CN110407924A

CN110407924A - Target binding protein and its application of CD133

Info

Publication number: CN110407924A
Application number: CN201910672222.0A
Authority: CN
Inventors: 魏星; 陈柔
Original assignee: Jinan University
Current assignee: Jinan University; University of Jinan
Priority date: 2019-07-24
Filing date: 2019-07-24
Publication date: 2019-11-05
Anticipated expiration: 2039-07-24
Also published as: CN113912694A; CN113912697B; CN110407924B; CN113912695B; CN113912697A; CN113912696B; CN113912695A; CN113912696A; CN113912694B

Abstract

The invention discloses a kind of binding protein for targeting CD133 and its applications.The present invention screens the albumen in conjunction with CD133 extracellular fragment from source of people protein library, using display technique of bacteriophage, and finishing screen selects 7 binding proteins；By this 7 binding-protein gene amplifications, sequencing, and it is cloned into expression vector, it is combined the purifying of albumen pronucleus system expression, then carry out ELISA detection, MTT, Apoptosis, Transwell Matrigel, 7 binding proteins and antigen binding capacity are stronger as the result is shown, can effectively inhibit tumor cell proliferation, invasion, and being capable of inducing apoptosis of tumour cell.Albumen provided by the present invention is full humanization binding protein, and immunogenicity will not be generated in human body by applying, and has the function of inhibiting tumour cell in vitro.This 7 binding proteins can lay the foundation for later development oncoprotein drug.

Description

Target binding protein and its application of CD133

Technical field

The present invention relates to binding protein field, in particular to a kind of binding protein for targeting CD133 and its application.

Background technique

Immortality that tumour cell has, migration and the characteristics of lose contact inhibition, so as to become the mankind extremely difficult for tumour One of disease of healing.Oncotherapy means mainly have operation to cut off at present, radiotherapy or chemotherapy, but in tumour there are one There is group the tumor stem cell of similar stem cell properties to cause tumor recurrence rate after treatment high.2006, american cancer research Association is by tumor stem cell is defined as: with self-renewal capacity and can generate the cell of heterogeneous cell in tumour.This Kind cell can be chronically at dormant state in vivo, and the external world with a variety of drug resistance molecules and to killing tumor cell is physical and chemical Factor is insensitive, causes after conventional tumor therapeuticing method eliminates most of Common tumors cell, tumour recurs again.Based on swollen The characteristic of tumor stem cell develops the albumen of targeting tumor stem cells surface specific marker molecule by technique for gene engineering, or Perhaps it will become the Neoma Foam for the treatment of cancer.

As one of stem cell and tumor stem cell surface specific marker's albumen, CD133 is that Yin etc. makes from CD34 earliest In hemocytoblast by artificial AC133 monoclonal antibody separate come.CDl33 belongs to one of Prominin family member, gene Due to No. 4 chromosomes for being located at people, size about 152kb includes at least 37 exons.CD133 albumen is by 865 amino acid groups At molecular weight about 120kDa includes: extracellular NH₂End, 5 transmembrane domains, 2 extracellular cyclic structures, 2 are rich in half Guang The cyclic structure intracellular of propylhomoserin ,-COOH structure intracellular.Existing research shows that CD133 is that a kind of candidate stem cell and nerve cord are thin The surface marker of born of the same parents；CD133 has table in the tumor stem cell of the tumours such as glioma, colon cancer, malignant mela noma It reaches, and in brain tumor, breast cancer and colon cancer, CD133⁺Cell ratio CD133^-Cell Tumor formation is strong；By genetic chip and Clinical analysis discovery, CDl33⁺Tumour cell shows stronger proliferative capacity, and prognosis is poor.The above equal table of result of study Bright, expression of the CDl33 in tumor stem cell is of great significance to the development of cancer.It therefore, can be anti-using CD133 as exploitation The novel targets of tumour medicine.

Compared with traditional monoclonal antibody, only have molecular weight small by the binding protein that a heavy chain variable region forms, Blood-brain barrier is facilitated penetration of, the advantages that immunogenicity is low, high specificity.Existing correlative study the result shows that, binding protein can have Effect ground targeting antigen, and antitumous effect is all had in vivo and in vitro.Currently, not yet about with tumor stem cell labelled protein The research for the albumen that CD133 is combined.

Summary of the invention

The shortcomings that it is a primary object of the present invention to overcome the prior art and deficiency, provide a kind of targeting tumor stem cells mark The binding protein of will molecule CD133.

Another object of the present invention is to provide the protein-bonded preparation methods of above-mentioned targeting CD133.

A further object of the present invention is to provide the protein-bonded applications of above-mentioned targeting CD133.

The purpose of the invention is achieved by the following technical solution:

It is a kind of target CD133 binding protein, be CD133-2B1 albumen, CD133-2C4 albumen, CD133-2C10 albumen, One of CD133-3B4 albumen, CD133-3B12 albumen, CD133-4B9 albumen and CD133-4B12 albumen or at least two Combine the binding protein formed；The binding protein of the targeting CD133 is made of 1 heavy chain variable region；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGYKITAEFMGWVRQAPGKGLEWVSTISRHSGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASVGKFPWVWVSEATVNYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGFKFISEYMGWVRQAPGKGLEWVSSITNADGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAVYVFPFGLADEVRYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGDSISPESMSWVRQAPGKGLEWVSTIDGPNGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARVRSAVLGLRLSANVSYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGYMLINQDMTWVRQAPGKGLEWVSGILDKDGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVSKWSKDAMSFWGQGTLVTVSSAAA；

The amino acid sequence of the CD133-3B12 albumen is following (also as shown in SEQ ID NO.5):

MAQVQLLESGGGLVQPGGSLRLSCAASGVRINNQDMGWVRQAPGKGLEWVSGIRTGDGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDAAVYYCAGFVMWAWEVWSSHPMWKPYLRYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGDSITSENMAWVRQAPGKGLEWVSTIKAHNGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHIAMKGTWKNWHPQSLHYWGQGTLVTVSSAAA；

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

MAQVQLLESGGGLVQPGGSLRLSCAASGYTISPEAMTWVRQAPGKGLEWVSTIYMRDGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGRGWSGWSWNSNVKYWGQGTLVTVSSAAA。

The protein-bonded nucleotide sequence of the coding targeting CD133 is as follows:

The nucleotide sequence of the coding CD133-2B1 albumen is following (also as shown in SEQ ID NO.8):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC CTGTGCAGCCTCCGGATATAAGATTACCGCTGAGTTTATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAG TGGGTATCAACCATTTCGAGGCATAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGC GGCATCTGTGGGGAAGTTTCCGTGGGTTTGGGTTTCGGAGGCCACGGTCAACTATTGGGGTCAGGGAACCTTGGTC ACCGTCTCGAGCGCGGCCGCA；

The nucleotide sequence of the coding CD133-2C4 albumen is following (also as shown in SEQ ID NO.9):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC CTGTGCAGCCTCCGGATTTAAGTTTATCTCTGAGTATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAG TGGGTATCAAGCATTACTAACGCAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGC GGCAGTTTATGTTTTTCCGTTTGGGTTGGCCGACGAGGTCAGGTATTGGGGTCAGGGAACCCTGGTCACCGTCTCG AGCGCGGCCGCA；

The nucleotide sequence of the coding CD133-2C10 albumen is following (also as shown in SEQ ID NO.10):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC CTGTGCAGCCTCCGGAGATAGCATTAGCCCTGAGTCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAG TGGGTATCAACCATTGATGGCCCAAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGC GGCAAGGGTTCGTTCTGCGGTTCTGGGTTTGAGGCTGTCCGCGAACGTGAGCTATTGGGGTCAGGGAACCCTGGTC ACCGTCTCGAGCGCGGCCGCA；

The nucleotide sequence of the coding CD133-3B4 albumen is following (also as shown in SEQ ID NO.11):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC CTGTGCAGCCTCCGGATATATGCTTATCAATCAGGATATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAG TGGGTATCAGGCATTCTGGACAAAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGC GAGAGATGTTTCGAAGTGGTCGAAGGACGCCATGTCGTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCG GCCGCA；

The nucleotide sequence of the coding CD133-3B12 albumen is following (also as shown in SEQ ID NO.12):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC CTGTGCAGCCTCCGGAGTTAGGATTAACAATCAGGATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAG TGGGTATCAGGCATTCGGACGGGTGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACGCCGCGGTATATTATTGCGC GGGGTTCGTCATGTGGGCTTGGGAGGTTTGGAGTAGTCATCCGATGTGGAAGCCGTACCTGAGGTATTGGGGTCAG GGAACCCTGGTCACCGTCTCGAGCGCGGCCGCA；

The nucleotide sequence of the coding CD133-4B9 albumen is following (also as shown in SEQ ID NO.13):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC CTGTGCAGCCTCCGGAGATAGCATTACCTCTGAGAATATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAG TGGGTATCAACCATTAAGGCCCATAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGC GACACATATTGCTATGAAGGGGACTTGGAAGAATTGGCATCCCCAGTCGTTGCACTATTGGGGTCAGGGAACCCTG GTCACCGTCTCGAGCGCGGCCGCA；

The nucleotide sequence of the coding CD133-4B12 albumen is following (also as shown in SEQ ID NO.14):

ATGGCCCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC CTGTGCAGCCTCCGGATATACGATTAGCCCTGAGGCTATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAG TGGGTATCAACCATTTATATGCGAGACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGC GAGAGTTGGGCGGGGGTGGAGTGGGTGGAGTTGGAACTCCAACGTGAAGTATTGGGGTCAGGGAACCCTGGTCACC GTCTCGAGCGCGGCCGCA。

As it can be seen that the nucleotide sequence of CD133-2B1 albumen described in coding and CD133-2C10 albumen is by 393 bases Composition, encodes 131 amino acid；The nucleotide sequence of the coding CD133-2C4 albumen is by 384 base compositions, coding 128 amino acid；The nucleotide sequence of the coding CD133-3B4 albumen encodes 126 amino by 378 base compositions Acid；The nucleotide sequence of the coding CD133-3B12 albumen encodes 135 amino acid by 405 base compositions；Coding institute The nucleotide sequence for the CD133-4B9 albumen stated encodes 132 amino acid by 396 base compositions；The coding CD133- The nucleotide sequence of 4B12 albumen encodes 130 amino acid by 390 base compositions.

The protein-bonded preparation method of above-mentioned targeting CD133, includes the following steps: that the targeting will be encoded The protein-bonded nucleotide sequence of CD133, which is cloned into expression vector, constructs recombinant expression plasmid, then by recombinant expression plasmid It is transferred to host cell and carries out protein expression, purifying, the binding protein of the targeting CD133 can be obtained；Or pass through albumen Synthetic method obtains the binding protein of the targeting CD133.

The binding protein application in preparation of anti-tumor drugs of above-mentioned targeting CD133.

The tumour is prostate cancer or breast cancer.

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

(1) present invention based on display technique of bacteriophage, filtered out from source of people albumen phage library 7 with The binding protein that CD133 extracellular fragment combines only can carry out protein expression by prokaryotic system, can significantly simplify production work Skill reduces protein production cost, and this albumen is immunized human body without using antigen and is achieved with.

(2) binding protein provided by the present invention is only made of a heavy chain variable domain, has molecular weight small, group It is strong to knit permeability, the advantages that stable structure.

(3) binding protein provided by the present invention is applied to that immunogenicity will not be generated in human body, can be used for developing in the future Oncoprotein drug.

(4) binding protein provided by the present invention, which is detected, there is significant inhibition to make prostate cancer and breast cancer cell With laying a good foundation for later development oncoprotein drug.

Detailed description of the invention

Fig. 1 is that result figure of the binding protein of 7 purifying in conjunction with CD133 extracellular fragment is detected by ELISA；Wherein, BSA For blank control, HER2 and EGFR are irrelevant antigen control, and CD133 is related antigen, using 7 binding proteins as one It is anti-, using HRP-protein A as secondary antibody, carry out ELISA detection；* p < 0.05, relative to BSA control group (n=3).

Fig. 2 is that binding protein is detected by MTT method to the result figure of tumor cell proliferation capacity；Wherein, using 7 A albumen (CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133- Tumour cell DU145, MCF-7 4B12) are cultivated under different concentration (0,25,50,100 μ g/mL), 30 μ are added in every hole after 72h The MTT of L is incubated for 4h, and the DMSO for adding 200 μ L dissolves first a ceremonial jade-ladle, used in libation sufficiently, detects OD570 light absorption value with microplate reader later；Figure In, A is the result figure influenced on tumour cell DU145 proliferative capacity, and B is the knot influenced on tumour cell MCF-7 proliferative capacity Fruit figure；* p < 0.05, relative to 0 μ g/mL (n=3).

Fig. 3 is to detect binding protein to tumour cell by stream type cell analyzer and the bis- transfection reagent boxes of Annexin V/PI The result figure of Apoptosis；Wherein, 7 albumen (CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133- 3B12, CD133-4B9, CD133-4B12) co-incubation tumour cell DU145 and MCF-7 under 50 μ g/mL concentration, it sets simultaneously Set a PBS control group；After cultivating 48h, cell is detected using the bis- transfection reagent boxes of Annexin V/PI and stream type cell analyzer Apoptosis；In figure, A is induction tumour cell DU-145 apoptosis as a result, B is the result for inducing tumour cell MCF-7 apoptosis；*p< 0.05, relative to no binding protein control group (n=3).

Fig. 4 is to invade method by Transwell to detect the result figure that binding protein influences tumor cell invasion；Its In, 7 albumen (CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12 tumour cell DU145 and MCF-7) are cultivated under different concentration (0,25,50,100 μ g/mL), then with containing 20% blood serum medium inducing cell is invaded for 24 hours, is taken pictures to cell dyeing and under the microscope with 0.5% crystal violet later, 33% acetic acid is incorporated into the elution of the crystal violet on cell, collects eluent, and microplate reader detects OD570 light absorption value；In figure, A It is the influence that albumen invades tumour cell MCF-7 to tumour cell DU-145, B for albumen；* p < 0.05, relative to 0 μ g/mL (n=3).

Specific embodiment

Below with reference to examples and drawings, the present invention is described in detail, and embodiments of the present invention are not limited thereto.If Unspecified, routinely experiment condition or the specification referring to kit manufacturer carry out embodiment.Except non-specifically Illustrate, agents useful for same and material of the present invention can pass through commercially available acquisition.

The preparation of reagents method used in embodiment is as follows:

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

Reagent is dissolved in 800mL deionized water, 950mL, 121 DEG C of high pressure steam sterilization 30min are settled to.Wait cultivate Base is cooled to 60 DEG C or so, and 20% glucose of 100 μ g/mL ampicillins and 1mL of 20 μ L is added in every 19mL culture medium, mixes A kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after even, 4 DEG C save backup.

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

Reagent is dissolved in deionized water, and is settled to 1L, 121 DEG C of high pressure steam sterilization 20min, room temperature long-term preservation.

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

By reagent, solution is dissolved in 800mL deionized water, then constant volume is at 1L, and when use is diluted to 1 × TAE running buffer Liquid, room temperature preservation.

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

Reagent is dissolved in 800mL deionized water, pH value is adjusted to 7.4, then constant volume, at 1L, 121 DEG C of high steams go out Bacterium 20min, room temperature long-term preservation.1mL Tween-20 is added in 1L PBS buffer solution, it is molten that PBST can be configured to after mixing well Liquid.

(5) PEG solution: NaCl 73g, 6000 PEG 100g.

Reagent is dissolved in deionized water, and is settled to 500mL, after 0.2 μM of filter bacterium, 4 DEG C are saved backup.

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

Reagent is dissolved in deionized water, and is settled to 200mL, 121 DEG C of high pressure steam sterilization 20min, 4 DEG C of preservations are standby With.

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

Reagent is dissolved in deionized water, is settled to 190mL, 121 DEG C of high pressure steam sterilization 30min.It is cooling to culture medium To about 60 DEG C, 20% glucose of 100 μ g/mL ampicillins and 1mL of 20 μ L of every 19mL culture medium addition, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after mixing, 4 DEG C save backup.

(8) 5 × SDS-PAGE electrophoretic buffers: glycine 47g, Tris alkali 15.1g, SDS 2.5g.

Reagent is dissolved in 400mL deionized water, constant volume is at 500mL later, and when use is diluted to 1 × SDS-PAGE electrophoresis Buffer, room temperature long-term preservation.

(9) 5 × SDS-PAGE Loading Buffer:1M Tris-HCl (pH 6.8) 1.25mL, glycerol 2.5mL, bromine Phenol indigo plant 25mg, SDS 0.5g.

Reagent and solution are mixed, constant volume is distributed into L/ parts of 500 μ at 5mL, and 25 μ L β-sulfydryl is added using preceding every aliquot Ethyl alcohol, room temperature long-term preservation.

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

By reagent, solvent is mixed and is sufficiently dissolved, and room temperature long-term preservation is spare.

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

Solvent is mixed, room temperature preservation.

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

Mentioned reagent is mixed with 1L water and is sufficiently dissolved, -4 DEG C of preservations, addition 100 × PMSF liquid storage, PMSF when use Final concentration of 1 × PMSF liquid storage of liquid storage.Sample-loading buffer: with broken bacterium buffer；Wash miscellaneous buffer: it is ready-to-use, it measures 9.9mL breaks bacterium buffer, and 100 μ L 2M imidazoles are added, mix well；Elution buffer: it is ready-to-use, it measures 9mL and breaks bacterium buffering Liquid is added 2M imidazoles 1mL, mixes well.

(13) kanamycins solution (50mg/mL): weighing 1g kanamycins powder and be completely dissolved in 20mL deionized water, mixes 0.2 μM of filter filtration sterilization of liquid is closed, is distributed into every pipe 1mL, -20 DEG C save backup.

(14) it ampicillin solution (100mg/mL): weighs 1g ampicillin powder and is completely dissolved in 10mL deionization Water, 0.2 μM of filter filtration sterilization of mixed liquor, is distributed into every pipe 1mL, -20 DEG C save backup.

(15) 2%BSA-PBS buffer: weighing 1g bovine serum albumin(BSA) powder and be completely dissolved in 50mL PBS buffer solution, 0.2 μM of filter filtration sterilization of mixed liquor, ready-to-use or -20 DEG C of long-term preservations.

(16) 1mg/mL trypsin solution: weighing 10mg trypsase powder and be completely dissolved in 10mL PBS buffer solution ,- 20 DEG C of long-term preservations.

(17) 20% glucose solutions: weighing 200g glucose powder and be completely dissolved in deionized water, and be settled to 1L, mixes 0.2 μM of filter filtration sterilization of liquid is closed, -4 DEG C save backup.

(18) 1M sulfuric acid solution: graduated cylinder measures the 9.8mL concentrated sulfuric acid, is slowly added in 187mL deionized water, mixes well, Room temperature preservation is spare.

(19) 10% ammonium persulfates (APS): it weighs 0.1g ammonium persulfate powder and is dissolved in 1mL deionized water, be distributed into The 200 every pipes of μ L, -20 DEG C of preservations.

(20) it IPTG solution (500mM): weighs IPTG powder 11.915g and is completely dissolved in deionized water, be settled to 100mL, 0.2 μM of filter filtration sterilization of mixed liquor, is distributed into every pipe 1mL, -20 DEG C of long-term preservations.

(21) 30% glycerites: graduated cylinder measures 15mL glycerol and is added in 35mL deionized water, with 0.22 μ after mixing well M filter filtration sterilization, -4 DEG C save backup.

(22) 100 × PMSF liquid storages: weighing 1.74g PMSF powder and be completely dissolved in 100mL isopropanol, mix well ,- 20 DEG C of long-term preservations.

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

Embodiment 1 prepares helper phage

(1) in a culture dish containing TYE solid medium, by the TG1 glycerol stock bought from the green skies with 3rd area Method of scoring coating, is placed in 37 DEG C of constant incubator cultures 12~16 hours for culture dish later；

(2) the TG1 single colonie grown on picking TYE solid medium is inoculated in 2 × TY fluid nutrient medium of 5mL, is placed in 37 DEG C of constant-temperature tables, 250rpm are cultivated 12~16 hours；

(3) inoculum in step (2) is forwarded to 2 × TY fluid nutrient medium of another pipe 5mL in 1:100 ratio, 37 DEG C of constant-temperature tables are placed in, 250rpm is cultivated to bacterium solution OD₆₀₀About 0.5；

(4) with PBS by helper phage KM13 gradient dilution (from 10¹²/ mL~10⁴/mL)；

(5) bacterium solution in 200 μ L steps (3) is taken, the helper phage KM13 that 10 μ L have diluted is added thereto, mixes postposition In 37 DEG C water-bath water-bath 30 minutes, obtain mixture A；

(6) the top agar culture medium for taking 3mL to melt is taped against the culture containing TYE solid medium preheated in advance On ware, being stored at room temperature makes its solidification；Specific steps are as follows: top agar is first heated to melting completely, then before use It is incubated in water and is cooled to 42 DEG C, then the mixture A obtained with step (5) is mixed.It is stored at room temperature to culture medium and solidifies completely, it will Culture dish is placed in 37 DEG C of constant incubator cultures 12~16 hours；

(7) with a small plaque on culture medium in sterile pipette tip picking step (6), it is inoculated in bacterium in 5mL step (3) Liquid (OD₆₀₀=0.5, prepared by method in step (3)), 37 DEG C of constant-temperature tables cultivate 2~3 hours under the conditions of 250rpm；

(8) by bacterium solution that step (7) obtains, 1:100 ratio is forwarded to another 2 × TY liquid containing 500mL by volume In the conical flask of culture medium, 37 DEG C of constant-temperature tables, 250rpm cultivates 2~3 hours；

(9) kanamycins is added in the culture solution obtained to step (8) to 50 μ g/mL of final concentration, 30 DEG C of constant-temperature tables, 250rpm is cultivated 12~16 hours；

(10) step (9) cultured culture solution is centrifuged in 12000g, takes supernatant liquid filtering, adds and be equivalent to supernatant The PEG solution (polyethylene glycol) of 1/5 volume of liquid, 4 DEG C of static a few hours, purifying obtain helper phage；

(11) 10 sensibility of the helper phage of detection preparation to pancreatin: are added in 1mL trypsin solution¹⁰It is a auxiliary Helper phage KM13 is incubated at room temperature 30 minutes.Then gradient dilution helper phage KM13 is carried out (from 10 with PBS¹⁰To 10²It is a Bacteriophage), take the KM13 grade dilution of 10 μ L to infect 200 μ L TG1 bacterium (OD respectively₆₀₀=0.5, prepare and infect method It is such as aforementioned), (kanamycins comprising 50 μ g/mL of final concentration), 37 DEG C of constant incubator cultures are coated on TYE solid medium Overnight.After the Phage Infection bacterium of pancreatin processing, clone's number of acquisition should be at least fewer by 10 than clone's number of untreated fish group⁶ Times.Otherwise the helper phage prepared product is abandoned, another plaque of picking is prepared again.

Embodiment 2 expresses phagocytosis binding protein precursor library

(1) appropriate source of people binding protein phage library (Source Bioscience, Human Domain is taken Antibody Library (DAb), London, UK) entering 2 × TY of 500mL fluid nutrient medium, (culture medium additionally contains 100 μ g/mL Ampicillin, 4% (w/v) glucose), 37 DEG C of constant-temperature tables, 250rpm is cultivated to bacterium OD₆₀₀=0.5；

(2) embodiment 1 is prepared 2 × 10 are added¹²The bacterium solution that a helper phage obtains to step (1), after mixing It is placed in 37 DEG C of water-baths 30 minutes.This 500mL culture is distributed into the every pipe of 50mL, 3200g is centrifuged 10 minutes, is abandoned supernatant, will be sunk It forms sediment and is resuspended, (culture medium additionally contains 0.1% (w/v) grape in the conical flask of 2 × TY fluid nutrient medium containing 500mL for switching Sugar, 100 μ g/ml ampicillins, 50 μ g/ml kanamycins).It is placed in 25 DEG C of constant-temperature tables, 250rpm shake culture 16~20 Hour.

3 purified phage binding protein library of embodiment

(1) overnight culture in embodiment 2 is distributed into the every pipe of 50mL, room temperature 3200g is centrifuged 20 minutes；

(2) centrifuged supernatant is taken to be transferred to another clean 50mL centrifuge tube, the ratio of every pipe 1:4 by volume is added 20%PEG solution is incubated for 1 hour on ice；

(3) centrifuge tube in step (2) is placed in 4 DEG C, 3200g centrifugation 30 minutes, abandons supernatant, is resuspended and is precipitated with 5mL PBS, 1mL 20%PEG solution is added thereto again, is incubated for 10 minutes on ice；

(4) by step (3) be placed in 4 DEG C, 3200g be centrifuged 30 minutes, abandon supernatant, then be resuspended precipitating with 1mL PBS, then 4 DEG C, 3200g be centrifuged 5 minutes, 0.45 μM of filter filtration sterilization of supernatant；

(5) by light absorption value at measurement 260nm, phage library titre is estimated: by above-mentioned purified phage library 100 times are diluted with PBS, phage library titre (PFU/mL) is estimated according to empirical equation below: bacteriophage/mL=OD₂₆₀× 100×22.14×10¹⁰.The phage library prepared can 4 DEG C save 2 weeks, can also Long-term Cryopreservation in -80 DEG C.

Embodiment 4 screens the albumen in conjunction with CD133 extracellular fragment from phagocytosis binding protein precursor library

(1) Biotechnology Co., Ltd, upper hypo Thailand synthesize CD133 Extracellular domain protein (sequence see GenBANK number: NP_006008.1), NUNC be immunized in pipe be added 4mL with PBS buffer solution dissolve, the CD133 of final concentration of 100 μ g/mL it is extracellular Section diluted protein solution is placed in 4 DEG C overnight；

(2) it abandons and stays overnight coating buffer in step (1), softly rinsed with PBS buffer solution immune inside pipe wall 3 times, be then added 4mL2%BSA-PBS confining liquid is placed in room temperature 2 hours；

(3) confining liquid in step (2) is abandoned, then is softly rinsed with PBS buffer solution immune inside pipe wall 3 times, 4mL 2% is added BSA-PBS confining liquid (wherein includes 5 × 10¹²The bacteriophage that a embodiment 3 is prepared), it is placed in room temperature 1 hour；

(4) confining liquid in step (3) is abandoned, is softly rinsed with PBST buffer immune inside pipe wall 10 times, adds 4mL's The trypsin solution of 1mg/ml is placed in room temperature 1 hour, digests bacteriophage thoroughly from immune pipe, obtains digestive juice A；

(5) the picking TG1 bacterium solution from TG1 bacterial strain glycerin storage object, lines TYE solid medium by three zoning collimation methods On, 37 DEG C of constant incubators are placed in, are cultivated 14~16 hours；

(6) TG1 single colonie is inoculated in 5mL 2 × TY fluid nutrient medium on culture medium in picking step (5), and 37 DEG C of constant temperature shake Bed, 250rpm overnight incubation；

(7) by bacterium solution in step (6) by volume 1:100 ratio be seeded to it is another containing 5mL 2 × TY fluid nutrient medium Test tube in, 37 DEG C of constant-temperature tables, 250rpm is cultivated to bacterium solution OD₆₀₀=0.5.

(8) bacterium solution that takes step (7) to obtain is added in the digestive juice A of step (4) preparation, 37 DEG C water bath with thermostatic control 1 hour, so 3200g is centrifuged 5 minutes afterwards；

(9) supernatant is abandoned, precipitating is resuspended with 2 × TY of 1mL fluid nutrient medium, obtains re-suspension liquid A；6 are taken to contain TYE solid The culture dish (contain 100 μ g/ml ampicillins, the glucose of 4% (w/v)) of culture medium, each plate take 166 μ L re-suspension liquid A into Row coating, the culture dish coated are placed in 37 DEG C of constant incubator overnight incubations；

(10) culture dish of step (9) overnight incubation is taken, every piece of ware is added 2 × TY of 2mL fluid nutrient medium, utilizes coating Stick scrapes bacterium on plate；

(11) by the bacterium solution scraped be transferred to containing 500mL 2 × TY fluid nutrient medium conical flask (culture medium contain 4% (w/ V) glucose, 100 μ g/mL ampicillins), 37 DEG C of constant-temperature tables are placed in, 250rpm is cultivated to bacterium solution OD₆₀₀=0.5, then plus Enter the helper phage KM13 that embodiment 1 is prepared, infects bacterium solution, be placed in 30 DEG C of constant-temperature tables, 250rpm shake culture mistake Night；

(12) overnight culture for taking step (11) to obtain is added PEG solution and carries out bacteriophage purifying (with reference to step above Carry out), then the resulting phage library titre of screening is measured by gradient dilution coated plate；

(13) the phage library elutriation that subsequent 4 wheel is carried out with reference to the method (repeating step (1)~(12)) of front, according to It is secondary to be screened from last round of obtained bacteriophage.

The picking monoclonal from library of embodiment 5 carries out ELISA verifying

(1) after completing 5 wheel phage library elutriations, sterile 96 orifice plate (being named as A plate) is taken, the 2 of 200mL are added in every hole × TY fluid nutrient medium (culture medium contains 100 μ g/mL ampicillins, 4% (w/v) glucose) is sieved with sterile pipette tip from the 5th It selects what is obtained to be incubated overnight picking monoclonal on ware, is seeded in 96 orifice plates containing culture solution, 37 DEG C of constant-temperature tables, 250rpm Shake culture is stayed overnight；

(2) another sterile 96 orifice plate (being named as B plate) is taken, 2 × TY fluid nutrient medium (culture medium of 200 μ L is added in every hole Containing 100 μ g/mL ampicillins, the glucose of 4% (w/v)), the overnight culture that 5 μ L steps (1) obtain is drawn, is seeded to Plate is placed in 37 DEG C of constant-temperature tables later by 96 orifice plates, and 250rpm shake culture 3 hours；A plate is remaining overnight in step (1) Final concentration of 20% glycerol is added in the every hole of culture, glycerol bacterium solution storage object is made, Long-term Cryopreservation is in -80 DEG C；

(3) by every hole culture in the plate after in step (2) culture of B plate 3 hours, sterile 1.5mL EP is gone to respectively Pipe, 50 μ L of Guan Zhongyou include 4 × 10⁸2 × TY fluid nutrient medium of a helper phage KM13, it is soft to mix, EP pipe is placed in 37 DEG C constant-temperature incubation 1 hour；

(4) after being incubated for well, 1.5mL EP pipe 3200g is centrifuged 10 minutes, abandons supernatant；Precipitate 200 μ L 2 × TY liquid Body culture medium is resuspended (culture medium contains 100 μ g/mL ampicillins, 50 μ g/mL kanamycins, 0.1% (w/v) glucose), and 25 DEG C constant-temperature table, 250rpm shake culture are stayed overnight；

(5) overnight culture that step (4) obtains is gone to another new 1.5mL EP to manage, 3200g is centrifuged 10 minutes, is taken Supernatant is then transferred to another 96 new orifice plate, is placed in 4 DEG C of refrigerator preservations；

(6) PBS buffer solution that every hole is added that 100 μ L contain 0.2 μ g CD133 Extracellular domain protein is coated with 96 hole elisa plates, It is placed in 4 DEG C overnight；

(7) 250 μ L 2%BSA-PBS, room temperature closing is added with PBS board-washing 3 times, then to every hole in the elisa plate being coated with It is elisa plate 2 hours, spare with PBS board-washing 3 times later；

(8) the 25 μ L of supernatant to new 1.5mL EP of aspiration step (5) preparation is managed, and adds 75 μ L 2%BSA-PBS Middle preparation bacteriophage dilution is incubated at room temperature in the elisa plate washed in bacteriophage dilution to step (7) 1 hour；

(9) it uses PBST board-washing 5 times, 100 μ L are added in every hole, and the diluted HRP of 1:10000 marks anti-M13 conjugate by volume (2%BSA-PBS dilution), is incubated at room temperature 1 hour；

(10) it uses PBST board-washing 5 times again, 100 μ L tetramethyl benzidine (TMB) solution are added in every hole, become to liquid in hole Indigo plant, every hole are added the 50 μ L 1M concentrated sulfuric acids and terminate reaction, read light absorption value at 450nm, and record experimental result.

Embodiment 6 prepares DH5 α, BL21 (DE3) competent escherichia coli cell

(1) a small amount of bacillus coli DH 5 alpha and BL21 (DE3) bacterium solution is taken to be lined respectively with three zoning collimation methods containing LB solid On the culture dish of culture medium, culture dish is placed in 37 DEG C of constant incubator cultures 14~16 hours later；

(2) respectively from picking bacillus coli DH 5 alpha, BL21 (DE3) single colonie on the culture dish containing LB solid medium, It is inoculated in the test tube containing 5mL LB liquid medium respectively, 37 DEG C of constant-temperature tables, 220rpm shake culture about 12 hours；

(3) two kinds of inoculums are seeded to the cone containing 50mL LB liquid medium by the volume ratio of 1:100 respectively In shape bottle, 37 DEG C of constant-temperature tables, 220rpm shaken cultivation 2~3 hours to bacterium solution OD₆₀₀=0.5 or so；

(4) step (3) cultured bacterium solution is transferred to other 2 sterile 50mL centrifuge tubes respectively, is placed in ice water mixed liquor It is middle to stand 10 minutes；

(5) centrifuge tube in step (4) is placed in 4 DEG C of refrigerated centrifuge, 3000g is centrifuged 5 minutes；

(6) supernatant is abandoned, the 0.1mol/L CaCl for taking 10mL to be pre-chilled₂Bacterial sediment is softly resuspended in solution, later by centrifuge tube It is placed in ice water mixed liquor and stands 30 minutes；

(7) centrifuge tube is placed in 4 DEG C of refrigerated centrifuge, 3000g is centrifuged 5 minutes；

(8) supernatant is abandoned, the 0.1mol/L CaCl for taking 3mL to be pre-chilled₂Precipitating is softly resuspended in solution, obtains re-suspension liquid B；

(9) the re-suspension liquid B that step (8) obtain is added in the sterile glycerol for 30% (v/v) for taking 3mL to be pre-chilled, after soft mixing, Mixed liquor is distributed into every 100 μ L of pipe, -80 DEG C of Long-term Cryopreservations.

Embodiment 7 will express the protein-bonded recombinant plasmid transformed of CD133 to DH5 α competent cell

(1) the protein-bonded recombinant plasmid of CD133 is constructed

(1) the 1:1000 ratio inoculation by volume of the positive monoclonal in embodiment 5 is transferred to 2 × TY liquid of 5mL Culture medium (culture medium contains 100 μ g/mL ampicillins, 4% (w/v) glucose) overnight incubation；

(2) with the protein-bonded positive and reverse primer (forward primer: 5 '-ATGGCCCAGGTGCAGCTGT- of CD133 3'；Reverse primer: 5 '-TCTGCGGCCGCGCTCGAGAC-3 '), the PCR amplification system of 25 μ L is prepared, by CD133 binding protein Nucleotide sequence amplification；Wherein:

PCR reaction system are as follows: template (overnight culture that step (1) obtains) 1 μ L, primer (forward primer/reversely draw Object) each 1 μ L, 5 × PrimerStar buffer, 10 μ L, dNTP mixture (every kind of 2.5mM) 4 μ L, PrimerStarDNA polymerase 0.5 μ L, aseptic deionized water complement to 25 μ L；

PCR reaction condition are as follows: 96 DEG C of 10min；95℃10s；60℃10s；72 DEG C of 30s, 30 circulations；72℃5min；

(3) obtained CD133 protein nucleotide sequence PCR product is subjected to agarose gel electrophoresis, cuts gel later On target fragment carry out glue recycling, glue recovery product be stored in -20 DEG C it is spare；

(4) appropriate CD133 binding protein nucleotide sequence glue recovery product is taken, restriction enzyme NotI is added thereto Digestion is carried out with NocI, digestion products carry out agarose gel electrophoresis, and the target fragment cut on gel later carries out glue recycling, Glue recovery product be stored in -20 DEG C it is spare

(5) appropriate expression vector pET28a is taken, restriction enzyme NotI and NocI is added thereto and carries out digestion, digestion Product carries out agarose gel electrophoresis, and the target fragment cut on gel later carries out glue recycling, and glue recovery product is stored in -20 It is DEG C spare

(6) glue recovery product after appropriate CD133 binding protein nucleotide sequence digestion is taken, after expression vector pET28a digestion Ligase is added in glue recovery product, is placed in 37 DEG C of connections overnight, obtains connection product；Wherein:

Coupled reaction system are as follows: pET28a double enzyme digestion product 0.03pmol, target fragment double enzyme digestion product 0.3pmol, enzyme Even 2.5 μ L of buffer, 1 μ L of T4DNA ligase, sterile water complement to 25 μ L.

(2) by anti-CD133 recombinant plasmid transformed to DH5 α competent cell

(1) a pipe DH5 α competent cell is taken out from -80 DEG C of refrigerators, is thawed on ice；

(2) take 10 μ L connection products that 100 μ L DH5 α competent cells (embodiment 6 is prepared) are added, it is soft to mix, Stand 30 minutes on ice；

(3) mixture prepared by step (2) is placed in water-bath 90s in 42 DEG C of thermostat water baths, is transferred quickly to ice later Upper standing is 2~3 minutes cooling；

(4) take 900 μ L LB liquid mediums that the mixed liquor of step (3) preparation, 37 DEG C of constant-temperature tables, 200rpm shake is added Swing culture 1 hour；

(5) cultured mixed liquor is placed in centrifuge, 3000g is centrifuged collection in 1 minute and mixes liquid precipitate；

(6) 900 μ L supernatants are abandoned, bacterial sediment is resuspended with remaining 100 μ L culture solution；

(7) bacteria suspension of mixing is coated on (culture medium on the preprepared culture dish containing LB solid medium Containing 100 μ g/mL ampicillins, 1% (w/v) glucose)；

(8) culture dish is placed in 37 DEG C of constant incubator cultures 12~16 hours,

(9) several monoclonals on random picking culture dish, being seeded to 5mL LB liquid medium respectively, (culture medium contains 100 μ G/mL ampicillin), 37 DEG C of constant-temperature tables, 220rpm shake culture is stayed overnight.

(10) with the protein-bonded positive and reverse primer (forward primer: 5 '-ATGGCCCAGGTGCAGCTGT- of CD133 3'；Reverse primer: 5 '-TCTGCGGCCGCGCTCGAGAC-3 '), the PCR amplification system of 25 μ L is prepared, 1 μ L step is added thereto Suddenly the overnight culture (template) that (9) obtain carries out bacterium solution PCR verifying to monoclonal；Wherein, PCR reaction system and reaction item Part refers to step (1).

(11) PCR product carries out agarose gel electrophoresis, and target fragment occur is then positive colony.

(12) plasmid extraction is carried out to positive colony, the protein-bonded recombinant plasmid of CD133 can be obtained.

(13) obtained positive colony is sequenced, the results show that obtain seven albumen, be named as CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12 albumen, encoding nucleoside Acid sequence respectively as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, Shown in SEQ ID NO.13, SEQ ID NO.14.

The protein-bonded prokaryotic expression of embodiment 8, protein purification, PAGE gel electrophoresis and coomassie brilliant blue staining

(1) binding protein prokaryotic expression

(1) the protein-bonded recombinant plasmid transformed of CD133 embodiment 7 being prepared to BL21 competent cell (has Body step reference implementation example 7)；

(2) picking single colonie on the plate being coated with after step (1) conversion, is seeded to containing 5mL LB liquid medium (culture medium contains 100 μ g/mL ampicillins, 1% (w/v) glucose), 37 DEG C of constant-temperature tables, 220rpm shake culture in test tube 12 hours；

(3) step (2) cultured bacterium solution is taken, is seeded to by 1:100 volume ratio containing 100mL LB liquid medium In conical flask (culture medium contains 100 μ g/mL ampicillins), 37 DEG C of constant-temperature tables, 220rpm shake culture 2~3 hours；

(4) to bacterium solution OD₆₀₀It is 0.6~1.0, takes 1mL bacterium solution to be used to prepare and do not induce Escherichia coli holoprotein sample, remains Remaining bacterium solution carries out subsequent experimental；

(5) final concentration of 0.25mM IPTG, 25 DEG C of constant-temperature tables, 220rpm induction are added into the remaining bacterium solution of step (4) Express 6h；

(6) the cultured bacterium solution 1mL of step (5) is taken to be used to prepare the Escherichia coli holoprotein sample after induction, remaining bacterium Liquid is placed in 4 DEG C, 5000g centrifugation 5min collection bacterial sediment；

(7) supernatant is abandoned, the bacterial sediment that step (6) obtain is resuspended with the broken bacterium buffer of 20mL pre-cooling；

(8) bacteria suspension is transferred to 50mL beaker, places the beaker ultrasonication bacteria suspension in ice chest, ultrasonic disintegrator work Power: 40%, it works 4 seconds, stops 8 seconds, be crushed 40 minutes；

(9) bacteria suspension being crushed is transferred to a clean 50mL centrifuge tube, 4 DEG C, 15000g is centrifuged 40 minutes；

(10) supernatant obtained after collection step (9) centrifugation, therefrom takes 20 μ L supernatants to be used to prepare supernatant after bacterial cell disruption Sample, then (precipitating is broken bacterium buffer with 20 μ l and is resuspended) is precipitated after taking partly precipitated to be used to prepare bacterial cell disruption, remaining supernatant turns Enter in another clean 50mL centrifuge tube, 4 DEG C of preservations waited column purification.

(2) binding protein crosses column purification

(1) a Ni-NTA HisBind Resin purification column is taken, is rushed with the sample-loading buffer of 10~15 times of column volumes Wash purification column；

(2) purification column is added in the supernatant for obtaining step (1), allows supernatant to flow through purification column with natural flow velocity, during which It takes 20 μ L to cross column liquid and was used to prepare column liquid sample；

(4) purification column is all flowed through to supernatant, the sample-loading buffer that 10 times of column volumes are added rinses purification column；

(5) it being flow to end to liquid in step (4), the miscellaneous buffer of washing that 20 times of column volumes are added cleans foreign protein on purification column, Period takes 20 μ L to wash miscellaneous liquid and be used to prepare and washes miscellaneous liquid sample；

(6) it is flow to end to liquid in step (5), the elution buffers of 5 times of column volumes is added by destination protein from purification column During which elution collects eluent with 1.5mL EP pipe, every pipe collects eluent 1mL, collects 5 pipes altogether, then from every pipe eluent 20 μ L are taken to be used to prepare 1~5 sample of eluent respectively；

(7) it completes after destination protein collects, the 6M urea of 5 times of column volumes is added by albumen wash-out remaining on purification column, Add the distilled water cleaning purification column of 30 times of column volumes；

(8) water flow to be distilled is most, takes 20% ethyl alcohol of 5mL that purification column is added, and re-closed purifying pipe upper and lower ends will purify The column length phase is stored in 4 DEG C.

(3) PAGE gel electrophoresis and coomassie brilliant blue staining

(1) by specification formula prepares the separation gel 5mL of 12% (w/v), and encapsulating die is added, makes liquid level apart from mold top Then 1.5mL dehydrated alcohol is added in portion 2cm or so, it is solid to be stored at room temperature 30min gelling to be separated；

(2) dehydrated alcohol in mold is eliminated, by specification formula prepares the concentration glue 2mL of 5% (w/v), is added to filling Sealing rubber die is inserted into the mating comb of mold, it is solid to be stored at room temperature 30min gelling to be concentrated to top；

(3) prepared offset plate is put into electrophoresis tank, appropriate electrophoresis liquid is added, pull out comb, prepare loading；

(4) sample being collected into above-mentioned binding protein expression purification step is separately added into 5 μ L 5 × SDS-PAGE eggs White sample-loading buffer mixes, is placed in 100 DEG C of 5~10min of heating；

(5) take the 5 μ L of sample prepared in step (4) that PAGE gel comb hole, 80V electrophoresis, to sample is added Concentration glue was run, voltage is adjusted to 110V, electrophoresis to sample blue indicates that band is run to gel bottom；

(6) appropriate coomassie brilliant blue staining liquid is added into dye glue box, takes out PAGE gel and is placed in dye glue box, room temperature Dye 30min；

(7) dyeing liquor is abandoned, dyeing liquor on glue is cleaned using clear water, adds appropriate destainer and decolourize；

(8) high-visible to purpose band on SDS-PAGE glue, gel decolourizes to transparence, photographs to record under white light.

The experimental results showed that obtain the CD133-2B1 albumen of purifying, CD133-2C4 albumen, CD133-2C10 albumen, CD133-3B4 albumen, CD133-3B12 albumen, CD133-4B9 albumen, CD133-4B12 albumen.

The combination of CD133 binding protein and antigen that embodiment 9 is purified using ELISA detection

(1) take a 96 new holes that plate is immunized, every hole is separately added into the final concentration of 2 μ g/mL of 100 μ L, and (solvent is PBS buffering Liquid) antigen HER2, EGFR, (HER2, EGFR are purchased from hypo Thailand biotechnology to be had antigens c D133 Extracellular domain protein dilution Limit company), while 100 μ L 2%BSA-PBS buffers are coated with as blank control, plate is placed in 37 DEG C of constant incubators and stands 2 Hour；

(2) the immune plate being coated with is taken out, PBS buffer solution board-washing 3 times, eliminates remaining liq in plate, 250 μ L are added in every hole 2% (w/v) BSA-PBS buffer, 37 DEG C of constant incubators stand 2 hours；

(3) the immune plate closed is taken out, PBS buffer solution board-washing 3 times, eliminates remaining liq in plate, 100 μ L are added in every hole Immune plate is placed in incubation at room temperature 1 hour by the CD133 binding protein-PBS mixed liquor that 50 times are diluted with PBS；Binding protein point Not Wei CD133-2B1 albumen, CD133-2C4 albumen, CD133-2C10 albumen, CD133-3B4 albumen, CD133-3B12 albumen, CD133-4B9 albumen and CD133-4B12 albumen；

(4) immune plate is taken out, PBST buffer board-washing 3 times eliminates remaining liq in plate, by secondary antibody HRP-protein A It is diluted with 2%BSA-PBS buffer in 1:5000 ratio, the every hole of plate is immunized, 100 μ L secondary antibody diluents, incubation at room temperature is added 1 hour；

(5) immune plate is taken out, PBST buffer board-washing 5 times eliminates remaining liq in plate, and 100 bottoms μ L TMB are added in every hole Object developing solution, room temperature are protected from light incubation 10 minutes, and immune plate is placed in microplate reader detection OD 450nm light absorption value, record experiment later Data.

Experimental result is as shown in Figure 1, wherein BSA is that blank control, HER2 and EGFR compare for irrelevant antigen, it is seen then that CD133-2B1 albumen, CD133-2C4 albumen, CD133-2C10 albumen, CD133-3B4 albumen, CD133-3B12 albumen, CD133-4B9 albumen and CD133-4B12 albumen can be specifically bound with CD133 extracellular fragment.

Embodiment 10 detects influence of the binding protein to tumor cell proliferation ability after purification using MTT method

(1) one piece of 96 porocyte culture plates is taken, the 100 full culture mediums of μ L are added (containing 10% calf serum in every hole RPMI1640, similarly hereinafter), wherein containing tumour cell (Human Prostate Cancer Cells DU145 or human breast carcinoma in logarithmic growth phase Cell MCF-7) 5000, culture plate is placed in 37 DEG C of cell incubator, 5%CO₂Overnight incubation；

(2) after cell is completely adherent, culture medium complete in hole is changed into serum free medium (RPMI1640, similarly hereinafter), is trained Feeding plate is placed in cell incubator Nature enemy 4 hours；

(3) serum free medium after step (2) Nature enemy is absorbed, every hole is separately added into containing various concentration albumen 100 μ L, 1% blood serum medium of (0,25,50 and 100 μ g/mL), culture plate are placed in 37 DEG C of cell incubator, 5%CO₂Culture 72 hours；Albumen is respectively CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133- 4B9,CD133-4B12；

(4) liquid in the culture plate handled well is absorbed, every hole, which is added, contains 100 μ L serum free mediums and 20 μ L MTT The mixed liquor of solution is placed in 37 DEG C of cell incubator, 5%CO₂It is incubated for 4 hours；

(5) mixed liquor in culture plate is absorbed, every hole is added 200 μ L DMSO, culture plate is placed in shaking table later and is quickly shaken 10min makes to precipitate abundant dissolution；

(6) culture plate is placed in light absorption value at microplate reader detection 570nm, records testing result.

As a result as shown in Figure 2, it is seen then that CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133- 3B12, CD133-4B9, CD133-4B12 albumen can significantly inhibit DU145 and MCF-7 cell to increase in 50 μ L/mL concentration It grows.

Embodiment 11 detects binding protein inducing apoptosis of tumour cell using stream type cell analyzer

(1) one piece of 6 porocyte culture plates is taken, the full culture medium of 1mL is added in every hole, wherein containing logarithmic growth phase tumour is in Cell 5 × 10⁶It is a, culture plate is placed in 37 DEG C of cell incubator, 5%CO₂Overnight incubation；

(2) after cell is completely adherent, culture medium complete in hole is changed into serum free medium, culture plate is placed in cell culture Case Nature enemy 4 hours；

(3) culture medium in (2) mesoporous to be absorbed, serum free medium 1mL is added in every hole, wherein contain 50 μ g/mL albumen, Culture plate is placed in 37 DEG C of cell incubator, 5%CO₂Culture 48 hours；Albumen be respectively CD133-2B1, CD133-2C4, CD133-2C10,CD133-3B4,CD133-3B12,CD133-4B9,CD133-4B12；

(4) culture plate inner cell is digested with pancreatin, is collected in 1.5mL EP pipe, cell is cleaned 2 times with PBS buffer solution；

(5) 4 × combination buffer in Annexin V-FITC apoptosis kit is taken out, ddH is used₂O its be diluted to 1 ×, Take 195 μ 1 × combination buffers of L that cell density is adjusted to 5 × 10⁶A/mL；

(6) the 5 μ L of Annexin V-FITC in kit is taken, the cell mixture of step (5) preparation is added, room temperature is kept away Light is incubated for 10~15min；

(7) it takes 200 μ 1 × combination buffers of L to be added in 1.5mL EP pipe, the mixing with cells of step (6) preparation is added Liquid is soft to mix；

(8) 1.5mL EP pipe in step (7) is placed in centrifuge 1000g centrifugation 5min, abandons supernatant, take 190 1 × knots of μ L It closes buffer and cell is resuspended, add 10 μ L PI dyeing liquors, it is soft to mix.

(9) tumour cell of apoptosis occurs for stream type cell analyzer detection, records experimental result.

As a result as shown in figure 3, CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12 albumen induce DU145 cell and MCF-7 Apoptosis in 50 μ L/mL concentration in which can dramatically.

Embodiment 12Transwell Matrigel detects influence of the binding protein to tumor cell invasion ability

(1) the sterile cell Transwell (8 μm of apertures, 24 orifice plates) are put into 24 porocyte culture plates, draw 50 μ L matrix Small interior is added in glue (Matrigel), and plate is placed in cell incubator and stands a few hours, is solidified completely to Matrigel；

(2) later that tumour is thin by the tumour cell in logarithmic growth phase with serum free medium Nature enemy 4 hours Born of the same parents digest and collect spare；

(3) upper chamber of each cell Transwell is separately added into 200 μ L of serum free medium, wherein containing 5 × 10⁴It is a Containing for 500 μ L is added in the tumour cell and various concentration albumen (0,25,50 and 100 μ g/mL) that step (2) is handled well, lower room The plate handled well is placed in 37 DEG C of cell incubator, 5%CO by the cell culture medium of 20% (w/v) FBS₂Culture 24 hours；Albumen Respectively CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133- 4B12；

(4) cell in cultured culture plate is taken out, absorbs small indoor culture medium, and gently wipe small interior with cotton swab The cell that do not invaded on Matrigel；

(5) the fixed cell inner cell 10min of 4% poly methanol is added into culture plate；

(6) fixer is absorbed, 500 μ L, 0.5% crystal violet dye liquor is added into culture plate, 30min is dyed to cell room temperature；

(7) cell is taken out, 3 times wash with distilled water, then cell is just being placed on glass slide, note of taking pictures under microscope Record；

(8) cell for taking photograph is put into another clean 24 porocyte culture plates, 100 μ L 33% (w/v) is added into plate Acetic acid solution, the crystal violet that would be incorporated on cell elute, and collect eluent, are placed in extinction at microplate reader detection 570nm Value, and record experimental result.

As a result as shown in figure 4, CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12 albumen inhibit DU145 cell invasion in 25 μ L/mL concentration in which can dramatically；CD133-2B1, CD133-2C4, CD133-2C10, CD133-3B4, CD133-3B12, CD133-4B9, CD133-4B12 albumen are dense in 50 μ L/mL MCF-7 cell invasion can significantly be inhibited when spending.

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

<110>Ji'nan University

<120>binding protein and its application of CD133 are targeted

<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 albumen

<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 albumen

<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 albumen

<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 albumen

<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 albumen

<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 albumen

<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 albumen

<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 of CD133-2B1 albumen is encoded

<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 of CD133-2C4 albumen is encoded

<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 of CD133-2C10 albumen is encoded

<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 of CD133-3B4 albumen is encoded

<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 of CD133-3B12 albumen is encoded

<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 of CD133-4B9 albumen is encoded

<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 of CD133-4B12 albumen is encoded

<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 CD133 binding protein forward primer

<400> 15

atggcccagg tgcagctgt 19

<210> 16

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>PCR amplification CD133 binding protein reverse primer

<400> 16

tctgcggccg cgctcgagac 20

Claims

1. a kind of binding protein for targeting CD133, it is characterised in that: be CD133-2B1 albumen, CD133-2C4 albumen, CD133- One of 2C10 albumen, CD133-3B4 albumen, CD133-3B12 albumen, CD133-4B9 albumen and CD133-4B12 albumen or The binding protein that at least two combinations are formed；

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

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

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

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

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

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

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

2. encoding the protein-bonded nucleotide sequence of targeting CD133 described in claim 1, it is characterised in that: be coding institute Described in the nucleotide sequence of CD133-2C4 albumen described in the nucleotide sequence of the CD133-2B1 albumen stated, coding, coding Described in the nucleotide sequence of CD133-3B4 albumen described in the nucleotide sequence of CD133-2C10 albumen, coding, coding The nucleotide sequence and coding of CD133-4B9 albumen described in the nucleotide sequence of CD133-3B12 albumen, coding is described The nucleotide sequence that a kind of or at least two combinations are formed in the nucleotide sequence of CD133-4B12 albumen.

3. the protein-bonded nucleotide sequence of coding targeting CD133 according to claim 2, it is characterised in that:

The nucleotide sequence of the coding CD133-2B1 albumen is as shown in SEQ ID NO.8；

The nucleotide sequence of the coding CD133-2C4 albumen is as shown in SEQ ID NO.9；

The nucleotide sequence of the coding CD133-2C10 albumen is as shown in SEQ ID NO.10；

The nucleotide sequence of the coding CD133-3B4 albumen is as shown in SEQ ID NO.11；

The nucleotide sequence of the coding CD133-3B12 albumen is as shown in SEQ ID NO.12；

The nucleotide sequence of the coding CD133-4B9 albumen is as shown in SEQ ID NO.13；

The nucleotide sequence of the coding CD133-4B12 albumen is as shown in SEQ ID NO.14.

4. the protein-bonded preparation method of targeting CD133 described in claim 1, it is characterised in that: by Claims 2 or 3 Described in coding targeting CD133 protein-bonded nucleotide sequence be cloned into expression vector, construct recombinant expression plasmid, Recombinant expression plasmid is transferred to host cell again and carries out protein expression, purifying, the combination egg of the targeting CD133 can be obtained It is white；Or by albumen synthetic method, obtain the binding protein of the targeting CD133.

5. the binding protein application in preparation of anti-tumor drugs of targeting CD133 described in claim 1.

6. application according to claim 5, it is characterised in that: the tumour is prostate cancer or breast cancer.