CD133-2B1 protein, CD133-2C4 protein, CD133-2C10 protein, CD133-3B4 protein, CD133-3B12 protein, CD133-4B9 protein and CD133-4B12 protein; the binding protein targeting CD133 consists of 1 heavy chain variable region;

the amino acid sequence of the CD133-2B1 protein is as follows (also shown in SEQ ID NO. 1):

MAQVQLLESGGGLVQPGGSLRLSCAASGYKITAEFMGWVRQAPGKGLEWVSTISRHSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASVGKFPWVWVSEATVNYWGQGTLVTVSSAAA；

the amino acid sequence of the CD133-2C4 protein is as follows (also shown in SEQ ID NO. 2):

MAQVQLLESGGGLVQPGGSLRLSCAASGFKFISEYMGWVRQAPGKGLEWVSSITNADGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAVYVFPFGLADEVRYWGQGTLVTVSSAAA；

the amino acid sequence of the CD133-2C10 protein is as follows (also shown in SEQ ID NO. 3):

MAQVQLLESGGGLVQPGGSLRLSCAASGDSISPESMSWVRQAPGKGLEWVSTIDGPNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARVRSAVLGLRLSANVSYWGQGTLVTVSSAAA；

the amino acid sequence of the CD133-3B4 protein is as follows (also shown in SEQ ID NO. 4):

MAQVQLLESGGGLVQPGGSLRLSCAASGYMLINQDMTWVRQAPGKGLEWVSGILDKDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVSKWSKDAMSFWGQGTLVTVSSAAA；

the amino acid sequence of the CD133-3B12 protein is as follows (also shown as SEQ ID NO. 5):

MAQVQLLESGGGLVQPGGSLRLSCAASGVRINNQDMGWVRQAPGKGLEWVSGIRTGDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDAAVYYCAGFVMWAWEVWSSHPMWKPYLRYWGQGTLVTVSSAAA；

the amino acid sequence of the CD133-4B9 protein is as follows (also shown in SEQ ID NO. 6):

MAQVQLLESGGGLVQPGGSLRLSCAASGDSITSENMAWVRQAPGKGLEWVSTIKAHNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHIAMKGTWKNWHPQSLHYWGQGTLVTVSSAAA；

the amino acid sequence of the CD133-4B12 protein is as follows (also shown in SEQ ID NO. 7):

MAQVQLLESGGGLVQPGGSLRLSCAASGYTISPEAMTWVRQAPGKGLEWVSTIYMRDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGRGWSGWSWNSNVKYWGQGTLVTVSSAAA。

the nucleotide sequence of the binding protein for targeting CD133 is as follows:

the nucleotide sequence for coding the CD133-2B1 protein is as follows (also shown in SEQ ID NO. 8):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATAAGATTACCGCTGAGTTTATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTTCGAGGCATAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGCATCTGTGGGGAAGTTTCCGTGGGTTTGGGTTTCGGAGGCCACGGTCAACTATTGGGGTCAGGGAACCTTGGTCACCGTCTCGAGCGCGGCCGCA；

the nucleotide sequence for coding the CD133-2C4 protein is as follows (also shown in SEQ ID NO. 9):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTTAAGTTTATCTCTGAGTATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTACTAACGCAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGCAGTTTATGTTTTTCCGTTTGGGTTGGCCGACGAGGTCAGGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

the nucleotide sequence for coding the CD133-2C10 protein is as follows (also shown in SEQ ID NO. 10):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGATAGCATTAGCCCTGAGTCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTGATGGCCCAAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGCAAGGGTTCGTTCTGCGGTTCTGGGTTTGAGGCTGTCCGCGAACGTGAGCTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

the nucleotide sequence for coding the CD133-3B4 protein is as follows (also shown in SEQ ID NO. 11):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATATGCTTATCAATCAGGATATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAGGCATTCTGGACAAAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGAGAGATGTTTCGAAGTGGTCGAAGGACGCCATGTCGTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

the nucleotide sequence for coding the CD133-3B12 protein is as follows (also shown in SEQ ID NO. 12):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGTTAGGATTAACAATCAGGATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAGGCATTCGGACGGGTGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACGCCGCGGTATATTATTGCGCGGGGTTCGTCATGTGGGCTTGGGAGGTTTGGAGTAGTCATCCGATGTGGAAGCCGTACCTGAGGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

the nucleotide sequence for coding the CD133-4B9 protein is as follows (also shown in SEQ ID NO. 13):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGATAGCATTACCTCTGAGAATATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTAAGGCCCATAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACACATATTGCTATGAAGGGGACTTGGAAGAATTGGCATCCCCAGTCGTTGCACTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

the nucleotide sequence for coding the CD133-4B12 protein is as follows (also shown in SEQ ID NO. 14):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATACGATTAGCCCTGAGGCTATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTTATATGCGAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGAGAGTTGGGCGGGGGTGGAGTGGGTGGAGTTGGAACTCCAACGTGAAGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA。

as can be seen, the nucleotide sequences encoding the CD133-2B1 protein and the CD133-2C10 protein each consist of 393 bases and encode 131 amino acids; the nucleotide sequence for coding the CD133-2C4 protein consists of 384 bases and codes 128 amino acids; the nucleotide sequence for coding the CD133-3B4 protein consists of 378 bases and codes 126 amino acids; the nucleotide sequence for coding the CD133-3B12 protein consists of 405 bases and codes 135 amino acids; the nucleotide sequence for coding the CD133-4B9 protein consists of 396 bases and codes 132 amino acids; the nucleotide sequence for coding the CD133-4B12 protein consists of 390 bases and codes 130 amino acids.

The preparation method of the binding protein targeting CD133 comprises the following steps: cloning the nucleotide sequence of the binding protein of the targeting CD133 into an expression vector to construct a recombinant expression plasmid, and transferring the recombinant expression plasmid into a host cell for protein expression and purification to obtain the binding protein of the targeting CD 133; or the binding protein targeting CD133 is obtained by a protein synthesis method.

The binding protein targeting CD133 is applied to preparing antitumor drugs.

The tumor is prostate cancer or breast cancer.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention screens 7 binding proteins combined with CD133 extracellular domain from a human protein phage library based on phage display technology, can express proteins only by a prokaryotic system, can greatly simplify production process, reduce protein production cost, and can be obtained without using antigen to immunize human bodies.

(2) The binding protein provided by the invention only consists of one heavy chain variable region structural domain, and has the advantages of small molecular weight, strong tissue permeability, stable structure and the like.

(3) The binding protein provided by the invention can not generate immunogenicity when being applied to a human body, and can be used for developing tumor protein medicaments in the future.

(4) The binding protein provided by the invention has obvious inhibition effect on prostate cancer and breast cancer cells through detection, and lays a foundation for later-stage development of tumor protein medicines.

Drawings

FIG. 1 is a graph showing the results of detection of the binding of 7 purified binding proteins to the extracellular domain of CD133 by ELISA; wherein, BSA is a blank control, HER2 and EGFR are irrelevant antigen controls, CD133 is relevant antigen, 7 binding proteins are respectively used as primary antibodies, and HRP-protein A is used as a secondary antibody for ELISA detection; p <0.05, relative to BSA control (n-3).

FIG. 2 is a graph showing the results of detecting the effect of binding proteins on the proliferative capacity of tumor cells by the MTT method; wherein, tumor cells DU145 and MCF-7 are cultured by using 7 proteins (CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B12) at different concentrations (0, 25, 50 and 100. mu.g/mL), 30. mu.L of MTT is added into each well after 72 hours, the MTT is incubated for 4 hours, 200. mu.L of DMSO is added to fully dissolve formazan, and then OD570 absorbance is detected by a microplate reader; in the figure, A is a graph showing the results of the effect on the proliferation potency of tumor cell DU145, and B is a graph showing the results of the effect on the proliferation potency of tumor cell MCF-7; p <0.05, relative to 0 μ g/mL (n-3).

FIG. 3 is a graph showing the results of detecting the effect of binding protein on tumor cell apoptosis by flow cytometry and Annexin V/PI double staining kit; among them, 7 proteins (CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12) were co-cultured with tumor cells DU145 and MCF-7 at a concentration of 50. mu.g/mL, while a PBS control group was set; after culturing for 48h, detecting apoptosis by using an Annexin V/PI double-staining kit and a flow cytometry; in the figure, A is the result of inducing apoptosis of tumor cell DU-145, and B is the result of inducing apoptosis of tumor cell MCF-7; p <0.05, relative to the no binding protein control group (n-3).

FIG. 4 is a graph showing the results of detecting the effect of binding proteins on tumor cell invasion by the Transwell invasion method; among them, 7 proteins (CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12) cultured tumor cells DU145 and MCF-7 at different concentrations (0, 25, 50, 100. mu.g/mL), then induced cell invasion with 20% serum-containing medium for 24h, then stained with 0.5% crystal violet and photographed under a microscope, 33% acetic acid eluted the crystal violet bound to the cells, the eluate was collected, and OD570 absorbance was measured by a microplate reader; in the figure, A is the effect of protein on tumor cell DU-145, B is the effect of protein on tumor cell MCF-7 invasion; p <0.05, relative to 0 μ g/mL (n-3).

Detailed Description

The present invention will be described in detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise indicated, the examples were carried out under conventional experimental conditions or with reference to the instructions of the kit manufacturer. Unless otherwise indicated, reagents and materials used in the present invention are commercially available.

The reagents used in the examples were formulated as follows:

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

Dissolving the reagent in 800mL deionized water, diluting to 950mL, and sterilizing with high-pressure steam at 121 ℃ for 30 min. After the medium was cooled to about 60 ℃, 20. mu.L of 100. mu.g/mL ampicillin and 1mL of 20% glucose were added to each 19mL of the medium, mixed well, and then the plate was inverted and stored at 4 ℃ for further use.

(2)2 × TY liquid medium: 5g of NaCl, 16g of peptone and 10g of yeast extract.

Dissolving the reagent in deionized water, diluting to 1L, sterilizing with 121 deg.C high pressure steam for 20min, and storing at room temperature for a long time.

(3)50 × TAE DNA electrophoresis buffer: tris-base 242g, Na₂EDTA·2H₂37.2g of O and 57.1mL of glacial acetic acid.

The reagent and the solution are dissolved in 800mL of deionized water, then the volume is determined to be 1L, and the solution is diluted into 1 XTAE electrophoresis buffer solution when in use and is stored at room temperature.

(4) PBS buffer (pH 7.4): KH (Perkin Elmer)₂PO₄ 0.24g、NaCl 8g、KCl 0.2g、Na₂HPO₄·12H₂O 9.07g。

Dissolving the reagent in 800mL deionized water, adjusting pH to 7.4, diluting to 1L, sterilizing with high pressure steam at 121 deg.C for 20min, and storing at room temperature for a long time. 1mL of Tween-20 was added to 1L of PBS buffer, and mixed well to prepare PBST solution.

(5) PEG solution: 73g of NaCl and 6000100 g of PEG.

The reagent was dissolved in deionized water and the volume was adjusted to 500mL, and the bacteria were filtered using a 0.2. mu.M filter and stored at 4 ℃ until use.

(6) LB liquid medium: NaCl 2g, peptone 2g, and yeast extract 1 g.

Dissolving the reagent in deionized water, diluting to 200mL, sterilizing with high pressure steam at 121 deg.C for 20min, and storing at 4 deg.C.

(7) LB solid medium: 2g of NaCl, 2g of peptone, 1g of Yeast extract Yeast extract and 4g of agar powder.

Dissolving the reagent in deionized water, diluting to 190mL, and sterilizing with high-pressure steam at 121 deg.C for 30 min. After the medium was cooled to about 60 ℃, 20. mu.L of 100. mu.g/mL ampicillin and 1mL of 20% glucose was added to each 19mL of the medium, mixed well, and then the plate was inverted and stored at 4 ℃ for further use.

(8)5 XSDS-PAGE running buffer: 47g of glycine, 15.1g of Tris alkali and 2.5g of SDS.

The reagent was dissolved in 400mL of deionized water, and then the solution was made to 500mL, diluted to 1 XSDS-PAGE electrophoresis buffer at the time of use, and stored at room temperature for a long period of time.

(9)5 XSDS-PAGE Loading Buffer: 1.25mL of 1M Tris-HCl (pH 6.8), 2.5mL of glycerol, 25mg of bromophenol blue, and 0.5g of SDS.

Mixing the reagent and the solution uniformly, setting the volume to 5mL, subpackaging to 500 mu L/part, adding 25 mu L beta-mercaptoethanol into each small part before use, and storing at room temperature for a long time.

(10) Coomassie brilliant blue R-250 staining solution: coomassie brilliant blue R-2501 g, isopropanol 250mL, acetic acid 100mL, ddH₂O 650mL。

Mixing the reagent and the solvent uniformly, fully dissolving, and storing at room temperature for a long time for later use.

(11) Coomassie brilliant blue staining destaining solution: acetic acid 100mL, ethanol 50mL, ddH₂O 850mL。

Mixing the solvent, and storing at room temperature.

(12) And (3) breaking the bacteria buffer solution: 14.6g of NaCl and 2.42g of Tris alkali.

The reagents are mixed and fully dissolved by 1L of water, and the mixture is stored at the temperature of-4 ℃, 100 XPMSF stock solution is added when the mixture is used, and the final concentration of the PMSF stock solution is 1 XPMSF stock solution. Loading buffer solution: breaking the bacteria in the same buffer solution; washing with a miscellaneous buffer solution: when the preparation is used, 9.9mL of bacteria breaking buffer solution is measured, 100 mu L of 2M imidazole is added, and the mixture is fully and uniformly mixed; elution buffer: when the preparation is used, 9mL of bacteria breaking buffer solution is measured, 1mL of 2M imidazole is added, and the mixture is fully and uniformly mixed.

(13) Kanamycin solution (50 mg/mL): weighing 1g kanamycin powder fully dissolved in 20mL deionized water, the mixture with 0.2M filter sterilization, subpackage into each tube of 1mL, -20 ℃ for storage.

(14) Ampicillin solution (100 mg/mL): ampicillin powder 1g was weighed and dissolved in 10mL of deionized water, and the mixture was filtered through a 0.2 μ M filter to sterilize the solution and dispensed into 1mL tubes and stored at-20 ℃ for further use.

(15) 2% BSA-PBS buffer: weighing 1g bovine serum albumin powder, fully dissolving in 50mL PBS buffer solution, filtering the mixed solution with a 0.2 mu M filter for sterilization, and storing the mixture for use at the present or for a long time at the temperature of-20 ℃.

(16)1mg/mL trypsin solution: 10mg of trypsin powder was weighed and dissolved in 10mL of PBS buffer, and stored at-20 ℃ for a long period of time.

(17) 20% glucose solution: 200g of glucose powder is weighed and fully dissolved in deionized water, the volume is fixed to 1L, the mixed solution is filtered and sterilized by a 0.2 mu M filter, and the mixed solution is stored at the temperature of-4 ℃ for standby.

(18)1M sulfuric acid solution: 9.8mL of concentrated sulfuric acid is measured in a measuring cylinder, slowly added into 187mL of deionized water, fully and uniformly mixed, and stored at room temperature for later use.

(19) 10% Ammonium Persulfate (APS): 0.1g of ammonium persulfate powder was weighed and dissolved in 1mL of deionized water, and the solution was dispensed into 200. mu.L tubes and stored at-20 ℃.

(20) IPTG solution (500 mM): weighing 11.915g IPTG powder, fully dissolving in deionized water, diluting to 100mL, filtering the mixed solution with a 0.2 mu M filter for sterilization, subpackaging into 1mL per tube, and storing at-20 ℃ for a long time.

(21) 30% glycerol solution: 15mL of glycerol is weighed in a measuring cylinder and added into 35mL of deionized water, and after the glycerol and the deionized water are fully mixed, a 0.22 mu M filter is used for filtering and sterilizing, and the mixture is stored at the temperature of-4 ℃ for standby.

(22)100 × PMSF stock solution: 1.74g of PMSF powder was weighed and dissolved in 100mL of isopropanol, mixed well and stored at-20 ℃ for a long period.

(23)2M imidazole: 1.14g of imidazole powder was weighed out and dissolved in 10mL of the lysis buffer, and stored at-4 ℃ for further use.

EXAMPLE 1 preparation of helper phage

(1) Coating TG1 glycerobacteria purchased from Biyunyan in a culture dish containing TYE solid culture medium by a three-zone marking method, and then placing the culture dish in a constant-temperature incubator at 37 ℃ for culturing for 12-16 hours;

(2) selecting a TG1 single colony growing on a TYE solid culture medium, inoculating the single colony to 5mL of 2 × TY liquid culture medium, placing the single colony in a constant-temperature shaking table at 37 ℃, and culturing for 12-16 hours at 250 rpm;

(3) transferring the bacterial culture solution in the step (2) to another tube of 5mL of 2 XTY liquid culture medium according to the proportion of 1:100, placing the tube in a constant temperature shaking table at 37 ℃, and culturing at 250rpm until the bacterial solutionOD₆₀₀About 0.5;

(4) the helper phage KM13 was diluted in PBS in a gradient (from 10)¹²/mL～10⁴/mL)；

(5) Taking 200 mu L of the bacterial liquid obtained in the step (3), adding 10 mu L of diluted helper phage KM13 into the bacterial liquid, uniformly mixing, and placing the mixture in a water bath kettle at 37 ℃ for 30 minutes to obtain a mixture A;

(6) taking 3mL of melted top agar culture medium, spreading the top agar culture medium on a culture dish which is preheated in advance and contains TYE solid culture medium, and standing at room temperature to solidify the top agar culture medium; the specific operation steps are as follows: before use, the top agar is heated to be completely melted, then is incubated in water and cooled to 42 ℃, and then is mixed with the mixture A obtained in the step (5). Standing at room temperature until the culture medium is completely solidified, and placing the culture dish in a constant-temperature incubator at 37 ℃ for culturing for 12-16 hours;

(7) picking a small plaque on the medium in step (6) with a sterile tip, inoculating to 5mL of the bacterial solution (OD) in step (3)₆₀₀0.5, prepared by the method in the step (3), shaking at a constant temperature of 37 ℃ and culturing for 2-3 hours at 250 rpm;

(8) transferring the bacterial liquid obtained in the step (7) into another conical flask containing 500mL of 2 XTY liquid culture medium according to the volume ratio of 1:100, and culturing for 2-3 hours at a constant temperature of 37 ℃ and 250 rpm;

(9) adding kanamycin into the culture solution obtained in the step (8) to a final concentration of 50 mug/mL, and culturing for 12-16 hours at a constant temperature of 30 ℃ by using a shaking table at 250 rpm;

(10) centrifuging 12000g of the culture solution cultured in the step (9), taking the supernatant, filtering, adding a PEG solution (polyethylene glycol) with the volume equivalent to 1/5 of the supernatant, standing for several hours at 4 ℃, and purifying to obtain the helper phage;

(11) and (3) detecting the sensitivity of the prepared helper phage to pancreatin: add 10 to 1mL trypsin solution¹⁰The helper phages KM13 were incubated at room temperature for 30 minutes. The helper phage KM13 was then diluted in PBS in a gradient (from 10)¹⁰To 10²Phage), 10. mu.L of KM13 gradient dilutions were individually infected with 200. mu.L of TG1 bacteria (OD)₆₀₀0.5, preparation and application as described above), and coating on TYE solidOn the body medium (containing kanamycin at a final concentration of 50. mu.g/mL), the culture was carried out overnight in a 37 ℃ incubator. After the pancreatin-treated phage have infected the bacteria, the number of clones obtained should be at least 10 less than the number of clones in the untreated group⁶And (4) doubling. Otherwise, the helper phage preparation is discarded and another plaque is picked for re-preparation.

Example 2 expression of phage binding protein library

(1) An appropriate amount of the Human-derived binding protein phage Library (Source Bioscience, Human Domain Antibody Library (DAb), London, UK) was taken and cultured in 500mL of 2 XTY liquid medium (medium additionally containing 100. mu.g/mL ampicillin, 4% (w/v) glucose), with a 37 ℃ constant temperature shaker at 250rpm until the bacterial OD was reached₆₀₀＝0.5；

(2) 2X 10 of the product of example 1 was added¹²And (3) adding the helper phage to the bacterial liquid obtained in the step (1), uniformly mixing, and placing in a water bath at 37 ℃ for 30 minutes. The 500mL culture was aliquoted into 50mL per tube, centrifuged at 3200g for 10min, the supernatant discarded, and the pellet resuspended and transferred to a conical flask containing 500mL of 2 XTY liquid medium (medium additionally containing 0.1% (w/v) glucose, 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin). Placing the mixture in a constant temperature shaking table at 25 ℃, and shaking and culturing the mixture at 250rpm for 16-20 hours.

Example 3 purification of phage binding protein libraries

(1) The overnight culture of example 2 was split into 50mL per tube and centrifuged at 3200g at room temperature for 20 min;

(2) transferring the centrifuged supernatant into another clean 50mL centrifuge tube, adding 20% PEG solution into each tube according to the volume ratio of 1:4, and incubating for 1 hour on ice;

(3) placing the centrifuge tube in the step (2) at 4 ℃ and 3200g for centrifugation for 30 minutes, discarding the supernatant, resuspending the precipitate with 5mL of PBS, adding 1mL of 20% PEG solution, and incubating on ice for 10 minutes;

(4) centrifuging the solution obtained in the step (3) at 4 ℃ and 3200g for 30 minutes, discarding the supernatant, then resuspending the precipitate with 1mL of PBS, centrifuging the precipitate at 4 ℃ and 3200g for 5 minutes, and filtering the supernatant with a 0.45 mu M filter for sterilization;

(5) estimation of phage library titer by measuring absorbance at 260 nm: will be at the topThe purified phage library was diluted 100-fold with PBS and phage library titer (PFU/mL) was estimated according to the following empirical formula: phage/mL ═ OD₂₆₀×100×22.14×10¹⁰. The prepared phage library can be stored at 4 ℃ for 2 weeks, and can also be frozen at-80 ℃ for a long time.

Example 4 screening of phage-binding protein libraries for proteins that bind to the extracellular domain of CD133

(1) CD133 extracellular domain protein (sequence shown in GenBANK number: NP-006008.1) synthesized by Shanghai Boragi Biotech, Inc., 4mL of CD133 extracellular domain protein diluent dissolved in PBS buffer solution and having a final concentration of 100 μ g/mL was added into NUNC immune tube, and the mixture was left at 4 ℃ overnight;

(2) abandoning the overnight coating solution in the step (1), softly washing the inner wall of the immune tube for 3 times by using PBS buffer solution, then adding 4mL of 2% BSA-PBS blocking solution, and placing at room temperature for 2 hours;

(3) discard the blocking solution from step (2), gently wash the inner wall of the immune tube with PBS buffer solution 3 times, add 4mL of 2% BSA-PBS blocking solution (containing 5X 10¹²Phage prepared as in example 3), left at room temperature for 1 hour;

(4) discarding the blocking solution in the step (3), softly washing the inner wall of the immune tube for 10 times by using PBST buffer solution, adding 4mL of 1mg/mL trypsin solution, and standing at room temperature for 1 hour to ensure that the phage is completely digested from the immune tube to obtain a digestive juice A;

(5) selecting TG1 bacterial liquid from a TG1 strain glycerol storage, scribing on a TYE solid culture medium according to a three-region scribing method, placing the TYE solid culture medium in a constant-temperature incubator at 37 ℃, and culturing for 14-16 hours;

(6) selecting a single TG1 colony on the culture medium in the step (5) to inoculate in 5mL of 2 XTY liquid culture medium, shaking at the constant temperature of 37 ℃, and culturing overnight at 250 rpm;

(7) and (3) mixing the bacterial liquid obtained in the step (6) according to a volume ratio of 1: inoculating 100% of the culture solution into another test tube containing 5mL of 2 XTY liquid culture medium, shaking at 37 deg.C and 250rpm, and culturing to obtain bacterial solution OD₆₀₀＝0.5。

(8) Adding the bacterial liquid obtained in the step (7) into the digestive juice A prepared in the step (4), carrying out constant-temperature water bath at 37 ℃ for 1 hour, and centrifuging 3200g for 5 minutes;

(9) discarding the supernatant, and resuspending the precipitate with 1mL of 2 × TY liquid culture medium to obtain a resuspension solution A; taking 6 culture dishes (containing 100 mu g/ml ampicillin and 4% (w/v) glucose) containing TYE solid culture medium, coating 166 mu L of the heavy suspension A on each plate, and placing the coated culture dishes in a constant-temperature incubator at 37 ℃ for overnight culture;

(10) taking the culture dishes cultured overnight in the step (9), adding 2mL of 2 XTY liquid culture medium into each dish, and scraping off bacteria on the plate by using a coating rod;

(11) transferring the scraped bacterial solution into an Erlenmeyer flask containing 500mL of 2 XTY liquid culture medium (the culture medium contains 4% (w/v) glucose and 100 mu g/mL ampicillin), placing the Erlenmeyer flask in a constant temperature shaking table at 37 ℃, and culturing at 250rpm until the bacterial solution OD is reached₆₀₀Adding the auxiliary phage KM13 prepared in example 1, infecting the bacterial liquid, placing the bacterial liquid on a constant temperature shaking table at 30 ℃, shaking and culturing overnight at 250rpm, wherein the auxiliary phage KM13 is 0.5;

(12) taking the overnight culture obtained in the step (11), adding a PEG solution to purify the phage (refer to the previous step), and determining the titer of the phage library obtained by screening through gradient dilution plating;

(13) subsequent 4 rounds of phage library panning were performed with reference to the previous procedure (i.e. repeating steps (1) to (12)), and screening was performed sequentially from the phage obtained in the previous round.

Example 5 Single clones were picked from the library for ELISA validation

(1) After 5 rounds of phage library panning, a sterile 96-well plate (named as plate A) is taken, 200mL of 2 XTY liquid medium (the medium contains 100. mu.g/mL ampicillin and 4% (w/v) glucose) is added into each well, a single clone is picked from an overnight culture dish obtained by 5 th screening by using a sterile suction head, inoculated into the 96-well plate containing the culture solution, and subjected to shaking culture at 37 ℃ and 250rpm overnight;

(2) taking another sterile 96-well plate (named as a B plate), adding 200 mu L of 2 XTY liquid culture medium (the culture medium contains 100 mu g/mL ampicillin and 4% (w/v) glucose) into each well, sucking 5 mu L of the overnight culture obtained in the step (1), inoculating the overnight culture into the 96-well plate, and then placing the plate in a constant temperature shaking table at 37 ℃ for shaking culture at 250rpm for 3 hours; adding glycerol with the final concentration of 20% into each hole of the overnight culture remained on the plate A in the step (1) to prepare a glycerol bacterial liquid storage, and freezing and storing the glycerol bacterial liquid storage for a long time at-80 ℃;

(3) each well of the plate after 3 hours of culture in the B plate in step (2) was transferred to a sterile 1.5mL EP tube containing 50. mu.L of 4X 10⁸Gently mixing 2 XTY liquid culture medium of the helper phage KM13, and incubating the EP tube at 37 ℃ for 1 hour;

(4) after incubation, 1.5mL of EP tube 3200g was centrifuged for 10 minutes, and the supernatant was discarded; the pellet was resuspended in 200. mu.L of 2 XTY liquid medium (medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin, 0.1% (w/v) glucose), shaken at 25 ℃ and 250rpm overnight;

(5) transferring the overnight culture obtained in the step (4) to another new EP tube with the volume of 1.5mL, centrifuging the overnight culture for 10 minutes at 3200g, taking the supernatant, transferring the supernatant to another new 96-well plate, and storing the plate in a refrigerator at 4 ℃;

(6) add 100. mu.L of PBS buffer containing 0.2. mu.g of CD133 extracellular domain protein to each well to coat the 96-well ELISA plate, and leave it at 4 ℃ overnight;

(7) washing the coated ELISA plate with PBS for 3 times, adding 250 μ L of 2% BSA-PBS to each well, blocking the ELISA plate for 2 hours at room temperature, and then washing the plate with PBS for 3 times for later use;

(8) sucking 25 mu L of the supernatant prepared in the step (5) into a new 1.5mL EP tube, adding 75 mu L of 2% BSA-PBS to prepare a phage diluent, putting the phage diluent into the ELISA plate washed in the step (7), and incubating for 1 hour at room temperature;

(9) washing the plate 5 times with PBST, adding 100 μ L of HRP-labeled anti-M13 conjugate (diluted with 2% BSA-PBS) diluted at a volume ratio of 1:10000 to each well, and incubating at room temperature for 1 hour;

(10) the plate was washed again 5 times with PBST, 100. mu.L of Tetramethylbenzidine (TMB) solution was added to each well, after the liquid in the wells turned blue, 50. mu.L of 1M concentrated sulfuric acid was added to each well to stop the reaction, the absorbance was read at 450nm, and the experimental results were recorded.

EXAMPLE 6 preparation of DH5 alpha, BL21(DE3) E.coli competent cells

(1) Respectively scribing a small amount of escherichia coli DH5 alpha and BL21(DE3) bacterial liquid on a culture dish containing an LB solid culture medium by a three-region scribing method, and then placing the culture dish in a constant-temperature incubator at 37 ℃ for culturing for 14-16 hours;

(2) picking single colonies of escherichia coli DH5 alpha and BL21(DE3) from a culture dish containing LB solid culture medium respectively, inoculating the single colonies into a test tube containing 5mL of LB liquid culture medium respectively, and performing shake culture at 37 ℃ and 220rpm for about 12 hours;

(3) respectively inoculating the two bacterial culture solutions into a conical flask containing 50mL of LB liquid culture medium according to the volume ratio of 1:100, carrying out shaking culture at 37 ℃ for 2-3 hours at 220rpm until the OD of the bacterial solution₆₀₀About 0.5;

(4) respectively transferring the bacterial liquid cultured in the step (3) into another 2 sterile 50mL centrifuge tubes, and standing for 10 minutes in ice-water mixed liquid;

(5) placing the centrifugal tube in the step (4) in a refrigerated centrifuge for 5 minutes at 4 ℃ and 3000 g;

(6) discarding the supernatant, and taking 10mL of precooled 0.1mol/L CaCl₂The solution is gently resuspended in the thallus precipitate, and then the centrifuge tube is placed in ice-water mixed solution for standing for 30 minutes;

(7) placing the centrifugal tube in a refrigerated centrifuge for 5 minutes at 4 ℃ and 3000g for centrifugation;

(8) discarding the supernatant, and taking 3mL of precooled 0.1mol/L CaCl₂The solution is gently resuspended and precipitated to obtain a resuspension solution B;

(9) and (3) adding 3mL of precooled 30% (v/v) sterile glycerol into the heavy suspension B obtained in the step (8), gently mixing uniformly, and subpackaging the mixed solution into 100 mu L of each tube for long-term freezing storage at-80 ℃.

Example 7 transformation of recombinant plasmids expressing CD133 binding proteins into DH5 alpha competent cells

(I) construction of recombinant plasmid of CD 133-binding protein

(1) The positive monoclonal in example 5 was cloned in a volume ratio of 1: inoculating the strain at a ratio of 1000 into 5mL of 2 XTY liquid medium (the medium contains 100. mu.g/mL ampicillin and 4% (w/v) glucose) and culturing overnight;

(2) preparing 25 mu L PCR amplification system by using the forward primer and the reverse primer (forward primer: 5'-ATGGCCCAGGTGCAGCTGT-3'; reverse primer: 5'-TCTGCGGCCGCGCTCGAGAC-3') of the CD133 binding protein, and amplifying the nucleotide sequence of the CD133 binding protein; wherein:

the PCR reaction system is as follows: mu.L of template (overnight culture from step (1)), 1. mu.L of each primer (forward/reverse primer), 0.5. mu.L of 5 XPrimerstar buffer 10. mu. L, dNTP mix (2.5 mM each) 4. mu. L, PrimerStarDNA polymerase, sterile deionized water to 25. mu.L;

the PCR reaction conditions are as follows: 10min at 96 ℃; 10s at 95 ℃; 10s at 60 ℃; 30 cycles at 72 ℃ for 30 s; 5min at 72 ℃;

(3) carrying out agarose gel electrophoresis on the obtained CD133 protein nucleotide sequence PCR product, then cutting a target fragment on the gel for gel recovery, and storing the gel recovery product at-20 ℃ for later use;

(4) taking a proper amount of CD133 conjugated protein nucleotide sequence glue recovery product, adding restriction enzyme NotI and NocI for enzyme digestion, carrying out agarose gel electrophoresis on the enzyme digestion product, then cutting a target fragment on the gel for glue recovery, and storing the glue recovery product at-20 ℃ for later use

(5) Taking a proper amount of expression vector pET28a, adding restriction enzyme NotI and NocI into the expression vector pET28a for enzyme digestion, carrying out agarose gel electrophoresis on the enzyme digestion product, then cutting a target fragment on the gel for gel recovery, and storing the gel recovery product at-20 ℃ for later use

(6) Taking a proper amount of a gel recovery product obtained after the enzyme digestion of the CD133 binding protein nucleotide sequence, adding ligase into the gel recovery product obtained after the enzyme digestion of an expression vector pET28a, and connecting at 37 ℃ overnight to obtain a connecting product; wherein:

the connection reaction system is as follows: 0.03pmol of pET28a double-restriction enzyme product, 0.3pmol of target fragment double-restriction enzyme product, 1 μ L of enzyme linked buffer 2.5 μ L, T4DNA ligase, and sterile water to make up to 25 μ L.

(II) transforming the anti-CD 133 recombinant plasmid into DH5 alpha competent cells

(1) Removing a tube of DH5 alpha competent cells from the freezer at-80 deg.C, thawing on ice;

(2) add 10. mu.L of ligation product to 100. mu.L of DH 5. alpha. competent cells (prepared in example 6), mix gently, and let stand on ice for 30 min;

(3) placing the mixture prepared in the step (2) in a water bath kettle with a constant temperature of 42 ℃ for 90s, and then quickly transferring to ice to stand and cool for 2-3 minutes;

(4) adding 900 mu L of LB liquid culture medium into the mixed solution prepared in the step (3), shaking and culturing for 1 hour at a constant temperature of 37 ℃ and 200 rpm;

(5) placing the cultured mixed solution in a centrifuge, centrifuging at 3000g for 1 min, and collecting the mixed solution precipitate;

(6) discarding 900. mu.L of supernatant, and resuspending the pellet with the remaining 100. mu.L of culture medium;

(7) the mixed bacterial suspension was spread on a petri dish containing LB solid medium (medium containing 100. mu.g/mL ampicillin, 1% (w/v) glucose) prepared in advance;

(8) the culture dish is placed in a constant temperature incubator at 37 ℃ for 12-16 hours,

(9) several single clones were randomly picked from the petri dish and inoculated into 5mL LB liquid medium (medium containing 100. mu.g/mL ampicillin), respectively, shaking at 37 ℃ and shaking at 220rpm for overnight culture.

(10) Preparing 25 mu L of PCR amplification system by using forward and reverse primers (forward primer: 5'-ATGGCCCAGGTGCAGCTGT-3'; reverse primer: 5'-TCTGCGGCCGCGCTCGAGAC-3') of CD133 binding protein, adding 1 mu L of overnight culture (template) obtained in the step (9) into the PCR amplification system, and carrying out bacterial liquid PCR verification on the monoclonal; wherein, the PCR reaction system and the reaction condition refer to the step (I).

(11) And carrying out agarose gel electrophoresis on the PCR product, wherein if the target fragment appears, the clone is positive.

(12) And carrying out plasmid extraction on the positive clone to obtain the recombinant plasmid of the CD133 binding protein.

(13) Sequencing the obtained positive clone, and displaying that seven proteins, namely CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B12 proteins are obtained, and the coding nucleotide sequences of the seven proteins are respectively shown as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

Example 8 prokaryotic expression of binding proteins, protein purification, SDS-PAGE gel electrophoresis and Coomassie blue staining

Prokaryotic expression of binding protein

(1) Transforming the recombinant plasmid of the CD 133-binding protein prepared in example 7 into BL21 competent cells (the specific steps refer to example 7);

(2) picking single colony from the plate coated after the transformation in the step (1), inoculating the single colony into a test tube containing 5mL of LB liquid culture medium (the culture medium contains 100 mug/mL of ampicillin and 1% (w/v) of glucose), and performing shake culture at 37 ℃ for 12 hours at 220rpm by using a constant temperature shaking table;

(3) taking the bacterial liquid cultured in the step (2), and mixing the components according to the proportion of 1: inoculating 100 volume ratio into a conical flask containing 100mL LB liquid culture medium (the culture medium contains 100 mu g/mL ampicillin), shaking at the constant temperature of 37 ℃ for 2-3 hours with shaking at 220 rpm;

(4) OD of bacterial liquid to be treated₆₀₀Taking 1mL of bacterial liquid to prepare a whole protein sample of the non-induced escherichia coli, and carrying out subsequent experiments on the residual bacterial liquid, wherein the total protein sample is 0.6-1.0;

(5) adding 0.25mM IPTG into the residual bacterial liquid in the step (4), carrying out induction expression for 6h at a constant temperature of 25 ℃ by using a shaking table at 220 rpm;

(6) taking 1mL of the bacterial liquid cultured in the step (5) for preparing an induced escherichia coli holoprotein sample, centrifuging the residual bacterial liquid at 4 ℃ for 5min by 5000g, and collecting bacterial precipitates;

(7) discarding the supernatant, and resuspending the thallus precipitate obtained in the step (6) by using 20mL of precooled bacteria breaking buffer solution;

(8) transferring the bacterial suspension to a 50mL beaker, placing the beaker in an ice box to ultrasonically break the bacterial suspension, wherein the working power of an ultrasonic crusher is as follows: working for 4 seconds at 40 percent, stopping for 8 seconds, and crushing for 40 minutes;

(9) transferring the crushed bacterial suspension into a clean 50mL centrifuge tube, and centrifuging for 40 minutes at the temperature of 4 ℃ and at the speed of 15000 g;

(10) and (3) collecting the supernatant obtained after centrifugation in the step (9), taking 20 mu L of the supernatant from the supernatant to prepare a supernatant sample after thallus crushing, taking part of the precipitate to prepare a precipitate after thallus crushing (the precipitate is re-suspended by 20 mu L of the thallus crushing buffer solution), transferring the rest supernatant into another clean 50mL centrifuge tube, storing at 4 ℃, and waiting for column purification.

(II) column purification of binding proteins

(1) Taking a Ni-NTA His bond Resin purification column, and washing the purification column by using a loading buffer solution with the volume 10-15 times of the column volume;

(2) adding the supernatant obtained in the step (one) into a purification column, and allowing the supernatant to flow through the purification column at a natural flow rate, wherein 20 mu L of column-through liquid is taken for preparing a column-through liquid sample;

(4) after the supernatant completely flows through the purification column, adding a sample loading buffer solution with the volume 10 times that of the column to wash the purification column;

(5) after the liquid in the step (4) is drained, adding a washing buffer solution with the volume 20 times that of the column to wash and purify the foreign protein on the column, and taking 20 mu L of washing liquid to prepare a washing liquid sample in the period;

(6) after the liquid in the step (5) is drained, adding an elution buffer solution with the volume 5 times that of the column to elute the target protein from the purification column, collecting the eluent by using 1.5mL of EP tubes during the elution, collecting 1mL of the eluent by each tube, collecting 5 tubes in total, and respectively taking 20 mu L of the eluent from each tube to prepare 1-5 samples of the eluent;

(7) after the target protein is collected, adding 6M urea with 5 times of column volume to elute the residual protein on the purification column, and then adding distilled water with 30 times of column volume to clean the purification column;

(8) after the distilled water is drained, 5mL of 20% ethanol is added into the purification column, the upper end and the lower end of the purification tube are sealed, and the purification column is stored at 4 ℃ for a long time.

(III) SDS-PAGE gel electrophoresis and Coomassie Brilliant blue staining

(1) Preparing 5mL of 12% (w/v) separation gel according to a formula of a specification, adding the separation gel into a gel filling mold, enabling the liquid level to be about 2cm away from the top of the mold, then adding 1.5mL of absolute ethyl alcohol, and standing for 30min at room temperature until the separation gel is solidified;

(2) removing absolute ethyl alcohol in the mold, preparing 2mL of 5% (w/v) concentrated glue according to a prescription, adding the concentrated glue into a glue filling mold to the top, inserting a comb matched with the mold, and standing at room temperature for 30min until the concentrated glue is solidified;

(3) putting the prepared rubber plate into an electrophoresis tank, adding a proper amount of electrophoresis liquid, and pulling out a comb to prepare for sample loading;

(4) respectively adding 5 mu L of 5 xSDS-PAGE protein loading buffer solution into the samples collected in the step of expression and purification of the binding protein, uniformly mixing, and heating at 100 ℃ for 5-10 min;

(5) taking 5 mu L of the sample prepared in the step (4), adding SDS-PAGE gel into comb holes, performing electrophoresis at a voltage of 80V, adjusting the voltage to 110V when the sample runs through the concentrated gel, and performing electrophoresis until a blue indicator band of the sample runs to the bottom of the gel;

(6) adding appropriate amount of Coomassie brilliant blue staining solution into the staining kit, taking out SDS-PAGE gel, placing in the staining kit, and staining for 30min at room temperature;

(7) discarding the staining solution, washing the staining solution on the gel with clear water, and adding appropriate amount of decolorizing solution for decolorizing;

(8) and (5) when the bands on the SDS-PAGE gel are clearly visible, decoloring the gel to be transparent, and taking a picture under white light for recording.

The experimental result shows that purified CD133-2B1 protein, CD133-2C4 protein, CD133-2C10 protein, CD133-3B4 protein, CD133-3B12 protein, CD133-4B9 protein and CD133-4B12 protein are obtained.

Example 9 detection of binding of purified CD133 binding protein to antigen by ELISA

(1) Taking a new 96-well immune plate, respectively adding 100 mu L of antigen HER2, EGFR and antigen CD133 extracellular protein diluent (HER2 and EGFR purchased from Shanghai Betay Biotech Co., Ltd.) with the final concentration of 2 mu g/mL (solvent is PBS buffer) into each well, coating 100 mu L of 2% BSA-PBS buffer as a blank control, and placing the plate in a constant-temperature incubator at 37 ℃ for standing for 2 hours;

(2) taking out the coated immune plate, washing the plate for 3 times by using PBS (phosphate buffer solution), removing residual liquid in the plate, adding 250 mu L of 2% (w/v) BSA-PBS (bovine serum albumin-phosphate buffer solution) into each hole, and standing for 2 hours in a constant-temperature incubator at 37 ℃;

(3) taking out the sealed immune plate, washing the plate for 3 times by using PBS buffer solution, removing residual liquid in the plate, adding 100 mu L of CD133 binding protein-PBS mixed solution diluted by 50 times by using PBS into each hole, and incubating the immune plate for 1 hour at room temperature; the binding proteins are respectively CD133-2B1 protein, CD133-2C4 protein, CD133-2C10 protein, CD133-3B4 protein, CD133-3B12 protein, CD133-4B9 protein and CD133-4B12 protein;

(4) the immune plate was removed and washed 3 times with PBST buffer, the remaining liquid was removed from the plate, and secondary antibody HRP-protein a was washed with 2% BSA-PBS buffer at 1: diluting at a ratio of 5000, adding 100 mu L of second antibody diluent into each hole of an immune plate, and incubating for 1 hour at room temperature;

(5) and (3) taking out the immune plate, washing the plate for 5 times by using the PBST buffer solution, removing residual liquid in the plate, adding 100 mu L of TMB substrate developing solution into each hole, incubating for 10 minutes at room temperature in a dark place, then placing the immune plate in an enzyme labeling instrument to detect the OD 450nm light absorption value, and recording experimental data.

The experimental results are shown in FIG. 1, wherein BSA is blank control, HER2 and EGFR are irrelevant antigen controls, and it can be seen that CD133-2B1 protein, CD133-2C4 protein, CD133-2C10 protein, CD133-3B4 protein, CD133-3B12 protein, CD133-4B9 protein and CD133-4B12 protein can be specifically bound with CD133 extracellular domain.

Example 10 MTT assay to determine the Effect of purified binding proteins on tumor cell proliferation potency

(1) A96-well cell culture plate was prepared by adding 100. mu.L of whole culture medium (RPMI1640 containing 10% calf serum, the same below) containing 5000 tumor cells (human prostate cancer cell DU145 or human breast cancer cell MCF-7) in logarithmic growth phase to each well, placing the plate in a cell culture chamber at 37 ℃ and 5% CO₂Culturing overnight;

(2) after the cells are completely attached to the wall, the whole culture medium in the holes is changed into a serum-free culture medium (RPMI1640, the same applies below), and the culture plate is placed in a cell culture box for starvation treatment for 4 hours;

(3) the serum-free medium after the starvation treatment in step (2) was aspirated, 100. mu.L of 1% serum medium containing different concentrations of protein (0, 25, 50 and 100. mu.g/mL) was added to each well, and the plates were placed in a cell culture chamber at 37 ℃ with 5% CO₂Culturing for 72 hours; the proteins are respectively CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B 12;

(4) the liquid in the well-treated culture plate was aspirated, a mixed solution containing 100. mu.L of a serum-free medium and 20. mu.L of MTT solution was added to each well, and the mixture was placed in a cell culture chamber 37℃，5％CO₂Incubating for 4 hours;

(5) removing the mixed solution in the culture plate by suction, adding 200 mu L DMSO into each hole, and then placing the culture plate in a shaking table to shake quickly for 10min to fully dissolve the precipitate;

(6) and (5) placing the culture plate in an enzyme labeling instrument to detect the light absorption value at 570nm, and recording the detection result.

As shown in FIG. 2, it can be seen that the proteins CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B12 can significantly inhibit the proliferation of DU145 and MCF-7 cells at a concentration of 50. mu.L/mL.

Example 11 detection of binding protein-induced apoptosis in tumor cells Using flow cytometry

(1) A6-well cell culture plate containing 5X 10 tumor cells in logarithmic growth phase was prepared by adding 1mL of whole medium to each well⁶Respectively, placing the culture plate in a cell culture box at 37 ℃ and 5% CO₂Culturing overnight;

(2) after the cells are completely attached to the wall, the whole culture medium in the holes is changed into a serum-free culture medium, and the culture plate is placed in a cell culture box for starvation treatment for 4 hours;

(3) removing the medium in the wells in (2) by aspiration, adding 1mL of serum-free medium containing 50. mu.g/mL of protein per well, placing the culture plate in a cell culture chamber at 37 ℃ and 5% CO₂Culturing for 48 hours; the proteins are respectively CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B 12;

(4) digesting the cells in the culture plate by using pancreatin, collecting the cells in a 1.5mL EP tube, and washing the cells for 2 times by using PBS buffer solution;

(5) remove 4 × binding buffer from Annexin V-FITC apoptosis kit and add ddH₂O it was diluted to 1X, and the cell density was adjusted to 5X 10 in 195. mu.L of 1X binding buffer⁶Per mL;

(6) adding 5 mu L of Annexin V-FITC in the kit into the cell mixed solution prepared in the step (5), and incubating for 10-15 min at room temperature in a dark place;

(7) adding 200 mu L of 1 × binding buffer solution into a 1.5mL EP tube, adding the cell mixed solution prepared in the step (6), and gently mixing uniformly;

(8) placing the 1.5mL EP tube in the step (7) in a centrifuge for 5min at 1000g, discarding the supernatant, taking 190 μ L of 1 × binding buffer solution to resuspend the cells, adding 10 μ L of PI staining solution, and gently mixing.

(9) And detecting the tumor cells subjected to apoptosis by using a flow cytometry, and recording an experimental result.

As shown in FIG. 3, CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B12 proteins can remarkably induce the apoptosis of DU145 cells and MCF-7 cells at a concentration of 50 muL/mL.

Example 12Transwell invasion assay to examine the Effect of binding proteins on the invasive potential of tumor cells

(1) Placing a sterile Transwell chamber (with 8 μm aperture and 24-hole plate) into a 24-hole cell culture plate, sucking 50 μ L Matrigel (Matrigel) and adding into the chamber, placing the plate in a cell culture box and standing for several hours until the Matrigel is completely solidified;

(2) starving the tumor cells in the logarithmic growth phase with a serum-free medium for 4 hours, and then digesting and collecting the tumor cells for later use;

(3) 200. mu.L of serum-free medium containing 5X 10 cells was added to the upper chamber of each Transwell chamber⁴Step (2) treated tumor cells and different concentrations of protein (0, 25, 50 and 100. mu.g/mL), 500. mu.L of cell culture medium containing 20% (w/v) FBS was added to the lower chamber, and the treated plate was placed in a cell culture chamber at 37 ℃ and 5% CO₂Culturing for 24 hours; the proteins are respectively CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B 12;

(4) taking out the small chamber in the cultured culture plate, sucking out the culture medium in the small chamber, and gently erasing the uninvaded cells on Matrigel in the small chamber by using a cotton swab;

(5) adding 4% poly methanol into the culture plate to fix the cells in the chamber for 10 min;

(6) absorbing the fixing solution, adding 500 mu L of 0.5% crystal violet staining solution into the culture plate, and staining the cells for 30min at room temperature;

(7) taking out the small chamber, washing the small chamber for 3 times by using distilled water, placing the small chamber on a glass slide, and taking a picture under a microscope for recording;

(8) placing the photographed chamber into another clean 24-hole cell culture plate, adding 100 mu L of 33% (w/v) acetic acid solution into the plate, eluting the crystal violet bound on the cells, collecting the eluent, placing the eluent in an enzyme labeling instrument to detect the light absorption value at 570nm, and recording the experimental result.

As shown in FIG. 4, the CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B12 proteins can significantly inhibit the invasion of DU145 cells at a concentration of 25 μ L/mL; the proteins of CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B12 can obviously inhibit the MCF-7 cell invasion at the concentration of 50 mu L/mL.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

<110> river-south university

<120> CD 133-targeting binding proteins and uses thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of CD133-2B1 protein

<400> 1

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Lys Ile Thr

20 25 30

Ala Glu Phe Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Thr Ile Ser Arg His Ser Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Ala Ser Val Gly Lys Phe Pro Trp Val Trp Val Ser Glu

100 105 110

Ala Thr Val Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Ala Ala Ala

130

<210> 2

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of CD133-2C4 protein

<400> 2

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ile

20 25 30

Ser Glu Tyr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ser Ile Thr Asn Ala Asp Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Ala Val Tyr Val Phe Pro Phe Gly Leu Ala Asp Glu Val

100 105 110

Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<210> 3

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of CD133-2C10 protein

<400> 3

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Ile Ser

20 25 30

Pro Glu Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Thr Ile Asp Gly Pro Asn Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Ala Arg Val Arg Ser Ala Val Leu Gly Leu Arg Leu Ser

100 105 110

Ala Asn Val Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Ala Ala Ala

130

<210> 4

<211> 126

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of CD133-3B4 protein

<400> 4

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Met Leu Ile

20 25 30

Asn Gln Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Gly Ile Leu Asp Lys Asp Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Val Ser Lys Trp Ser Lys Asp Ala Met Ser Phe

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<210> 5

<211> 135

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of CD133-3B12 protein

<400> 5

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Arg Ile Asn

20 25 30

Asn Gln Asp Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Gly Ile Arg Thr Gly Asp Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ala Ala Val Tyr

85 90 95

Tyr Cys Ala Gly Phe Val Met Trp Ala Trp Glu Val Trp Ser Ser His

100 105 110

Pro Met Trp Lys Pro Tyr Leu Arg Tyr Trp Gly Gln Gly Thr Leu Val

115 120 125

Thr Val Ser Ser Ala Ala Ala

130 135

<210> 6

<211> 132

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of CD133-4B9 protein

<400> 6

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ser Ile Thr

20 25 30

Ser Glu Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Thr Ile Lys Ala His Asn Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Thr His Ile Ala Met Lys Gly Thr Trp Lys Asn Trp His

100 105 110

Pro Gln Ser Leu His Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

115 120 125

Ser Ala Ala Ala

130

<210> 7

<211> 130

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of CD133-4B12 protein

<400> 7

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Ser

20 25 30

Pro Glu Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Thr Ile Tyr Met Arg Asp Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Gly Arg Gly Trp Ser Gly Trp Ser Trp Asn Ser

100 105 110

Asn Val Lys Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala

130

<210> 8

<211> 393

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding CD133-2B1 protein

<400> 8

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatataag attaccgctg agtttatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaaccattt cgaggcatag cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcggca 300

tctgtgggga agtttccgtg ggtttgggtt tcggaggcca cggtcaacta ttggggtcag 360

ggaaccttgg tcaccgtctc gagcgcggcc gca 393

<210> 9

<211> 384

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding CD133-2C4 protein

<400> 9

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatttaag tttatctctg agtatatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaagcatta ctaacgcaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcggca 300

gtttatgttt ttccgtttgg gttggccgac gaggtcaggt attggggtca gggaaccctg 360

gtcaccgtct cgagcgcggc cgca 384

<210> 10

<211> 393

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding CD133-2C10 protein

<400> 10

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagatagc attagccctg agtctatgag ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaaccattg atggcccaaa cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcggca 300

agggttcgtt ctgcggttct gggtttgagg ctgtccgcga acgtgagcta ttggggtcag 360

ggaaccctgg tcaccgtctc gagcgcggcc gca 393

<210> 11

<211> 378

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding CD133-3B4 protein

<400> 11

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatatatg cttatcaatc aggatatgac ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaggcattc tggacaaaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

gatgtttcga agtggtcgaa ggacgccatg tcgttttggg gtcagggaac cctggtcacc 360

gtctcgagcg cggccgca 378

<210> 12

<211> 405

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding CD133-3B12 protein

<400> 12

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagttagg attaacaatc aggatatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaggcattc ggacgggtga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacgccg cggtatatta ttgcgcgggg 300

ttcgtcatgt gggcttggga ggtttggagt agtcatccga tgtggaagcc gtacctgagg 360

tattggggtc agggaaccct ggtcaccgtc tcgagcgcgg ccgca 405

<210> 13

<211> 396

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding CD133-4B9 protein

<400> 13

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagatagc attacctctg agaatatggc ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaaccatta aggcccataa cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaca 300

catattgcta tgaaggggac ttggaagaat tggcatcccc agtcgttgca ctattggggt 360

cagggaaccc tggtcaccgt ctcgagcgcg gccgca 396

<210> 14

<211> 390

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding CD133-4B12 protein

<400> 14

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatatacg attagccctg aggctatgac ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaaccattt atatgcgaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

gttgggcggg ggtggagtgg gtggagttgg aactccaacg tgaagtattg gggtcaggga 360

accctggtca ccgtctcgag cgcggccgca 390

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PCR amplification of CD133 binding protein Forward primer

<400> 15

atggcccagg tgcagctgt 19

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> reverse primer for PCR amplification of CD133 binding protein

<400> 16

tctgcggccg cgctcgagac 20

Claims

1. A binding protein that targets CD133, characterized by: is a CD133-2B1 protein; or a binding protein formed by combining at least one of CD133-2C4 protein, CD133-2C10 protein, CD133-3B4 protein, CD133-3B12 protein, CD133-4B9 protein and CD133-4B12 protein with CD133-2B1 protein;

the amino acid sequence of the CD133-2B1 protein is shown in SEQ ID NO. 1;

the amino acid sequence of the CD133-2C4 protein is shown in SEQ ID NO. 2;

the amino acid sequence of the CD133-2C10 protein is shown in SEQ ID NO. 3;

the amino acid sequence of the CD133-3B4 protein is shown in SEQ ID NO. 4;

the amino acid sequence of the CD133-3B12 protein is shown in SEQ ID NO. 5;

the amino acid sequence of the CD133-4B9 protein is shown in SEQ ID NO. 6;

the amino acid sequence of the CD133-4B12 protein is shown in SEQ ID NO. 7.

2. A nucleotide sequence encoding the CD133 targeted binding protein of claim 1, wherein: is a nucleotide sequence encoding said CD133-2B1 protein; or a nucleotide sequence formed by the combination of at least one of a nucleotide sequence for coding the CD133-2C4 protein, a nucleotide sequence for coding the CD133-2C10 protein, a nucleotide sequence for coding the CD133-3B4 protein, a nucleotide sequence for coding the CD133-3B12 protein, a nucleotide sequence for coding the CD133-4B9 protein and a nucleotide sequence for coding the CD133-4B12 protein and a nucleotide sequence for coding the CD133-2B1 protein.

3. The nucleotide sequence encoding a CD133 targeting binding protein according to claim 2, wherein:

the nucleotide sequence for coding the CD133-2B1 protein is shown as SEQ ID NO. 8;

the nucleotide sequence for coding the CD133-2C4 protein is shown as SEQ ID NO. 9;

the nucleotide sequence for coding the CD133-2C10 protein is shown as SEQ ID NO. 10;

the nucleotide sequence of the coded CD133-3B4 protein is shown as SEQ ID NO. 11;

the nucleotide sequence for coding the CD133-3B12 protein is shown as SEQ ID NO. 12;

the nucleotide sequence of the CD133-4B9 protein is shown in SEQ ID NO. 13;

the nucleotide sequence of the CD133-4B12 protein is shown in SEQ ID NO. 14.

4. The method of making a CD133 targeting binding protein of claim 1, wherein: cloning a nucleotide sequence of the binding protein of the targeting CD133 as claimed in claim 2 or 3 into an expression vector to construct a recombinant expression plasmid, and transferring the recombinant expression plasmid into a host cell for protein expression and purification to obtain the binding protein of the targeting CD 133; or the binding protein targeting CD133 is obtained by a protein synthesis method.

5. Use of the CD 133-targeted binding protein of claim 1 in the preparation of anti-CD 133⁺Application in tumor medicine.

6. Use according to claim 5, characterized in that: the tumor is prostate cancer or breast cancer.