Polypeptides targeting the epidermal growth factor receptor

Abstract

The present application relates to the technical field of biotechnology, in particular to a polypeptide targeting epidermal growth factor receptor; the amino acid sequence of the polypeptide comprises the sequence shown in any one of (i)-(iii): (i) the sequence shown in any one of SEQ ID NO. 1-4; (ii) the sequence with one or more amino acids substituted, deleted or added in the sequence shown in any one of SEQ ID NO. 1-4 and having the activity of targeting epidermal growth factor receptor; (iii) the sequence with at least 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the sequence shown in any one of SEQ ID NO. 1-4 and having the activity of targeting epidermal growth factor receptor. The polypeptide has a relatively small molecular weight, stable properties and extremely strong affinity to EGFR, which provides great help for the development of drugs targeting EGFR.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to polypeptides targeting epidermal growth factor receptor. BACKGROUND

[0002] The Human Epidermal Growth-Factor Receptor (HER) family includes four members: HER1 (ErbB1, EGFR), HER2 (ErbB2, NEU), HER3 (ErbB3) and HER4 (ErbB4), which play an important regulatory role in cell physiological processes. Among them, the Epidermal Growth Factor Receptor (EGFR) is the receptor for Epidermal Growth Factor (EGF) induced cell proliferation and signal transduction, which belongs to the tyrosine kinase type receptor. EGFR is a transmembrane glycoprotein composed of three parts: extracellular domain (N-terminal), transmembrane domain (hydrophobic alpha helix structure) and intracellular domain (C-terminal), with a molecular weight of about 170 KDa.

[0003] EGFR is activated by binding with ligands, which include EGF and Transforming Growth Factor alpha (TGFa). The ligand binds to the extracellular domain of EGFR to form a homodimer or a heterodimer (for example, converted from a monomer to a dimer, or polymerized with other members of the HER family such as ErbB2 / HER2 / NEU to form a dimer). After EGFR dimerization, receptor autophosphorylation occurs, including Y992, Y1045, Y1068, Y1148 and Y1173, etc. Autophosphorylation of these sites can guide downstream phosphorylation and activate its intracellular kinase pathway, including MAPK, Akt and JNK pathways, to induce cell proliferation.

[0004] EGFR is expressed on the surface of normal epithelial cells. Studies have shown that EGFR is highly expressed or abnormally expressed in many solid tumors, and overexpression of EGFR is associated with tumor cell proliferation, metastasis, infiltration and poor prognosis. Therefore, developing drugs targeting EGFR is of great significance for tumor treatment. Currently, the polypeptide targeting EGFR that is relatively widely used in the market is GE11 polypeptide, which has been shown to be an effective active targeting agent in tumor treatment.

[0005] In addition, compared with proteins with large molecular weight, peptides with small molecular weight have the advantages of small molecular size, easy synthesis, modification and optimization, high product purity, stable properties, specific biological activity, and are one of the hotspots in the field of biopharmaceutical research. At present, polypeptide-based drugs include polypeptide vaccines, anti-tumor polypeptides, polypeptide-guided drugs (or polypeptide-targeted drugs), cytokine-mimic peptides, antibacterial active peptides, and diagnostic polypeptides, etc. Among them, polypeptide-guided drugs are fused with polypeptides having binding ability to target points and cytotoxins or cytokines, etc. to guide them to the lesion site to play a therapeutic role, while reducing toxic side effects. The development of polypeptide-guided drugs is one of the current hotspots.

[0006] At present, the methods for obtaining polypeptides include searching for natural unknown polypeptides from nature or screening from artificially constructed polypeptide libraries, and the screening methods include, for example, phage display technology. Phage display technology is to insert the foreign gene encoding polypeptide or protein into the appropriate position of the structural gene of the coat protein of phage through genetic engineering technology, so that the foreign polypeptide or protein forms a fusion protein on the coat protein of phage, and is presented on the surface of phage through reassembly of the progeny phage, thereby forming a phage display library. Phage display technology can maintain the relative spatial structure and biological activity of the foreign polypeptide or protein. Then, using the target molecule, using appropriate elution methods, washing away the phage that does not specifically bind, and then eluting the phage that binds to the target molecule with acid and alkali or competitive molecules, the neutralized phage infects E. coli for amplification, and after 3-5 rounds of screening and enrichment, the polypeptide or protein that recognizes the target molecule can be finally obtained. Phage display technology is an operating system for expressing foreign genes, which has the advantages of simplicity, effectiveness and easy control, can realize the unity of phenotype and genotype, and has a wide application prospect. SUMMARY

[0007] Problem to be solved by the invention

[0008] The overexpression of EGFR is closely related to the occurrence, development and prognosis of tumors. In view of the market demand for developing polypeptide-guided drugs targeting EGFR, the present application provides a polypeptide with high affinity to EGFR, which helps the development of EGFR-targeted drugs.

[0009] Solution to the problem

[0010] [1] A polypeptide, characterized in that the amino acid sequence of the polypeptide comprises a sequence as shown in any one of (i) to (iii) below:

[0011] (i) a sequence as shown in any one of SEQ ID NO. 1 to 4;

[0012] (ii) a sequence in which one or more amino acids are substituted, deleted, or added in the sequence shown in any one of SEQ ID NO. 1 to 4 and has an activity of targeting epidermal growth factor receptor;

[0013] (iii) a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to the sequence shown in any one of SEQ ID NO. 1 to 4 and has an activity of targeting epidermal growth factor receptor.

[0014] [2] The polypeptide according to [1], characterized in that the amino acid sequence of the polypeptide comprises the sequence shown in any one of SEQ ID NO. 1 to 4.

[0015] [3] The polypeptide according to [1] or [2], characterized in that the amino acid sequence of the polypeptide is the sequence shown in any one of SEQ ID NO. 1 to 4.

[0016] [4] A polynucleotide, characterized in that the polynucleotide encodes the polypeptide according to any one of [1] to [3].

[0017] [5] A recombinant expression vector, characterized in that the recombinant expression vector comprises the polynucleotide according to [4];

[0018] Preferably, the recombinant expression vector includes a plasmid vector and a viral vector;

[0019] More preferably, the plasmid vector includes a pET series vector, and / or, more preferably, the viral vector includes a bacteriophage.

[0020] [6] A recombinant host cell, characterized in that the recombinant host cell comprises the recombinant expression vector according to [5];

[0021] Preferably, the recombinant host cell includes a eukaryotic cell and a prokaryotic cell;

[0022] More preferably, the eukaryotic cell includes a yeast, and / or, more preferably, the prokaryotic cell includes an Escherichia coli.

[0023] [7] A method for preparing the polypeptide according to any one of [1] to [3], characterized in that the method comprises the step of culturing the recombinant host cell according to [6] and expressing the polypeptide.

[0024] [8] A medicament, characterized in that the medicament comprises the polypeptide according to any one of [1] to [3];

[0025] Preferably, the medicament targets epidermal growth factor receptor;

[0026] More preferably, the drug targeting epidermal growth factor receptor is used for preventing and / or treating a tumor.

[0027] Even more preferably, the tumor comprises at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer and prostate cancer; more preferably, the tumor is non-small cell lung cancer.

[0028] [9] A detection or diagnosis kit containing the polypeptide according to any one of [1] to [3].

[0029]

[10] Use of the polypeptide according to any one of [1] to [3] in the preparation of a medicament.

[0030] Preferably, the medicament targets epidermal growth factor receptor.

[0031] More preferably, the drug targeting epidermal growth factor receptor is used for preventing and / or treating a tumor.

[0032] Even more preferably, the tumor comprises at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer and prostate cancer; more preferably, the tumor is non-small cell lung cancer.

[0033] Effects of the invention

[0034] The present application provides a polypeptide targeting epidermal growth factor receptor, which has a relatively small molecular weight, stable properties, and extremely strong affinity for EGFR, and greatly helps the development of drugs targeting EGFR. DETAILED DESCRIPTION

[0035] The embodiments of the present application are described below, but the present application is not limited thereto. The present application is not limited to each of the configurations described below, and various modifications can be made within the scope of the present application, and embodiments obtained by appropriately combining the technical means disclosed in each of the different embodiments and examples are also included in the technical scope of the present application.

[0036] The first aspect of the present application provides a polypeptide capable of targeting epidermal growth factor receptor. Specifically, the present application uses phage display technology to screen polypeptides with extremely strong affinity for EGFR from an artificially constructed phage library (Ph.D. TM -12Phage Display Peptide Library Kit v2, NEB#E8210-1).

[0037] In the present application, the terms "polypeptide", "protein", "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having otherwise similar properties. In addition, the polypeptides described in the present application can be linear or cyclic.

[0038] In some embodiments, the present application provides a polypeptide having an amino acid sequence comprising any one of the sequences set forth in (i) to (iii) below:

[0039] (i) a sequence as set forth in any one of SEQ ID NOs. 1 to 4;

[0040] (ii) a sequence having one or more amino acids substituted, deleted or added in a sequence as set forth in any one of SEQ ID NOs. 1 to 4 and having an activity of targeting epidermal growth factor receptor;

[0041] (iii) a sequence having at least 85%, 90%, 95%, 97%, 98% or 99% sequence identity to a sequence as set forth in any one of SEQ ID NOs. 1 to 4 and having an activity of targeting epidermal growth factor receptor.

[0042] In the present application, the term "amino acid" can include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids and abbreviations and English abbreviations in the present application are as follows: histidine (His, H); serine (Ser, S); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); isoleucine (Ile, I); arginine (Arg, R); alanine (Ala, A); glutamine (Gln, Q); valine (Val, V); tryptophan (Trp, W); leucine (Leu, L); methionine (Met, M); asparagine (Asn, N); cysteine (Cys, C); lysine (Lys, K); proline (Pro, P).

[0043] In the present application, "addition" of an amino acid means adding an amino acid at any position in the C-terminus, N-terminus or between the C-terminus and N-terminus of an amino acid sequence, and the added amino acids can be all or part adjacent to each other, or none of the added amino acids are adjacent to each other; "deletion" of an amino acid means deleting 1, 2 or more than 3 amino acids from an amino acid sequence; "substitution" of an amino acid means replacing an amino acid at a certain position in an amino acid sequence with another amino acid, as long as the changed sequence retains the activity of the original amino acid sequence completely or partially. The substitution of an amino acid can be a conservative amino acid substitution, which means that several amino acids are replaced with amino acids of similar or similar properties to form a peptide (conservative variant peptide) compared with the original amino acid sequence. The substitution of an amino acid can also be a non-conservative amino acid substitution.

[0044] In the present application, "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if both molecules share an adenine at a particular location, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions in the comparison times 100%. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. In general, the comparison is made when the two sequences are aligned for maximum percent identity.

[0045] In some specific embodiments, the amino acid sequence of the polypeptide provided by the present application comprises a sequence as shown in any one of SEQ ID NO. 1-4. In some specific embodiments, the amino acid sequence of the polypeptide provided by the present application comprises a sequence as shown in any one of SEQ ID NO. 1-3.

[0046] In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present application comprises a sequence as shown in SEQ ID NO. 1. In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present application comprises a sequence as shown in SEQ ID NO. 2. In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present application comprises a sequence as shown in SEQ ID NO. 3. In some more specific embodiments, the amino acid sequence of the polypeptide provided by the present application comprises a sequence as shown in SEQ ID NO. 4.

[0047] In some preferred embodiments, the amino acid sequence of the polypeptide provided by the present application is a sequence as shown in any one of SEQ ID NO. 1-4. In some specific embodiments, the amino acid sequence of the polypeptide provided by the present application is a sequence as shown in any one of SEQ ID NO. 1-3.

[0048] In some more preferred embodiments, the polypeptide provided by the present application has an amino acid sequence as set forth in SEQ ID NO. 1. In some more preferred embodiments, the polypeptide provided by the present application has an amino acid sequence as set forth in SEQ ID NO. 2. In some more preferred embodiments, the polypeptide provided by the present application has an amino acid sequence as set forth in SEQ ID NO. 3. In some more preferred embodiments, the polypeptide provided by the present application has an amino acid sequence as set forth in SEQ ID NO. 4.

[0049] The polypeptide targeting EGFR provided by the present application has a small molecular weight, which can consist of only 12 amino acid residues, and is stable in nature, and has good affinity and targeting property to EGFR. In some embodiments, the polypeptide can form a cyclic polypeptide by forming a disulfide bond through the sulfhydryl group in the intramolecular cysteine.

[0050] The second aspect of the present application provides a polynucleotide capable of encoding the polypeptide of the first aspect of the present application.

[0051] In the present application, the terms "polynucleotide", "nucleic acid molecule", "polynucleic acid", and "nucleic acid" are used interchangeably, and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. The polynucleotide can have any three-dimensional structure, and can perform any known or unknown function. In addition, the nucleotides of the present application can be linear or circular. The polynucleotide of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0052] The polynucleotide encoding the polypeptide of the present application includes: a coding sequence encoding only the polypeptide; a coding sequence of the polypeptide and various additional coding sequences; a coding sequence of the polypeptide (and optional additional coding sequences) and non-coding sequences. Among them, the "polynucleotide encoding the polypeptide" can be a polynucleotide including a coding sequence encoding the polypeptide, or a polynucleotide further including additional coding and / or non-coding sequences.

[0053] The third aspect of the present application provides a recombinant expression vector comprising the polynucleotide of the second aspect of the present application.

[0054] In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. The vector is well known to those skilled in the art, including but not limited to: plasmid, bacteriophage, cosmid, etc. Further, the "recombinant expression vector" is an expression vector into which a foreign gene is introduced.

[0055] In some embodiments, the recombinant expression vector provided by the present application further comprises a suitable promoter or control sequence, etc., which can be used to transform a suitable host cell, so that the host cell is capable of expressing a protein.

[0056] In some embodiments, the recombinant expression vector provided by the present application includes a plasmid vector or a viral vector; for example, the plasmid vector includes pET series vectors, and the viral vector includes bacteriophages.

[0057] The fourth aspect of the present application provides a recombinant host cell, which introduces or contains the recombinant expression vector of the third aspect of the present application.

[0058] In the present application, the term "host cell" refers to a cell into which an expression vector has been introduced. The host cell can include a bacterium, a microorganism, a plant cell or an animal cell. Bacteria that are easy to transform include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells. Further, the "recombinant host cell" is a host cell that is different from the parent cell after the introduction of a foreign polynucleotide or a recombinant expression vector, and the recombinant host cell can be obtained by transformation.

[0059] In some embodiments, the host cell provided by the present application includes a eukaryotic cell or a prokaryotic cell; for example, the eukaryotic cell includes yeast, and the prokaryotic cell includes E. coli.

[0060] The fifth aspect of the present application provides a method for preparing the polypeptide of the first aspect of the present application, which comprises the steps of culturing the recombinant host cell of the fourth aspect of the present application and expressing the polypeptide. The preparation method provided by the present application can further comprise the step of isolating and purifying the polypeptide from the culture.

[0061] The sixth aspect of the present application provides a medicament comprising the polypeptide of the first aspect of the present application.

[0062] In some embodiments, the drug is a drug targeting EGFR; illustratively, the drug is a polypeptide directed drug.

[0063] In some embodiments, the drug targeting EGFR is used for preventing and / or treating a tumor.

[0064] In the present application, "treatment" means: before suffering from a disease, by contacting (e.g. administering) a drug comprising the polypeptide of the present application to a subject, etc., thereby delaying or weakening the progress of the disease or condition, including alleviating, relieving or reducing one or more symptoms of the condition or state, and does not mean that the symptoms of the disease must be completely inhibited.

[0065] In the present application, "prevention" means: before suffering from a disease, by contacting (e.g. administering) a drug comprising the polypeptide of the present application to a subject, etc., thereby alleviating the symptoms after suffering from the disease compared with not contacting, and does not mean that the disease must be completely inhibited.

[0066] In some embodiments, the tumor comprises at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer and prostate cancer.

[0067] In some preferred embodiments, the tumor is non-small cell lung cancer.

[0068] The seventh aspect of the present application provides a detection or diagnosis kit comprising the polypeptide of the first aspect of the present application.

[0069] The kit provided by the present application can be used for detecting EGFR or for diagnosing a disease or finding a lesion related to high expression of EGFR, etc. by virtue of the EGFR targeting property of the polypeptide.

[0070] The eighth aspect of the present application provides a method for detecting EGFR, comprising contacting the polypeptide of the first aspect of the present application with a sample to be tested and detecting the binding of the two, thereby determining whether EGFR exists in the sample to be tested.

[0071] In some embodiments, the method for detecting EGFR is a method for non-diagnostic or therapeutic purposes. In some embodiments, the method for detecting EGFR is a method for diagnostic or therapeutic purposes.

[0072] The ninth aspect of the present application provides the use of the polypeptide of the first aspect of the present application in the preparation of a drug, particularly a drug targeting EGFR.

[0073] In some embodiments, the drug targeting EGFR is used for preventing and / or treating a tumor.

[0074] In some embodiments, the tumor comprises at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer.

[0075] In some preferred embodiments, the tumor is non-small cell lung cancer.

[0076] The tenth aspect of the present application provides a method for treating a tumor, comprising the step of administering to a subject in need thereof an effective amount of a medicament comprising the polypeptide of the first aspect of the present application.

[0077] In the present application, "administering" means administering by a nurse, a health care provider, a patient, or any other individual, including self-administration. The administration includes not only delivery into the body, but also prescribing, dispensing, or otherwise assisting in the delivery in any way.

[0078] In the present application, "subject", "patient" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0079] In some embodiments, the medicament is an EGFR-targeting drug.

[0080] In some embodiments, the tumor comprises at least one of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer, bladder cancer, and prostate cancer.

[0081] In some preferred embodiments, the tumor is non-small cell lung cancer.

[0082] Example

[0083] The present application is further illustrated by the following examples, which are not intended to be limiting. The specific materials used in the examples of the present application are provided below, along with their sources. However, it is understood that these are merely exemplary and that materials of the same or similar type, make, quality, property, or function as the following reagents and instruments can be used to practice the present application. Unless otherwise indicated, the experimental methods used in the following examples are routine methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, are commercially available.

[0084] Example 1: Screening of polypeptides binding to EGFR using phage display technology

[0085] Take 10 μL of phage library (12 peptide library (2 x 1011pfu / mL, Ph.D. 13 pfu / mL, Ph.D. TM-12 Phage Display Peptide Library Kit v2, NEB #E8210-1))(2x10 11 PFU) into 790 μΐ of 2% Milk-PBS (2% milk added to PBS) for blocking, and shake at room temperature for 1 h. Take 100 μΐ of streptavidin magnetic beads (Dynabeads TM M-280, Invitrogen, Cat# 11206D), wash 3 times with PBS (1 mL each time), then block with 2% Milk-PBS, shake at room temperature for 2 h. Add Biotin-EGFR (Yi Qiao 10001-H27H-B) to the blocked phage milk mixture, shake at room temperature for 1.5 h. Remove the 2% Milk-PBS used for blocking in the magnetic beads, resuspend with 200 μΐ of 0.1% PBST-2% Milk (0.1% PBST, 2% milk). Add the resuspended magnetic beads to the incubated phage-antigen mixture, shake at room temperature for 30 min. Place the mixture in a magnetic stand, remove the liquid in the tube after the magnetic beads are adsorbed to the tube wall, then wash the magnetic beads: 0.1% PBST for 10 times. Adsorb the magnetic beads to the tube wall and remove the PBST in the tube, add 1 mL of elution buffer (0.2 M Gly-HCl, 1 mg / mL BSA, pH 2.2), shake at room temperature for 30 min, then immediately neutralize with 150 μΐ of neutralization buffer (1 M Tris-HCl, pH 9.1, filter sterilized). Reserve 5-10 μΐ of the eluate for titration, immediately amplify the rest of the eluate.

[0086] Inoculate E. coli K12 ER2738 (ER2738 (NEB #E4104S) into 20 mL of LB medium, shake at 37°C, 250 rpm for 1 h. Add the phage eluate, continue to shake at 37°C, 250 rpm for 4.5-5 h. Centrifuge at 4°C, 5000 g for 10 min, resuspend the supernatant with PEG / NaCl (20% (w / v) PEG-8000, 2.5 M NaCl), mix gently, and let the phage precipitate at 4°C for 2 h or overnight. Centrifuge, discard the supernatant, resuspend with PBS, centrifuge, transfer the supernatant to a fresh microcentrifuge tube, add 200 μΐ of PEG / NaCl, the solution will become turbid quickly, incubate on ice for 0.5-1 h. Centrifuge, discard the supernatant, resuspend the precipitate in 0.5 mL of PBS. Centrifuge for 1 min to precipitate any residual insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.

[0087] Titer the amplified eluate on LB / IPTG / Xgal plates (LB medium + 15 g / L agar powder. Autoclave, cool to below 70°C, add 1 ml IPTG / Xgal, mix and pour plates). Count the number of blue plaques on the plates to determine the titer.

[0088] Repeat the above steps for the second, third, and fourth rounds of panning. After the fourth round of panning, do not amplify the eluate, but titer the eluate to obtain individual plaques for sequencing in the phage sequencing reaction. Determine the titer of the eluate from the fourth round of panning without amplification on LB / IPTG / Xgal plates. The plaques obtained in the titer determination can be used for sequencing.

[0089] Example 2: Determination of phage titer

[0090] Inoculate a single ER2738 colony into 5-10 mL of LB medium and incubate with shaking until mid-log phase (OD600=0.5). While the cells are growing, microwave a top layer of agarose gel and dispense into sterile culture tubes, 3 mL per tube. Keep at 45°C until needed. Pre-warm LB / IPTG / Xgal plates at 37°C until needed. Dilute the phage 10-fold in LB medium. Once the ER2738 culture has reached mid-log phase, dispense 200 μL per tube into microfuge tubes. Add the diluted phage supernatant to the microfuge tubes containing the bacterial culture, 10 μL of one dilution per tube, vortex briefly, and incubate at room temperature for 1-5 minutes. Transfer to the tubes containing the 45°C top layer of agarose gel, vortex briefly, and immediately pour onto the pre-warmed LB / IPTG / Xgal plates, rocking the plates gently to distribute the top layer of gel evenly. Allow the plates to cool for 5 minutes and then incubate inverted at 37°C overnight. Count the plaques in a plate with approximately 100 plaques, and multiply the number of plaques by the dilution factor to obtain the titer in plaque forming units (pfu) per 10 μL of phage.

[0091] Example 3: Amplification of plaques

[0092] Dilute the ER2738 overnight culture 1:100 into LB medium and inoculate 200 μL per well into a 96-deep well bacterial culture plate. Using a sterile toothpick or pipette tip, pick a blue plaque into the 96-deep well plate, one well per plaque to be identified. Note: Pick from a plate with less than 100 total plaques to ensure that each picked plaque contains only one DNA sequence. After 4.5-5 hours incubation at 37°C on a shaker, store the plate at 4°C until needed.

[0093] Example 4: Detection of binding of selected polypeptides to target molecules using ELISA

[0094] 0.1M NaHCO3(pH 8.6) coated antigen: Coat 100 μL of 100 μg / mL of target molecule Recombinant Human EGFR Protein (ECD, His Tag) (Sino, Cat: 10001-H08H) to ELISA plate, concentration 10 μg / ml, 100 μl / well, 4°C coating overnight. Phage treatment: Pack phage plate 4°C, 3000g centrifugal 10 min. Take 100 μl supernatant to new 96-well plate (100 μl / well 2% milk-PBS in advance), incubate for 1 h; 96-well enzyme-labeled plate coated with antigen, wash plate 3 times with 1 x PBS / 1‰ Tween (Biosharp PBS buffer (ready-to-use dry powder) prepared to 10 mM, pH 7.2-7.4, item number BL601A; Tween 20, Sigma, item number P9416-100 ml); 2% milk-PBS blocking for 30 min, 350 μl / well; PBST washing 3 times; take the blocked phage and add to the enzyme-labeled plate, incubate at room temperature for 1 h; PBST washing 3 times; dilute HRP-labeled anti-M13 antibody (Sino, 11973-MM05T-H) at a ratio of 1:2000. Add 100 μl of diluted antibody to each well, shake at room temperature for 1 h. PBST wash 6 times; color development: add 100 μl of TMB color developing solution, color develop for 2 min. Stop: add 100 μl / well TMB stop solution to stop the reaction. Reading: detect OD450nm with a microplate reader. Pick positive monoclonal phage liquid for sequencing analysis and polypeptide synthesis.

[0095] Pick positive monoclonal phage liquid for sequencing analysis and polypeptide synthesis.

[0096] Example 5: Polypeptide affinity determination

[0097] The binding affinity of the polypeptide and EGFR antigen was obtained by SPR analysis using a surface plasmon resonance instrument.

[0098] Antigen immobilization: Take recombinant target protein Recombinant Human EGFR Protein (ECD, His Tag), Sino, Cat: 10001-H08H, dilute it to 25 μg / mL using 10 mM sodium acetate solution (pH 4.0), and couple it to a dextran surface chip (manufacturer Reichert, item number 13206066) so that the final chip antigen binding amount is 1500 μRIU.

[0099] Sample preparation: The prepared polypeptide ligand mother liquor (5 mM) was diluted 100 times to 50 μM initial concentration with PBST (0.05% Tween 20) + 1% DMSO, and then 2-fold gradient dilution was performed with PBST (0.05% Tween 20) + 1% DMSO, i.e. 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μM, a total of 7 concentration gradients. Every 3 samples were inserted with a PBST (0.05% Tween 20) + 1% DMSO sample as a blank control.

[0100] Running program: The detection sample was injected with the following parameters: set the binding time to 3 min, the dissociation time to 5 min, the regeneration reagent (10 mM Gly-Hcl pH 3.0) binding time to 30 s, the regeneration reagent dissociation time to 1 min, set the Loop repetition number according to the sample number, the flow rate to 25 μL / min, and the running buffer to PBST (0.05% Tween 20) + 1% DMSO. All experiments were carried out at room temperature 25°C, and the sample was kept at 4°C before flowing through the chip. The software TraceDrawer (Reichert Technologies) was used to determine the steady-state fitting according to Langmuir 1:1 binding.

[0101] Table 1 is the binding affinity of the EGFR polypeptide ligand screened by the present application and the known GE11 polypeptide (SEQ ID NO. 5) to human EGFR protein. The experimental results show that the EGFR polypeptide ligand screened by the present application has a comparable affinity, and the dissociation equilibrium constant KD value is between 2.80 x 10 -5 ~ 1.69 x 10 -4 M, which can meet the requirements of developing conjugated ligands.

[0102] The affinity detection results are shown in Table 1 as follows:

[0103] Table 1 Polypeptide and target molecule affinity detection results

[0104]

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is as shown in any one of SEQ ID NO. 2 to 4.

2. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide according to claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vectors include plasmid vectors and viral vectors.

5. The recombinant expression vector according to claim 4, characterized in that, The plasmid vector includes pET series vectors, and / or the viral vector includes bacteriophages.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant expression vector according to any one of claims 3 to 5.

7. The recombinant host cell according to claim 6, characterized in that, The recombinant host cells include eukaryotic cells and prokaryotic cells.

8. The recombinant host cell according to claim 7, characterized in that, The eukaryotic cells include yeast, and / or the prokaryotic cells include Escherichia coli.

9. The method for preparing the polypeptide according to claim 1, characterized in that, The method includes the steps of culturing recombinant host cells according to any one of claims 6 to 8 and expressing polypeptides.

Images