CN113912694B

CN113912694B - CD 133-targeting binding proteins and uses

Info

Publication number: CN113912694B
Application number: CN202111041203.1A
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: 2023-05-30
Anticipated expiration: 2039-07-24
Also published as: CN113912694A; CN113912697B; CN110407924B; CN113912695B; CN113912697A; CN113912696B; CN113912695A; CN110407924A; CN113912696A

Abstract

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

Description

CD 133-targeting binding proteins and uses

The application is a divisional application of Chinese patent application with the application number of 2019106722220 and the invention name of CD133 targeting binding protein and application thereof.

Technical Field

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

Background

The characteristics of the tumor cells such as immortality, mobility and loss of contact inhibition make the tumor one of the diseases which are extremely difficult to cure for human beings. At present, the tumor treatment means mainly comprise surgical excision, 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 a tumor have self-renewing ability and are capable of producing 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 physical and chemical 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 development of proteins targeting tumor stem cell surface specific marker molecules by genetic engineering techniques might become new eosin for the treatment of cancer.

As one of the stem cell and tumor stem cell surface specific marker proteins, CD133 was originally isolated from CD34 hematopoietic stem cells by artificial AC133 monoclonal antibodies, yin and the like. CDl33 belongs to one of the Prominin family members, genes are located on human chromosome 4, are approximately 152kb in size, and contain at least 37 exons. The CD133 protein consists of 865 amino acids and has a molecular weight of about 120kDa and comprises: extracellular NH ₂ -a terminal, 5-fold transmembrane domain, 2 extracellular loop structures, 2 cysteine-rich intracellular loop structures, intracellular-COOH structures. CD133 has been shown to be a surface marker for hematopoietic and neural stem cells; CD133 is expressed in tumor stem cells of tumors such as brain glioma, colon cancer, malignant melanoma, etc., and CD133 is expressed in brain tumor, breast cancer and colon cancer ⁺ Cell ratio CD133 ^- The cell has strong tumorigenicity; through gene chip and clinical analysis, CDl33 is found ⁺ Tumor cells exhibit greater proliferation capacity and have a poorer prognosis. The above results show that CDl33 expression in tumor stem cells has important significance for cancer development. Therefore, CD133 can be used as a new target for developing anti-tumor 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 crossing of blood brain barrier, low immunogenicity, strong specificity and the like. The related research results show that the binding protein can effectively target antigen and has anti-tumor effect in vitro and in vivo. Currently, there is no study on the protein binding to the tumor stem cell marker protein CD 133.

Disclosure of Invention

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

It is another object of the present invention to provide a method for preparing the CD 133-targeting binding protein described above.

It is a further object of the present invention to provide the use of a CD 133-targeting binding protein as described above.

The aim of the invention is achieved by the following technical scheme:

a binding protein targeting CD133, which is the CD133-2C4 protein; or a binding protein formed by combining at least one of CD133-2B1 protein, CD133-2C10 protein, CD133-3B4 protein, CD133-3B12 protein, CD133-4B9 protein and CD133-4B12 protein with CD133-2C4 protein; the CD 133-targeting binding protein consists of 1 heavy chain variable region;

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

MAQVQLLESGGGLVQPGGSLRLSCAASGYKITAEFMGWVRQAPGKGLEWVSTISRHSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASVGKFPWVWVSEATVNYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGFKFISEYMGWVRQAPGKGLEWVSSITNADGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAVYVFPFGLADEVRYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGDSISPESMSWVRQAPGKGLEWVSTIDGPNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARVRSAVLGLRLSANVSYWGQGTLVTVSSAAA；

the amino acid sequence of the CD133-3B4 protein is as follows (also shown as 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 as SEQ ID NO. 6):

MAQVQLLESGGGLVQPGGSLRLSCAASGDSITSENMAWVRQAPGKGLEWVSTIKAHNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHIAMKGTWKNWHPQSLHYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGYTISPEAMTWVRQAPGKGLEWVSTIYMRDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGRGWSGWSWNSNVKYWGQGTLVTVSSAAA。

the nucleotide sequence encoding the CD 133-targeting binding protein is the nucleotide sequence encoding the CD133-2C4 protein; or a nucleotide sequence formed by combining at least one of a nucleotide sequence encoding the CD133-2B1 protein, a nucleotide sequence encoding the CD133-2C10 protein, a nucleotide sequence encoding the CD133-3B4 protein, a nucleotide sequence encoding the CD133-3B12 protein, a nucleotide sequence encoding the CD133-4B9 protein and a nucleotide sequence encoding the CD133-4B12 protein with a nucleotide sequence encoding the CD133-2C4 protein.

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

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATAAGATTACCGCTGAGTTTATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTTCGAGGCATAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGCATCTGTGGGGAAGTTTCCGTGGGTTTGGGTTTCGGAGGCCACGGTCAACTATTGGGGTCAGGGAACCTTGGTCACCGTCTCGAGCGCGGCCGCA；

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

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTTAAGTTTATCTCTGAGTATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTACTAACGCAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGCAGTTTATGTTTTTCCGTTTGGGTTGGCCGACGAGGTCAGGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

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

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGATAGCATTAGCCCTGAGTCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTGATGGCCCAAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGCAAGGGTTCGTTCTGCGGTTCTGGGTTTGAGGCTGTCCGCGAACGTGAGCTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

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

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATATGCTTATCAATCAGGATATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAGGCATTCTGGACAAAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGAGAGATGTTTCGAAGTGGTCGAAGGACGCCATGTCGTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

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

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGTTAGGATTAACAATCAGGATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAGGCATTCGGACGGGTGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACGCCGCGGTATATTATTGCGCGGGGTTCGTCATGTGGGCTTGGGAGGTTTGGAGTAGTCATCCGATGTGGAAGCCGTACCTGAGGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

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

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGATAGCATTACCTCTGAGAATATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTAAGGCCCATAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACACATATTGCTATGAAGGGGACTTGGAAGAATTGGCATCCCCAGTCGTTGCACTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

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

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATACGATTAGCCCTGAGGCTATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAACCATTTATATGCGAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGAGAGTTGGGCGGGGGTGGAGTGGGTGGAGTTGGAACTCCAACGTGAAGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA。

it can be seen that the nucleotide sequences encoding the CD133-2B1 protein and the CD133-2C10 protein are each composed of 393 bases encoding 131 amino acids; the nucleotide sequence for encoding the CD133-2C4 protein consists of 384 bases and encodes 128 amino acids; the nucleotide sequence for encoding the CD133-3B4 protein consists of 378 bases and encodes 126 amino acids; the nucleotide sequence for encoding the CD133-3B12 protein consists of 405 bases and encodes 135 amino acids; the nucleotide sequence for encoding the CD133-4B9 protein consists of 396 bases and encodes 132 amino acids; the nucleotide sequence encoding the CD133-4B12 protein consists of 390 bases and encodes 130 amino acids.

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

The above binding protein targeting CD133 is used for preparing anti-CD 133 ⁺ Application of tumor medicine.

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 segments from a human protein phage library based on phage display technology, can carry out protein expression only through a prokaryotic system, can greatly simplify the production process, reduces the protein production cost, and can be obtained without immunizing human body with antigen.

(2) The binding protein provided by the invention is composed of only one heavy chain variable region 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 human bodies, and can be used for developing tumor protein medicines in future.

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

Drawings

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

FIG. 2 is a graph of the results of detecting the effect of binding proteins on the proliferative capacity of tumor cells by the MTT method; wherein 7 proteins (CD 133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9 and CD133-4B 12) are used for culturing tumor cells DU145 and MCF-7 at different concentrations (0, 25, 50 and 100 mu g/mL), 30 mu L of MTT is added into each hole after 72 hours for incubation for 4 hours, 200 mu L of DMSO is added for fully dissolving formazan, and an enzyme-labeling instrument is used for detecting OD570 absorbance; in the figure, A is a result graph of influence on proliferation capacity of tumor cells DU145, and B is a result graph of influence on proliferation capacity of tumor cells MCF-7; * p <0.05, relative to 0 μg/mL (n=3).

FIG. 3 is a graph showing the results of detection of the effect of binding proteins on tumor apoptosis by flow cytometric analysis and Annexin V/PI double-stain kit; wherein 7 proteins (CD 133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B 12) co-culture tumor cells DU145 and MCF-7 at a concentration of 50. Mu.g/mL, while setting a PBS control group; after 48 and h are cultured, detecting apoptosis by using an Annexin V/PI double-dyeing kit and a flow cytometry; in the figure, A is the result of inducing apoptosis of tumor cells DU-145, and B is the result of inducing apoptosis of tumor cells MCF-7; * p <0.05, relative to the no binding protein control (n=3).

FIG. 4 is a graph of the results of detection of binding proteins by the Transwell invasion method on tumor cell invasion; wherein 7 proteins (CD 133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B 12) were cultured on tumor cells DU145 and MCF-7 at various concentrations (0, 25, 50, 100. Mu.g/mL), followed by induction of cell invasion 24 h with 20% serum-containing medium, followed by staining of the cells with 0.5% crystal violet and photographing under a microscope, elution of the crystal violet bound to the cells with 33% acetic acid, collection of the eluate, detection of OD570 absorbance by a microplate reader; in the figure, A is the influence of protein on tumor cell DU-145, and B is the influence of protein on tumor cell MCF-7 invasion; * p <0.05, relative to 0 μg/mL (n=3).

Detailed Description

The present invention will be described in detail with reference to examples and drawings, but 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. The reagents and materials used in the present invention are commercially available unless otherwise specified.

The preparation method of the reagent used in the examples is as follows:

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

The reagents were dissolved in 800mL deionized water, and the volume was set to 950mL and autoclaved at 121℃for 30min. After the culture medium is cooled to about 60 ℃, 20 mu L of 100 mu g/mL ampicillin and 1mL of 20% glucose are added into each 19mL of culture medium, the mixture is poured into a plate after uniform mixing, and the plate is preserved at 4 ℃ for standby.

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

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

(3) 50×TAE DNA electrophoresis buffer: 242g of Tris-base, na ₂ EDTA•2 H ₂ O37.2 g, glacial acetic acid 57.1mL.

The reagent, solution, was dissolved in 800mL deionized water and then sized to 1L, diluted to 1 x TAE running buffer when used, and stored at room temperature.

(4) PBS buffer (ph=7.4): KH (KH) ₂ PO ₄ 0.24g、NaCl 8g、KCl 0.2g、Na ₂ HPO ₄ • 12H ₂ O 9.07g。

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

(5) PEG solution: 73g of NaCl and 6000 g of PEG.

The reagents were dissolved in deionized water and fixed to a volume of 500 mL, and after bacteria were filtered using a 0.2 μm filter, stored at 4 ℃ for use.

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

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

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

The reagents were dissolved in deionized water, fixed to 190mL, and autoclaved at 121℃for 30min. After the medium was cooled to about 60℃and 20. Mu.L of 100. Mu.g/mL ampicillin and 1mL of 20% glucose were added per 19mL of medium, the mixture was poured into a plate and stored at 4℃for further use.

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

The reagents were dissolved in 400 mL deionized water and then sized 500 mL, diluted to 1 XSDS-PAGE running buffer for use, and stored for long periods at room temperature.

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

Mixing the reagent and the solution uniformly, metering the volume to 5mL, subpackaging the mixture into 500 [ mu ] L/serving, adding 25 [ mu ] L of beta-mercaptoethanol per serving before use, and preserving the mixture at room temperature for a long time.

(10) Coomassie brilliant blue R-250 staining solution: coomassie brilliant blue R-250 g, isopropanol 250mL, acetic acid 100mL, ddH ₂ O 650mL。

Mixing the reagents and the solvent uniformly, dissolving fully, and preserving for a long time at room temperature for standby.

(11) Coomassie brilliant blue staining and decolorizing solution: 100mL of acetic acid, 50mL of ethanol and ddH ₂ O 850mL。

Mixing the solvents, and preserving at room temperature.

(12) Bacteria-destroying buffer solution: 14.6g of NaCl and 2.42g of Tris base.

Mixing the above reagents with 1L water, dissolving thoroughly, preserving at-4deg.C, and adding 100×PMSF stock solution with final concentration of 1×PMSF stock solution. Loading buffer solution: the same bacteria breaking buffer solution; washing impurity buffer solution: at present, 9.9mL of bacteria-destroying buffer solution is measured, 100 mu L of 2M imidazole is added, and the materials are fully and uniformly mixed; elution buffer: at present, 9mL of the bacteria-destroying 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 dissolving in 20 mL deionized water, filtering the mixed solution with a 0.2 mu M filter for sterilization, subpackaging into 1mL for each tube, and preserving at-20 ℃ for later use.

(14) Ampicillin solution (100 mg/mL): weighing 1g ampicillin powder, fully dissolving in 10 mL deionized water, filtering the mixed solution with 0.2 mu M filter for sterilization, subpackaging into 1mL per tube, and preserving at-20deg.C for later use.

(15) 2% BSA-PBS buffer: 1g of bovine serum albumin powder is weighed and fully dissolved in 50mL of PBS buffer solution, and the mixed solution is filtered and sterilized by a 0.2 mu M filter, and is prepared for use or is stored for a long time at minus 20 ℃.

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

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

(18) 1M sulfuric acid solution: the measuring cylinder is used for measuring 9.8 of mL of concentrated sulfuric acid, the concentrated sulfuric acid is slowly added into 187 of mL of deionized water, and the mixture is fully and uniformly mixed and stored at room temperature for standby.

(19) 10% Ammonium Persulfate (APS): weighing 0.1 g ammonium persulfate powder, dissolving in 1 mL deionized water, subpackaging into 200 mu L of each tube, and preserving at-20 ℃.

(20) IPTG solution (500 mM): weighing IPTG powder 11.915 g, fully dissolving in deionized water, fixing volume to 100 mL, filtering the mixed solution with 0.2 μm filter for sterilization, subpackaging into 1-mL tubes, and long-term storing at-20deg.C.

(21) 30% glycerol solution: 15 g mL glycerin is measured by a measuring cylinder and added into 35 g mL deionized water, and after being fully mixed, the mixture is filtered and sterilized by a 0.22 mu M filter, and the mixture is preserved at the temperature of minus 4 ℃ for standby.

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

(23) 2M imidazole: 1.14g of imidazole powder is weighed and fully dissolved in 10mL of bacteria breaking buffer solution, and the mixture is preserved at the temperature of minus 4 ℃ for standby.

Example 1 preparation of helper phage

(1) Coating TG1 glycerol bacteria purchased from Biyun days on a culture dish containing TYE solid culture medium by a three-area lineation method, and then placing the culture dish in a 37 ℃ constant temperature incubator for culturing for 12-16 hours;

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

(3) Transferring the bacterial culture solution in the step (2) to another tube of 5mL 2 XTY liquid culture medium according to the ratio of 1:100, placing the tube in a constant temperature shaking table at 37 ℃ for culturing at 250rpm until the bacterial solution OD ₆₀₀ About 0.5;

(4) Helper phage KM13 was diluted gradient with PBS (from 10 ¹² /mL ~10 ⁴ /mL）；

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

(6) Spreading 3mL of melted 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 culture dish; the specific operation steps are as follows: before use, the top agar is heated to be completely melted, then incubated in water and cooled to 42 ℃, and then mixed with the mixture A obtained in the step (5). Standing at room temperature until the culture medium is completely coagulated, 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 pipette, inoculating 5mL of the bacterial solution (OD) ₆₀₀ =0.5, cultured for 2-3 hours at 37 ℃ with a constant temperature shaker at 250rpm according to the method in step (3);

(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 ℃ by a shaking table at 250 rpm;

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

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

(11) Detecting the sensitivity of the prepared helper phage to pancreatin: to 1mL trypsin solution was added 10 ¹⁰ The helper phage KM13 was incubated at room temperature for 30 minutes. The helper phage KM13 was then gradient diluted with PBS (from 10 ¹⁰ To 10 ² Phage), 200 μl TG1 bacteria (OD ₆₀₀ =0.5, preparation and infection methods as described above) were spread on TYE solid medium (containing kanamycin at a final concentration of 50 μg/mL) and incubated overnight at 37 ℃. After infection of the bacteria with pancreatin-treated phages, the number of clones obtained should be at least 10 less than in the untreated group ⁶ Multiple times. Otherwise, the helper phage preparation is discarded and another plaque is picked for re-preparation.

EXAMPLE 2 expression of phage binding protein library

(1) Taking a proper amount of human binding protein phage library (Source Bioscience, human Domain Antibody Library (DAb), london, UK) into 500 mL 2×TY liquid medium (the medium additionally contains 100 μg/mL ampicillin and 4% (w/v) glucose), culturing at 37deg.C with a constant temperature shaker at 250rpm until the bacteria OD ₆₀₀ =0.5；

(2) 2X 10 prepared in example 1 was added ¹² And (3) adding auxiliary phage into the bacterial liquid obtained in the step (1), uniformly mixing, and placing in a water bath at 37 ℃ for 30 minutes. The 500 mL cultures were aliquoted into 50 mL tubes per tube, 3200. 3200 g centrifuged for 10 minutes, the supernatant discarded, the pellet resuspended and transferred to an Erlenmeyer flask containing 500 mL 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 shaking table at a constant temperature of 25 ℃ and shaking and culturing at 250rpm for 16-20 hours.

EXAMPLE 3 purification of phage binding protein library

(1) The overnight cultures from example 2 were aliquoted into 50mL tubes and centrifuged at room temperature 3200g for 20 minutes;

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

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

(4) Centrifuging 3200g at 4 ℃ for 30 minutes, discarding the supernatant, re-suspending the precipitate by using 1mL of PBS, centrifuging 3200g at 4 ℃ for 5 minutes, and filtering and sterilizing the supernatant by using a 0.45 mu M filter;

(5) Phage library titers were estimated by measuring absorbance at 260 nm: the 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 = OD ₂₆₀ ×100×22.14×10 ¹⁰ . The prepared phage library can be stored at 4 ℃ for 2 weeks or frozen at-80 ℃ for a long time.

Example 4 screening of phage binding protein library for proteins that bind to the extracellular segment of CD133

(1) CD133 extracellular domain protein (sequence see GenBANK number: NP 006008.1) was synthesized at the same time by the company of Haibotai Biotechnology, 4 mL was added to NUNC immune tube and dissolved in PBS buffer, and the final concentration was 100. Mu.g/mL of CD133 extracellular domain protein dilution, and left at 4℃overnight;

(2) Discarding the coating liquid in the step (1) overnight, gently flushing the inner wall of the immune tube with PBS buffer solution for 3 times, then adding 4mL of 2% BSA-PBS blocking solution, and standing at room temperature for 2 hours;

(3) Discarding the blocking solution in step (2), gently washing the inner wall of the immune tube 3 times with PBS buffer, adding 4mL of 2% BSA-PBS blocking solution (containing 5×10 ¹² Phage prepared in example 3), and left at room temperature for 1 hour;

(4) Discarding the sealing solution in the step (3), gently flushing the inner wall of the immune tube by using PBST buffer solution for 10 times, adding 4mL of 1mg/mL trypsin solution, and standing at room temperature for 1 hour to enable phage to be thoroughly digested from the immune tube to obtain digestive juice A;

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

(6) Selecting a single colony of TG1 on the culture medium in the step (5), inoculating the single colony on 5mL 2 xTY liquid culture medium, and culturing the single colony at a constant temperature of 37 ℃ on a shaking table at 250rpm for overnight;

(7) And (3) the bacterial liquid in the step (6) is prepared according to the volume ratio of 1:100 proportion is inoculated into another test tube containing 5mL 2 xTY liquid culture medium, a constant temperature shaking table at 37 ℃ is used for culturing at 250rpm until the bacterial liquid OD ₆₀₀ =0.5。

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

(9) Discarding the supernatant, and re-suspending the precipitate with 1mL of 2×TY liquid medium to obtain a re-suspension A; 6 culture dishes containing TYE solid medium (containing 100 mug/ml ampicillin and 4% (w/v) glucose) were coated with 166 uL of heavy suspension A per plate, and the coated dishes were placed in a 37℃incubator for overnight incubation;

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

(11) Transferring the scraped bacterial liquid into a conical flask containing 500mL of 2 xTY liquid culture medium (the culture medium contains 4% (w/v) glucose and 100 mug/mL ampicillin), placing the conical flask in a constant temperature shaking table at 37 ℃ for culture until the bacterial liquid OD600 = 0.5, adding the auxiliary phage KM13 prepared in the example 1, infecting bacterial liquid, placing the conical flask in a constant temperature shaking table at 30 ℃ for shake culture at 250rpm for overnight;

(12) Taking the overnight culture obtained in the step (11), adding a PEG solution for phage purification (refer to the previous step), and measuring the titer of the phage library obtained by screening by gradient dilution plating;

(13) Subsequent 4 rounds of phage library panning were performed with reference to the previous method (i.e., repeating steps (1) - (12)) and the phage from the previous round was screened sequentially.

EXAMPLE 5 selection of monoclonal from library for ELISA validation

(1) After completion of 5 rounds of phage library panning, a sterile 96-well plate (designated as A plate) was used, 200mL of 2 XTY liquid medium (medium containing 100. Mu.g/mL ampicillin, 4% (w/v) glucose) was added to each well, and the single clone was picked from the overnight dish obtained by the 5 th screening with a sterile tip, inoculated into a 96-well plate containing the culture solution, incubated at 37℃on a thermostatic shaker, and shake-cultured at 250rpm overnight;

(2) Taking another sterile 96-well plate (named B plate), adding 200 mu L of 2 XTY liquid medium (the 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 to the 96-well plate, and placing the plate on a constant temperature shaking table at 37 ℃ for shaking culture at 250rpm for 3 hours; adding 20% glycerol to the remaining overnight culture of the A plate in the step (1) to obtain glycerol bacterial liquid stock, and freezing at-80deg.C for a long time;

(3) Each well of the culture in the plate after 3 hours of culturing in the plate B in the step (2) was transferred to a sterile 1.5mL EP tube containing 50. Mu.L of 4X 10 ⁸ 2 XTY liquid medium of helper phage KM13, gently mixed, and the EP tube was incubated at 37℃for 1 hour;

(4) After incubation, 1.5mL EP tube 3200g was centrifuged for 10 min and the supernatant 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), incubated at 25℃on a shaking table at 250rpm overnight;

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

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

(7) Washing the coated ELISA plate with PBS for 3 times, adding 250 mu L of 2% BSA-PBS into each hole, sealing the ELISA plate for 2 hours at room temperature, and washing the ELISA plate with PBS for 3 times for later use;

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

(9) Wash plate 5 times with PBST, add 100 μl of HRP-labeled anti-M13 conjugate (diluted with 2% BSA-PBS) at a volume ratio of 1:10000 per well, incubate for 1 hour at room temperature;

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

EXAMPLE 6 preparation of DH 5. Alpha. BL21 (DE 3) E.coli competent cells

(1) Respectively streaking a small amount of escherichia coli DH5 alpha and BL21 (DE 3) bacterial liquid on a culture dish containing an LB solid culture medium by a three-zone streaking method, and then placing the culture dish in a constant-temperature incubator at 37 ℃ for culturing for 14-16 hours;

(2) E.coli DH5 alpha and BL21 (DE 3) single colonies are respectively picked from a culture dish containing LB solid culture medium, respectively inoculated into test tubes containing 5 mL LB liquid culture medium, and shake-cultured at a constant temperature of 37 ℃ and a shaking table at 220rpm for about 12 hours;

(3) Respectively inoculating the two bacterial culture solutions into conical flasks containing 50 mL of LB liquid culture medium according to the volume ratio of 1:100, and carrying out shaking culture at a constant temperature of 37 ℃ and a shaking table at 220rpm for 2-3 hours until the bacterial liquid OD ₆₀₀ =about 0.5;

(4) Transferring the bacterial solutions cultured in the step (3) into 2 other sterile 50 mL centrifuge tubes respectively, and placing the bacterial solutions in ice water mixed solution for standing for 10 minutes;

(5) Placing the centrifuge tube in the step (4) in a refrigerated centrifuge at 4 ℃ for 5 minutes by 3000 g;

(6) Discarding supernatant, collecting 10. 10 mL precooled 0.1 mol/L CaCl ₂ The solution gently resuspends the bacterial precipitate, and then the centrifuge tube is placed in ice water mixed solution for standing for 30 minutes;

(7) Centrifuging the centrifuge tube at a temperature of 3000 g for 5 minutes at a temperature of 4 ℃ of a refrigerated centrifuge;

(8) Discarding supernatant, collecting 3. 3mL precooled 0.1 mol/L CaCl ₂ The solution is gently resuspended and precipitated to obtain a resuspension B;

(9) 3mL of precooled 30% (v/v) sterile glycerol is added into the heavy suspension B obtained in the step (8), and after gentle mixing, the mixed solution is split into 100 mu L of each tube and frozen for a long time at-80 ℃.

Example 7 transformation of recombinant plasmid expressing CD133 binding protein into DH 5. Alpha. Competent cells

Construction of recombinant plasmid of CD 133-binding protein

(1) The positive monoclonal in example 5 was used in a volume ratio of 1:1000 ratio inoculation was transferred to 5mL of 2 XTY liquid medium (medium containing 100. Mu.g/mL ampicillin, 4% (w/v) glucose) for overnight incubation;

(2) 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 the CD133 binding protein, and amplifying the nucleotide sequence of the CD133 binding protein; wherein:

The PCR reaction system is as follows: 1. Mu.L of template (overnight culture from step (1)), 1. Mu.L of primers (forward primer/reverse primer) each, 10. Mu. L, dNTP mixture of 5 XPrimestar buffer (2.5 mM each) 4. Mu. L, primerStarDNA polymerase 0.5. Mu.L, sterile deionized water to 25. Mu.L;

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

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

(4) Taking a proper amount of CD133 binding protein nucleotide sequence gel recovery product, adding restriction enzymes NotI and NocI into the gel for enzyme digestion, performing 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 the temperature of-20 ℃ for standby

(5) Taking proper amount of expression vector pET28a, adding the expression vector pET28a into the expression vectorRestriction enzymeNotI andNoci, enzyme digestion is carried out, the enzyme digestion product is subjected to agarose gel electrophoresis, then target fragments on the gel are cut and are subjected to gel recovery, and the gel recovery product is stored at the temperature of minus 20 ℃ for standby

(6) Taking a proper amount of CD133 binding protein nucleotide sequence, performing enzyme digestion on the obtained product, performing enzyme digestion on the expression vector pET28a, performing enzyme digestion on the obtained product, adding ligase, and standing at 37 ℃ for overnight connection to obtain a connection product; wherein:

the connection reaction system is as follows: pET28a double cleavage product 0.03pmol, target fragment double cleavage product 0.3pmol, enzyme ligation buffer 2.5. Mu. L, T4DNA ligase 1. Mu.L, sterile water make up to 25. Mu.L.

(II) transformation of anti-CD 133 recombinant plasmid into DH 5. Alpha. Competent cells

(1) Taking out a tube of DH5 alpha competent cells from the refrigerator at-80 ℃ and thawing on ice;

(2) 10. Mu.L of the ligation product was added to 100. Mu.L of DH 5. Alpha. Competent cells (prepared in example 6), gently mixed, and allowed to stand on ice for 30 minutes;

(3) Placing the mixture prepared in the step (2) in a constant-temperature water bath kettle at 42 ℃ for 90 seconds, quickly transferring the mixture to ice, standing and cooling for 2-3 minutes;

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

(5) Placing the cultured mixed solution in a centrifugal machine, centrifuging 3000g for 1 minute, and collecting mixed solution sediment;

(6) Discarding 900 mu L of supernatant, and re-suspending bacterial sediment by using the rest 100 mu L of culture solution;

(7) The mixed bacterial suspension is coated on a prepared culture dish containing LB solid medium (the culture medium contains 100 mug/mL ampicillin and 1% (w/v) glucose);

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

(9) Several single clones were randomly picked up on dishes and inoculated into 5mL of LB liquid medium (medium containing 100. Mu.g/mL ampicillin) respectively, shaking at 37℃on a constant temperature shaker, and shaking at 220rpm overnight.

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

(11) And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the positive clone appears on the target fragment.

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

(13) The obtained positive clones were sequenced, and the results showed that seven proteins were obtained, designated as CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, and CD133-4B12, and the coding nucleotide sequences thereof were 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, respectively.

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

Prokaryotic expression of binding proteins

(1) Transforming the recombinant plasmid of the CD133 binding protein prepared in example 7 into BL21 competent cells (for specific steps, refer to example 7);

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

(3) Taking the bacterial liquid cultured in the step (2), and mixing the bacterial liquid with the bacterial liquid according to the formula of 1: inoculating 100 volume ratio into a conical flask containing 100 mL of LB liquid medium (the medium contains 100 mug/mL ampicillin), shaking and culturing for 2-3 hours at 220rpm by a constant temperature shaking table at 37 ℃;

(4) Waiting bacteria liquid OD ₆₀₀ 0.6 to 1.0, 1mL of bacterial liquid is taken to be used for preparing the large non-induction bacterial liquidCarrying out subsequent experiments on the whole protein sample of enterobacteria and the residual bacterial liquid;

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

(6) Taking the bacterial liquid 1mL cultured in the step (5) for preparing an induced escherichia coli whole protein sample, placing the rest bacterial liquid at 4 ℃, centrifuging at 5000g for 5min, and collecting bacterial precipitate;

(7) Discarding the supernatant, and re-suspending the bacterial pellet obtained in the step (6) by using 20 mL precooled bacteria-destroying buffer solution;

(8) The bacterial suspension was transferred to a 50mL beaker, the bacterial suspension was sonicated in an ice box, and the ultrasonic pulverizer operating power: 40%, working for 4 seconds, stopping for 8 seconds, and crushing for 40 minutes;

(9) Transferring the crushed bacterial suspension to a clean 50mL centrifuge tube, and centrifuging at 4 ℃ for 40 minutes at 15000 g;

(10) Collecting the supernatant obtained after centrifugation in the step (9), taking 20 mu L of the supernatant from the supernatant for preparing a supernatant sample after thallus breakage, taking part of the precipitate for preparing a precipitate after thallus breakage (the precipitate is resuspended by 20 mu L of a bacteria breaking buffer solution), transferring the rest of the supernatant into another clean 50mL centrifuge tube, preserving at 4 ℃ and waiting for column purification.

(II) purification of binding proteins by column

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

(2) Adding the supernatant obtained in the step (I) 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 passing liquid is taken for preparing a column passing liquid sample;

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

(5) After the liquid in the step (4) is completely drained, adding 20 times of the volume of the impurity washing buffer solution to wash and purify the impurity proteins on the column, and taking 20 mu L of the impurity washing solution for preparing an impurity washing solution sample;

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

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

(8) And (3) taking 5mL of 20% ethanol to be added into a purification column after distilled water is drained, sealing the upper end and the lower end of the purification tube, and storing the purification column at 4 ℃ for a long time.

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

(1) Preparing 5mL of separating gel with the concentration of 12% (w/v) according to the specification formula, adding a gel filling mold to ensure that the liquid level is about 2 cm away from the top of the mold, then adding 1.5mL of absolute ethyl alcohol, and standing for 30 min at room temperature to solidify the separating gel;

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

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

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

(5) Adding 5 mu L of the sample prepared in the step (4) into an SDS-PAGE gel comb hole, carrying out electrophoresis at 80V voltage, adjusting the voltage to 110V when the sample runs through concentrated gel, and carrying out electrophoresis until a blue indication band of the sample runs to the bottom of the gel;

(6) Adding a proper amount of coomassie brilliant blue staining solution into the staining gel box, taking out the SDS-PAGE gel, placing the SDS-PAGE gel into the staining gel box, and staining for 30 minutes at room temperature;

(7) Discarding the staining solution, cleaning the staining solution on the gel by using clear water, and adding a proper amount of decoloring solution for decoloring;

(8) The target band on the SDS-PAGE gel is clearly visible, the gel is decolorized to be transparent, and the gel is photographed under white light for recording.

Experimental results show 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 purified CD133 binding protein binding to antigen Using ELISA

(1) Taking a new 96-well immune plate, respectively adding 100 mu L of antigen HER2, EGFR and antigen CD133 extracellular protein diluent (HER 2 and EGFR are purchased from Shanghai Biotechnology Co., ltd.) with a final concentration of 2 mu g/mL (a 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 buffer solution, removing residual liquid in the plate, adding 250 mu L of 2% (w/v) BSA-PBS 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 which is diluted by 50 times by using PBS into each hole, and incubating the immune plate at room temperature for 1 hour; the binding proteins are CD133-2B1 protein, CD133-2C4 protein, CD133-2C10 protein, CD133-3B4 protein, CD133-3B12 protein, CD133-4B9 protein and CD133-4B12 protein, respectively;

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

(5) Taking out the immune plate, washing the plate for 5 times by using PBST buffer solution, removing residual liquid in the plate, adding 100 mu L 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-labeled instrument to detect OD 450 nm absorbance value, and recording experimental data.

The results of the experiment are shown in FIG. 1, wherein BSA is a blank, HER2 and EGFR are independent 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 combined with the extracellular segment of CD 133.

Example 10 use of MTT method to examine the effect of purified binding proteins on tumor cell proliferation potency

(1) Taking a 96-well cell culture plate, adding 100 mu L of whole culture medium (RPMI 1640 containing 10% calf serum, the same applies below) containing 5000 tumor cells (human prostatic cancer cells DU145 or human breast cancer cells MCF-7) in logarithmic growth phase into each well, placing the culture plate in a cell culture box at 37deg.C, and 5% CO ₂ Culturing overnight;

(2) After the cells are completely adhered, the whole culture medium in the holes is changed into a serum-free culture medium (RPMI 1640, the same applies below), and the culture plates are placed in a cell culture box for starvation treatment for 4 hours;

(3) Sucking the serum-free culture medium after starvation treatment in the step (2), adding 100 mu L of 1% serum culture medium containing different concentrations of proteins (0, 25, 50 and 100 mu g/mL) into each hole, placing the culture plates in a cell culture box at 37 ℃ and 5% CO ₂ Culturing for 72 hours; the proteins are CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, and CD133-4B12, respectively;

(4) Sucking the liquid in the treated culture plate, adding mixed solution containing 100 mu L of serum-free culture medium and 20 mu L of MTT solution into each hole, and placing the mixed solution in a cell culture box at 37 ℃ and 5% CO ₂ Incubating for 4 hours;

(5) Sucking out mixed liquid in the culture plate, adding 200 mu L of DMSO into each hole, and then placing the culture plate in a shaking table for rapid shaking for 10min to fully dissolve the precipitate;

(6) The culture plate is placed in an enzyme-labeled instrument to detect the absorbance at 570nm, and the detection result is recorded.

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

Example 11 detection of tumor apoptosis induced by binding proteins Using flow cytometric analysis

(1) Taking a 6-well cell culture plate, adding 1mL of whole culture medium to each well, wherein the whole culture medium contains 5×10 tumor cells in logarithmic growth phase ⁶ The plates were placed in a cell incubator at 37℃with 5% CO ₂ Culturing overnight;

(2) After the cells are completely adhered, 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) Sucking out the medium in the holes in the step (2), adding 1mL of serum-free medium containing 50 mug/mL of protein into each hole, placing the culture plate in a cell culture box at 37 ℃ and 5% CO ₂ Culturing for 48 hours; the proteins are CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, and CD133-4B12, respectively;

(4) Cells in the culture plate were digested with pancreatin, collected in 1.5mL EP tubes, and washed 2 times with PBS buffer;

(5) The 4 Xbinding buffer in the Annexin V-FITC apoptosis kit was removed and ddH was used ₂ O was diluted to 1X, 195. Mu.L of 1X binding buffer was used to adjust the cell density to 5X 10 ⁶ individual/mL;

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

(7) 200 mu L of 1 Xbinding buffer solution is added into a 1.5mL EP tube, and the cell mixture prepared in the step (6) is added and mixed gently;

(8) The EP tube (1.5 mL) in step (7) was placed in a centrifuge 1000 g and centrifuged for 5 min, the supernatant was discarded, 190. Mu.L of 1 Xbinding buffer was taken to resuspend the cells, and 10. Mu.L of PI staining solution was added and gently mixed.

(9) The flow cytometry detects tumor cells undergoing apoptosis, and records experimental results.

As shown in FIG. 3, CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12 proteins significantly induced apoptosis of DU145 cells and MCF-7 cells at a concentration of 50. Mu.L/mL.

Example 12 Transwell invasion assay to examine the Effect of binding proteins on tumor cell invasion Capacity

(1) Placing a sterile Transwell chamber (8 μm aperture, 24-well plate) into a 24-well cell culture plate, sucking 50. Mu.L Matrigel (Matrigel) into the chamber, placing the plate into a cell culture box, standing for several hours, and completely solidifying the Matrigel;

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

(3) 200 mu L of serum-free culture medium is added into the upper chamber of each Transwell chamber respectively, wherein the culture medium contains 5 multiplied by 10 ⁴ Tumor cells treated in step (2) and proteins with different concentrations (0, 25, 50 and 100. Mu.g/mL), adding 500. Mu.L of cell culture medium containing 20% (w/v) FBS into a lower chamber, placing the treated plates in a cell culture incubator at 37 ℃ and 5% CO ₂ Culturing for 24 hours; the proteins are CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, and CD133-4B12, respectively;

(4) Taking out the cells in the cultured culture plate, sucking the culture medium in the cells, and gently wiping uninfected cells on Matrigel in the cells by using a cotton swab;

(5) Adding 4% poly-methanol into the culture plate to fix the cells in the cell for 10 min;

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

(7) Taking out the cell, cleaning with distilled water for 3 times, placing the cell on a glass slide, and photographing and recording under a microscope;

(8) The well-photographed chamber was placed in another clean 24-well cell culture plate, 100. Mu.L of 33% (w/v) acetic acid solution was added to the plate, crystal violet bound to the cells was eluted, the eluate was collected, and the absorbance at 570 nm was measured by an ELISA reader, and the experimental results were recorded.

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

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

<110> and university of south China

<120> CD133 targeting binding proteins and uses

<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> PCR amplified CD133 binding protein reverse primer

<400> 16

tctgcggccg cgctcgagac 20

Claims

1. A CD 133-targeting binding protein, characterized in that: is CD133-2C4 protein;

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

2. A nucleic acid encoding a CD 133-targeting binding protein of claim 1, wherein: is a nucleic acid encoding the CD133-2C4 protein.

3. The nucleic acid encoding a CD 133-targeting binding protein of claim 2, wherein: the sequence of the nucleic acid for encoding the CD133-2C4 protein is shown as SEQ ID NO. 9.

4. The method for preparing the CD 133-targeting binding protein of claim 1, wherein: cloning nucleic acid encoding the CD 133-targeted binding protein of claim 2 or 3 into an expression vector to construct a recombinant expression plasmid, transferring the recombinant expression plasmid into a host cell to perform protein expression and purification, and obtaining the CD 133-targeted binding protein; or by protein synthesis to obtain the binding protein targeting CD 133.

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

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