Polypeptide targeting human endothelial cell receptor protein tyrosine kinase as well as screening method and application thereof

Abstract

The invention provides a polypeptide targeting human endothelial cell receptor protein tyrosine kinase as well as a screening method and application thereof. A one-bead-one compound (OBOC) combinatorial chemical method is used for screening the polypeptide targeting the EphA2 protein, the screened polypeptide is high in binding capacity with the EphA2 protein, can be specifically bound with EphA2 positive tumor cells and has the self-assembly property, a new compound is provided for treatment and detection of the targeting tumor-related receptor EphA2, and the application has the advantages that the polypeptide targeting the EphA2 protein is provided for treatment and detection of the targeting tumor-related receptor EphA2. The development of the polypeptide in the fields of biomedicine and material application is promoted.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology and relates to a polypeptide targeting human endothelial cell receptor protein tyrosine kinase and a screening method and application thereof. Background Art

[0002] The high mortality rate of cancer makes it crucial to scientifically conquer the mechanism of cancer, early diagnosis and treatment, and the development of targeted drugs. EphA2 protein is a transmembrane receptor protein and a member of the tyrosine kinase family. EphA2 protein is highly expressed in many cancers such as breast cancer, bladder cancer, lung cancer, prostate cancer, etc. The high expression of EphA2 in clinical practice is correlated with poor prognosis and higher recurrence rate. This receptor interacts with the ligand, activates downstream pathways, and promotes the adhesion, proliferation and metastasis of tumor cells under certain conditions. This shows that drugs targeting EphA2 receptor proteins have good application prospects in preventing tumor cell adhesion, proliferation and metastasis.

[0003] The polypeptide has good biocompatibility, degradability and low immunogenicity. The self-assembly property of the polypeptide is conducive to the design of new biomaterials with specific functions. For example, CN110563830A discloses an ANXA1-derived polypeptide, which synthesizes the ANXA1 N-terminal 20-30 amino acid polypeptide in vitro, enters tumor cells with the help of the transmembrane peptide TAT, and can significantly block the binding of ANXA1 and EphA2 in tumor cells, increase the binding of Cbl and EphA2, promote the ubiquitination degradation of EphA2, inhibit the growth of tumor cells in vitro and in vivo, and has a significant anti-tumor effect.

[0004] The "one bead one object" chemical peptide library (OBOC library) is a strategy widely used in high-throughput discovery of molecules such as cell surface receptors, ligands for target proteins, host molecules for small molecules, protease substrates, etc. In the process of chemical synthesis, in addition to natural amino acids, the "one bead one object" chemical peptide library can also further increase the library capacity and create more structural possibilities by introducing organic molecules and non-natural amino acids. Its synthesis process is simple and stable, and the products are easy to obtain. In previous studies, it has been reported that the "one bead one object" chemical peptide library was used to successfully screen self-assembling peptides. The screened peptides were characterized and it was found that all the peptides were successfully assembled into nanoparticles or nanofibers. It has been proved that the "one bead one object" chemical peptide library has a larger capacity and is more efficient than computational screening and dynamic combinatorial peptide libraries.

[0005] In summary, the development of new peptides targeting EphA2 protein and corresponding screening methods are of great significance in the field of tumor treatment. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides polypeptides targeting human endothelial cell receptor protein tyrosine kinase and screening methods and applications thereof, designs a strategy for high-throughput screening of self-assembling polypeptides targeting EphA2, and is capable of high-throughput, rapid and efficient screening of self-assembling polypeptide sequences that can closely interact with the extracellular domain of the EphA2 protein in a physiological environment, which is of great significance in the development of new nanostructures in the fields of biomedicine and material applications.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a polypeptide targeting human endothelial cell receptor protein tyrosine kinase, wherein the polypeptide amino acid sequence comprises:

[0009] (1) a sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6, or,

[0010] (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1), or,

[0011] (3) An amino acid sequence that has at least 95% sequence homology (e.g., 96%, 97%, 98% or 99%, etc.) with the sequence described in (1) or (2), and has the same or similar function as the sequence described in (1).

[0012] In the present invention, a self-assembling polypeptide targeting EphA2 protein was screened based on the one-bead-one-substance method. The polypeptide has high affinity and specificity, specifically targets the extracellular domain of EphA2 protein, and can self-assemble into nanostructures (nanofibers or nanoparticles) in a physiological environment (PBS), and has a good therapeutic effect in vivo.

[0013] It can be understood that the present application screens for self-assembling polypeptides targeting EphA2 protein. Therefore, polypeptides obtained by replacing, deleting or adding one or at least two amino acid residues to the sequences shown in SEQ ID NO:1 to SEQ ID NO:6 using general technical means in the art, and having the same or similar functions as the original polypeptide, should also be within the scope of protection of the present application.

[0014] SEQ ID NO: 1: LNDSITWLKISA.

[0015] SEQ ID NO:2: LNDSIKLLTKYV.

[0016] SEQ ID NO:3: LNDSIIAQAAIE.

[0017] SEQ ID NO:4: LNDSIKLIATFK.

[0018] SEQ ID NO:5: LNDSILVLLPDW.

[0019] SEQ ID NO:6: LNDSIKILSWAK.

[0020] In a second aspect, the present application provides a nucleic acid molecule encoding the polypeptide targeting human endothelial cell receptor protein tyrosine kinase as described in the first aspect.

[0021] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0022] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell expresses the polypeptide targeting human endothelial cell receptor protein tyrosine kinase according to the first aspect.

[0023] In a fifth aspect, the present invention provides a method for screening a polypeptide targeting human endothelial cell receptor protein tyrosine kinase according to the first aspect, the screening method comprising the following steps:

[0024] (1) using a one-bead-one-substance separation-mixing method to synthesize a peptide library in a solid phase; the peptide library contains a known self-assembly sequence;

[0025] (2) The EphA2 protein extracellular domain is mixed with a buffer solution, labeled with FITC fluorescent molecules, and incubated with the peptide library of step (1), and the resin microbeads with the strongest fluorescence are selected for sequence analysis to obtain peptides that can bind to the EphA2 protein.

[0026] In the present invention, a one-bead-one-compound (OBOC) combinatorial chemistry method is used to synthesize a polypeptide library, and the polypeptide library is used to interact with the EphA2 protein to screen out polypeptides with targeting activity of the EphA2 protein as shown in SEQ ID NO:1 to SEQ ID NO:6. The polypeptides can be fused with other polypeptides and / or proteins to construct drugs that target and kill EphA2 overexpressing cells.

[0027] Preferably, the method for synthesizing the polypeptide library in step (1) specifically comprises:

[0028] (1-1) Firstly, the leader sequence and the self-assembling polypeptide sequence are synthesized on the resin microbeads;

[0029] (1-2) the resin beads are evenly divided, the Fmoc-protected amino acid is activated and added to the resin beads to undergo a condensation reaction with the previously deprotected amino acid;

[0030] (1-3) mixing the resin beads and performing Fmoc deprotection;

[0031] (1-4) Repeat steps (1-2) and (1-3) until the last amino acid is synthesized;

[0032] (1-5) Fmoc deprotection is performed followed by side chain deprotection to obtain the polypeptide library.

[0033] In the present invention, during the solid phase synthesis process, the resin microbeads are mixed and evenly distributed multiple times so that each resin microbead carries a unique random polypeptide sequence, thereby constructing a polypeptide library.

[0034] Preferably, the length of the leader sequence in step (1-1) is 2 to 4 aa; and the self-assembly sequence includes the sequence shown in SEQ ID NO:7.

[0035] Preferably, the leader sequence comprises methionine (M).

[0036] In the present invention, a leader sequence containing methionine is first synthesized on the resin microbeads, and the main function is that the CNBr3 ethanol solution cleaves methionine to generate homoserine, so that the functional polypeptide is cleaved from the resin microbeads.

[0037] According to the present invention, the leader sequence further contains any one or more of the other 19 amino acids, preferably glycine (G), which is used to space the resin microbeads from the functional polypeptide.

[0038] Preferably, the activation reagent in step (1-2) includes any one of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), N,N-dimethylformamide (DMF), methanol, dichloromethane, N-methylmorpholine, N,N'-diisopropylcarbodiimide (DIC) or 1-hydroxybenzotriazole (HOBT) or a combination of at least two thereof, preferably a mixture of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT).

[0039] Preferably, the deprotection reagent in step (1-3) comprises a DMF solution containing hexahydropyridine or a DMF solution containing piperazine and 1,8-diazabicycloundec-7-ene, preferably a DMF solution containing 20% ​​hexahydropyridine.

[0040] Preferably, the side chain deprotection reagent in step (1-5) contains trifluoroacetic acid, benzene and water.

[0041] Preferably, the length of the polypeptides in the polypeptide library in step (1) is 7 to 15 aa, including but not limited to 8aa, 9aa, 10aa, 11aa, 12aa, 13aa or 14aa, etc.

[0042] In the present invention, polypeptides with a length of 7 to 15aa are synthesized to construct a polypeptide library. The random sequence of 7aa not only enriches the sequence diversity of polypeptides and increases the number of polypeptides in the polypeptide library. Theoretically, the polypeptide library contains 197 polypeptides (the synthesized polypeptides do not contain methionine), but also ensures the efficiency of screening functional polypeptides. The design of the library also includes a 5aa assembly sequence, which helps to efficiently screen polypeptides with protein-targeted self-assembly capabilities; at the same time, the polypeptide is short in length and small in molecular weight, has the advantages of weak immunogenicity and high activity, and has broad application prospects in forming targeted nanomaterials in vivo and in vitro.

[0043] Preferably, the amino acid sequence of the extracellular domain of the EphA2 protein in step (2) includes the sequence shown in SEQ ID NO:8.

[0044] Preferably, the buffer in step (2) comprises PBS buffer.

[0045] Preferably, the incubation time in step (2) is 2-4 hours.

[0046] SEQ ID NO:7: LNDSI.

[0047] SEQ ID NO:8:

[0048] EVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITV SSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSP GFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEP HMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSP.

[0049] As a preferred technical solution, the method for screening polypeptides targeting human endothelial cell receptor protein tyrosine kinase comprises the following steps:

[0050] S1. Firstly, a leader sequence containing methionine and having a length of 2 to 4 aa and a self-assembly sequence as shown in SEQ ID NO: 7 are synthesized on resin microbeads;

[0051] S2. After the resin beads are evenly divided, an Fmoc-protected amino acid is activated and added to the resin beads to undergo a condensation reaction with the previously deprotected amino acid;

[0052] S3. The resin beads were mixed and Fmoc deprotection was performed using a DMF solution containing hexahydropyridine;

[0053] S4. Repeat steps (S.2) and (S.3) until the last amino acid is synthesized to obtain a polypeptide library, wherein the length of the polypeptides in the polypeptide library is 7 to 15 aa;

[0054] S5. Add the fluorescently labeled EphA2 protein with the amino acid sequence shown in SEQ ID NO: 8 to the polypeptide library, incubate for 2 to 4 hours, and screen for polypeptides that specifically bind to the HER2 protein based on the fluorescence intensity.

[0055] In the present invention, artificially labeled EphA2 protein is co-cultured with a polypeptide library to screen and obtain a polypeptide resin targeting the EphA2 protein, and the polypeptide is cleaved from the resin and sequenced and identified to obtain a polypeptide sequence.

[0056] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a drug or contrast agent molecule connected to the polypeptide targeting human endothelial cell receptor protein tyrosine kinase as described in the first aspect.

[0057] In the present invention, the cytotoxic drug connected to the polypeptide described in the first aspect uses the polypeptide to target EphA2-positive tumor cells, so that the cytotoxic drug exerts the molecular targeted therapy effect, specifically kills EphA2-positive tumor cells, and avoids affecting the surrounding normal tissue cells. The contrast agent connected to the polypeptide described in the first aspect enables the contrast agent to exert the function of molecular targeted imaging, specifically imaging at the location of EphA2-positive tumors, and improving the signal-to-noise ratio between normal tissues and normal tissues.

[0058] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent, and the dosage form of the pharmaceutical composition is preferably an injection preparation.

[0059] In a seventh aspect, the present invention provides use of the polypeptide targeting human endothelial cell receptor protein tyrosine kinase described in the first aspect and / or the pharmaceutical composition described in the sixth aspect in the preparation of imaging agents and / or therapeutic drugs for diseases.

[0060] Preferably, the disease comprises an EphA2-positive tumor.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] (1) The polypeptide screened by the present invention has a strong ability to bind to the EphA2 protein and can specifically bind to EphA2-positive tumor cells, and has important application prospects in the field of tumor treatment and / or detection;

[0063] (2) The polypeptides screened by the present invention have the property of self-assembly and can aggregate into supramolecular structures, thereby increasing the local molecular concentration and promoting the aggregation of target receptors, and have the potential to improve the therapeutic effect;

[0064] (3) Compared with antibody-directed drugs, the polypeptides screened by the present invention have small molecular weight, weak immunogenicity, high activity, low preparation cost and easy modification;

[0065] (4) The polypeptide screening method of the present invention combines self-assembling polypeptides into the polypeptide library, and has the advantages of high throughput and high screening efficiency for screening targeted self-assembling polypeptides. It establishes the connection between protein and polypeptide, provides a simple and easy method for discovering polypeptides that bind to EphA2 protein, and provides a basis for further research and development of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 It is a fluorescence microscopy image of the co-incubation of peptide library microbeads and fluorescently labeled EphA2;

[0067] Figure 2 Comparison chart of the binding of the screened peptides to proteins (ELISA results);

[0068] Figure 3 This is the result of the test of the binding force between peptide and protein;

[0069] Figure 4 TEM image of peptide self-assembly;

[0070] Figure 5 This is a diagram of the interaction between peptides and cells. The scale bar is 15 microns. DETAILED DESCRIPTION

[0071] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0072] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0073] The present invention uses a one-bead-one-compound (OBOC) combinatorial chemistry method to screen polypeptides that target specific proteins. A polypeptide library synthesized in solid phase using a chemical method is cultured with a fluorescently labeled EphA2 protein in an in vitro environment. When the protein and polypeptide interact, the fluorescent protein will bind to the resin. The polypeptides screened by contrasting the fluorescence intensity and preferably using a resin with a high fluorescence intensity are the polypeptide sequences that can specifically bind to EphA2 in vitro. The polypeptide library for screening includes some known self-assembly sequences, which have been verified to have self-assembly properties, and can promote the aggregation of the screened polypeptides into nanostructures, thereby improving the efficiency of targeting receptors and providing the possibility of better activating downstream functions. The present invention provides new compounds for the treatment and detection of the tumor-related receptor EphA2, and promotes the development of polypeptides in the fields of biomedicine and material applications.

[0074] Example 1 Establishment of polypeptide library

[0075] In this example, a peptide library was synthesized on resin beads using a one-bead-one-substance method, and the steps are as follows:

[0076] (1) Take 0.9 g of resin beads and place them in a synthesis tube. Add a certain amount of DMF to immerse the resin to swell the resin and soak for more than 2 hours;

[0077] (2) Add Fmoc-protected methionine (M) to the synthesis tube, and add a mixture of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT), and react on a shaking table for more than 2 hours; rinse with methanol and DMF alternately three times; perform a ninhydrin boiling water bath test on the resin microbeads, and the resin microbeads are colorless;

[0078] (3) Add deprotection agent (20% hexahydropyridine + 80% DMF) to carry out Fmoc deprotection for 10 min, and rinse with methanol and DMF alternately three times; then, test the resin microbeads in a boiling water bath with ninhydrin, and the beads turn dark blue-purple;

[0079] (4) Add Fmoc-protected glycine (G) to the synthesis tube and repeat steps (2) and (3);

[0080] (5) Referring to steps (2) and (3), five amino acids LNDSI were synthesized and the Fmoc protection was removed;

[0081] (6) Divide the resin in the synthesis tube into 19 parts, add the corresponding dose of Fmoc-protected amino acids (19 natural amino acids except methionine) to each synthesis tube, dissolve them in DMF solution of HOBT (1-hydroxybenzotriazole) / DIC (N,N'-diisopropylcarbodiimide), and shake the reaction for more than 2 hours;

[0082] (7) Rinse with methanol and DMF alternately three times and mix thoroughly;

[0083] (8) Check the synthesis efficiency of several amino acids, take out the resin beads, soak them in ethanol, and then test them in a boiling water bath with ninhydrin, repeat steps (5)-(7) until the beads become colorless, mix the resin, and remove the Fmoc protection;

[0084] (9) Repeat steps (5)-(8) until the last random amino acid is synthesized;

[0085] (10) After removing the Fmoc protecting group with a deprotecting agent (20% hexahydropyridine + 80% DMF), the resin was repeatedly rinsed with DMF and methanol, and dried at 25°C in the dark to obtain a relatively dry resin; the dry resin was transferred to a small reaction bottle (dried in advance), a magnetic bar was added, and all acid-responsive side chain protecting groups were removed with an acidic deprotecting agent (TFA 82.5%, water 15%, triisopropylsilane 2.5%), and the reaction was carried out at 25°C for 4 hours to construct a peptide library.

[0086] Example 2 FITC labeling of EphA2 protein

[0087] In this example, the EphA2 protein (amino acid sequence as shown in SEQ ID NO: 8) was labeled with FITC, and the steps were as follows:

[0088] (1) dissolving the EphA2 protein in water so that the final concentration of the EphA2 protein is 10 μg / mL;

[0089] (2) adding FITC (5%) to the EphA2 protein solution prepared in step (1), placing in an ice bath, and magnetically stirring overnight in a dark environment;

[0090] (3) placing the mixed solution in a dialysis bag with a molecular weight cut-off of 500 and dialyzing it in deionized water for three days;

[0091] (4) Collect the dialyzed solution, freeze-dry it, and obtain FITC-labeled EphA2 protein.

[0092] Example 3 Screening of polypeptides targeting EphA2 protein

[0093] The FITC-labeled EphA2 protein was dissolved at a concentration of 2 μg / mL and incubated with the peptide library in PBS buffer for 2 h. The color of the peptide resin under 405 nm excitation was observed under a fluorescence microscope. Figure 1 As shown, the peptide resin with obvious green fluorescence was selected.

[0094] The selected peptide resin was cleaved overnight with 30% CNBr3 ethanol solution, and the supernatant was collected by centrifugation and desalted for peptide sequencing.

[0095] Example 4 ELISA further confirms and screens polypeptides

[0096] (1) The six peptides were diluted to 2 μg / mL with ELISA coating buffer, and 100 μL of the diluted peptide was added to each well of a 96-well plate for overnight coating at 4°C.

[0097] (2) Add 300 μL of ELISA special washing solution to each well, let it stand for 20 seconds, and then remove it. Repeat this step three times.

[0098] (3) Plate coating and blocking: Add 100 μL of ELISA blocking solution containing BSA to each well and block at 25°C for 2 h. After blocking, repeat the plate washing step (2) three times to dry the plate.

[0099] (4) EphA2 protein labeling with biotin: 20 μg of protein was diluted with 200 μL PBS, and 2 μL 10 mg / mL (DMSO) biotin stock solution was added, and the mixture was incubated at 25°C for 30 min. The mixture was dialyzed with a 3500D dialysis bag for 2 days.

[0100] (5) The biotin-labeled EphA2 protein was diluted into 6 concentrations, 200 μL per well, incubated at 37°C for 2 h, and then washed three times with washing buffer.

[0101] (6) Avidin-labeled horseradish peroxidase was diluted 2000-fold, 80 μL was added to each well, incubated at 37°C for 1 h, and then washed three times with the washing solution.

[0102] (7) Add 100 μL of TMB to each well and incubate at 37°C in the dark for 30 min. Then add 50 μL of terminator to each well and measure the absorbance at 450 nM.

[0103] like Figure 2 As shown, six polypeptides (amino acid sequences are SEQ ID NO: 1 to SEQ ID NO: 6) can bind to EphA2 protein, and the binding of the six polypeptides to EphA2 protein and to BSA is significantly different, indicating that the polypeptides have the ability to specifically target proteins.

[0104] Example 5 Binding Constant Test

[0105] In this example, microscale thermophoresis (MST) was used to measure the binding force between the polypeptide and the EphA2 protein, and the steps were as follows:

[0106] (1) EphA2 protein was prepared into a 1.25 μM protein solution using PBS (pH=7.4), and labeled with the dye N-hydroxysuccinimide (NHS)-ester. After labeling, free dye was removed by gel column filtration;

[0107] (2) 12 peptide solutions with different concentration gradients (5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039, 0.0195, 0.01, 0.005, 0.0025 μM) were mixed with the dye-labeled EphA2 protein solution and then tested on the machine;

[0108] like Figure 3 As shown, the binding constant KD of the polypeptide shown in SEQ ID NO: 2 and EphA2 protein is 1.19×10 -6 , showing the highest affinity to EphA2 protein.

[0109] Example 6 Regulation of polypeptide self-assembly

[0110] The polypeptide with the sequence shown in SEQ ID NO: 2 was prepared into a 20 mM stock solution with DMSO, and then prepared into a 200 μM solution with water. After incubation at 25° C. for 4 h, the sample was prepared on a copper mesh carbon support film by the drop method, and the morphology of the polypeptide nanostructure was observed using a lanthanum hexaboride electron transmission microscope.

[0111] like Figure 4 As shown, the polypeptide with a sequence as shown in SEQ ID NO: 2 can be assembled into nanofibers.

[0112] Example 7 Cell experiment

[0113] Human hepatocellular carcinoma epithelial cell line HepG2 overexpressing EphA2 receptor was used, and after adherence, a 10 μM peptide solution labeled with fluorescent molecule FITC as shown in SEQ ID NO: 2 was added, and after 4 hours of culture, it was incubated with nuclear dye for 20 minutes, and the interaction between cells and peptide molecules was observed under a confocal microscope. As shown in Figure 5, the peptide can efficiently enter the cell after targeting the cell.

[0114] In summary, the present invention adopts the one-bead-one-substance method to synthesize a polypeptide library, screens out polypeptide sequences that can bind to EphA2 receptor protein, and self-assembles into nanostructures (nanofibers or nanoparticles) in a PBS environment. The polypeptide sequence proposed thereby provides support for the construction of a new type of nanomaterial targeting tumor cells that overexpress EphA2 receptors: the targeting sequence can specifically bind to the EphA2 receptor, and the assembly sequence can aggregate into a nanostructure on the surface of the tumor cell membrane; the polypeptide screening method of the present invention has the advantages of high throughput and high screening efficiency, and provides a simple and easy method for discovering EphA2 protein-targeted self-assembling polypeptides.

[0115] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A polypeptide targeting human endothelial cell receptor protein tyrosine kinase, characterized in that: The amino acid sequence of the polypeptide comprises: (1) a sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6, or, (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1), or, (3) An amino acid sequence that has at least 95% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide targeting human endothelial cell receptor protein tyrosine kinase according to claim 1.

3. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 2.

4. A recombinant cell, characterized in that The recombinant cell expresses the polypeptide targeting human endothelial cell receptor protein tyrosine kinase according to claim 1.

5. A method for screening polypeptides targeting human endothelial cell receptor protein tyrosine kinase according to claim 1, characterized in that: The screening method comprises the following steps: (1) using a one-bead-one-substance separation-mixing method to solid-phase synthesize a peptide library; the peptide library contains a known self-assembly sequence; (2) The EphA2 protein extracellular domain is mixed with a buffer solution, labeled with FITC fluorescent molecules, and incubated with the peptide library of step (1), and the resin microbeads with the strongest fluorescence are selected for sequence analysis to obtain peptides that can bind to the EphA2 protein.

6. The method for screening polypeptides targeting human endothelial cell receptor protein tyrosine kinase according to claim 5, characterized in that: The method for synthesizing the polypeptide library in step (1) specifically comprises: (1-1) Firstly, the leader sequence and the self-assembling polypeptide sequence are synthesized on the resin microbeads; (1-2) the resin beads are evenly divided, the Fmoc-protected amino acid is activated and added to the resin beads to undergo a condensation reaction with the previously deprotected amino acid; (1-3) mixing the resin beads and performing Fmoc deprotection; (1-4) Repeat steps (1-2) and (1-3) until the last amino acid is synthesized; (1-5) Fmoc deprotection is performed followed by side chain deprotection to obtain the polypeptide library.

7. The method for screening polypeptides targeting human endothelial cell receptor protein tyrosine kinase according to claim 6, characterized in that: The length of the leader sequence in step (1-1) is 2 to 4 aa; the self-assembly sequence includes the sequence shown in SEQ ID NO: 7; Preferably, the leader sequence comprises methionine; Preferably, the activation reagent in step (1-2) comprises any one of N,N-dimethylformamide, methanol, dichloromethane, N-methylmorpholine, N,N'-diisopropylcarbodiimide or 1-hydroxybenzotriazole, or a combination of at least two thereof; Preferably, the deprotection reagent in step (1-3) comprises a DMF solution containing hexahydropyridine or a DMF solution containing piperazine and 1,8-diazabicycloundec-7-ene; Preferably, the side chain deprotection reagent in step (1-5) contains trifluoroacetic acid, benzene and water; Preferably, the length of the polypeptides in the polypeptide library in step (1) is 7 to 15 aa; Preferably, the amino acid sequence of the extracellular domain of the EphA2 protein in step (2) includes the sequence shown in SEQ ID NO: 8; Preferably, the buffer in step (2) comprises PBS buffer; Preferably, the incubation time in step (2) is 2-4 hours.

8. The method for screening polypeptides targeting human endothelial cell receptor protein tyrosine kinase according to any one of claims 5 to 7, characterized in that: The method for screening polypeptides targeting human endothelial cell receptor protein tyrosine kinase comprises the following steps: S1. Firstly, a leader sequence containing methionine and having a length of 2 to 4 aa and a self-assembly sequence as shown in SEQ ID NO: 7 are synthesized on resin microbeads; S2. After the resin beads are evenly divided, an Fmoc-protected amino acid is activated and added to the resin beads to undergo a condensation reaction with the previously deprotected amino acid; S3. The resin beads were mixed and Fmoc deprotection was performed using a DMF solution containing hexahydropyridine; S4. Repeat steps (S.2) and (S.3) until the last amino acid is synthesized to obtain a polypeptide library, wherein the length of the polypeptides in the polypeptide library is 7 to 15 aa; S5. Add the fluorescently labeled EphA2 protein with the amino acid sequence shown in SEQ ID NO: 8 to the polypeptide library, incubate for 2 to 4 hours, and screen for polypeptides that specifically bind to the HER2 protein based on the fluorescence intensity.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a drug or contrast agent molecule connected with the polypeptide targeting human endothelial cell receptor protein tyrosine kinase according to claim 1.

10. Use of the polypeptide targeting human endothelial cell receptor protein tyrosine kinase according to claim 1 and / or the pharmaceutical composition according to claim 9 in preparing imaging agents and / or therapeutic drugs for diseases.

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