Affinity peptides targeting human cd81 protein and uses thereof

Abstract

The application discloses an affinity peptide targeting human CD81 protein and application thereof. The amino acid sequence of the affinity peptide provided by the application is shown as SEQ ID NO: 7. The affinity peptide provided by the application has high affinity and high selectivity with a small loop fragment of an extracellular CD81 protein, can recognize and combine with the CD81 protein in free and living cells, can be used for high-sensitivity detection and in-situ imaging of the CD81 protein, and can be further used as a selective inhibitor of a migration process of tumor cells expressing the CD81 protein. The affinity peptide provided by the application can specifically target and recognize the CD81 protein, is a specific probe for analysis and detection of the CD81 protein and functional research, and has application prospects in detection of diseases such as malignant tumors and development of targeted drugs. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to an affinity peptide targeting human CD81 protein and its applications. Background Technology

[0002] The four-transmembrane protein CD81 participates in important physiological processes such as protein transport, cell fusion, and signal transduction in the human body, and is closely related to the occurrence and development of various diseases. Studies have shown that CD81 interacts with other proteins to form a transmembrane protein interaction network, regulating processes such as tumor cell adhesion, proliferation, migration, and invasion, and is a key signaling protein in cancer development and metastasis. Furthermore, as a receptor for the hepatitis C virus (HCV) envelope glycoprotein, CD81 can mediate virus-cell fusion, playing an indispensable role in the process of viral invasion and infection of host cells. Therefore, CD81 is an ideal target for the detection and intervention of tumor metastasis and HCV infection. Screening for novel affinity recognition molecules targeting CD81 is crucial for disease detection and the development of new targeted drugs. However, CD81 lacks natural binding ligands, and research on its artificial affinity ligands is scarce, severely hindering its development into a novel biomarker and drug target. The discovery of CD81 ligand molecules with high affinity and high specificity promises new opportunities for the early detection of major diseases such as metastatic tumors and the development of targeted drugs.

[0003] Peptides, as endogenous physiologically active substances, participate in numerous molecular events within living organisms, playing vital physiological functions. Molecular probes using peptides as recognition units not only exhibit good biocompatibility and low immunogenicity but also possess strong designability and are easy to synthesize and modify, leading to their widespread application in the analysis and detection of physiologically active substances. Simultaneously, based on the specific binding of targeting peptides to target protein molecules, an increasing number of novel targeted drugs and vaccines for anticancer and antiviral purposes have been developed. Therefore, designing and constructing novel artificial peptide recognition molecules targeting key structural fragments of the CD81 protein using chemical methods, and enabling them to specifically bind to the CD81 protein, can not only develop highly selective and sensitive new methods for disease analysis and detection but also provide new and valuable ideas and means for regulating and intervening in CD81 function and developing novel targeted drugs. Summary of the Invention

[0004] The purpose of this invention is to provide an affinity peptide that targets the human CD81 protein.

[0005] This invention first protects a polypeptide whose amino acid sequence is shown in SEQ ID NO: 7.

[0006] The polypeptide can be synthesized using solid-phase peptide synthesis methods (BOC or FMOC methods), or using an automated polypeptide synthesizer, or by cloning the gene encoding the polypeptide into an expression vector for biosynthesis.

[0007] The present invention also protects the application of the said polypeptide, which may be at least one of the following (c1)-(c12):

[0008] (c1) Prepare a kit for detecting CD81 protein;

[0009] (c2) Prepare a kit for detecting peptide EL1;

[0010] (c3) Prepare a kit for detecting cells expressing CD81 protein;

[0011] (c4) Prepare a kit for inhibiting the migration and / or invasion of cells expressing CD81 protein;

[0012] (c5) Prepare a kit for inhibiting the migration and / or invasion of cancer tissues expressing CD81 protein;

[0013] (c6) Prepare a kit for inhibiting CD81 protein function;

[0014] (c7) Detection of CD81 protein;

[0015] (c8) Detection of peptide EL1;

[0016] (c9) Detection of cells expressing CD81 protein;

[0017] (c10) Inhibits the migration and / or invasion of cells expressing CD81 protein;

[0018] (c11) Inhibits the migration and / or invasion of cancer tissues expressing CD81 protein;

[0019] (c12) acts as an inhibitor of CD81 protein function;

[0020] The amino acid sequence of polypeptide EL1 is shown in SEQ ID NO: 10.

[0021] In the above applications, the cells expressing CD81 protein can be human breast cancer cells MCF-7, human liver cancer cells Huh-7, or human glioma cells U251.

[0022] The present invention also protects a conjugate obtained by conjugating the polypeptide and a substance.

[0023] In the conjugate, the substance may be a drug. The drug may have the following functions (1), (2), (3), and / or (4): (1) inhibiting cancer cell proliferation, migration, and / or invasion; (2) killing cancer cells; (3) inhibiting cancer tissue growth and / or metastasis; (4) treating cancer. In the conjugate, the polypeptide serves as a carrier for targeting the drug to the CD81 protein.

[0024] The drug may be a compound or a protein. The drug may be a toxin, cytokine, enzyme, or lectin.

[0025] In the conjugate, the substance may be a labeling group, meaning the conjugate is formed by coupling the polypeptide with the labeling group. This conjugate can be used as a detection probe to detect CD81 protein.

[0026] The labeling group may be a radioactive element, a fluorescent group, a quantum dot, or a chromophore with a high absorption coefficient. Specifically, the fluorescent group may be fluorescein thiocyanate (FITC) or tetraphenylethylene (TPE).

[0027] The conjugate may specifically be a conjugate obtained by conjugating the polypeptide with FITC or TPE.

[0028] The structural formula of any of the above-mentioned FITCs is shown in formula (Ⅰ);

[0029]

[0030] The structural formula of any of the above-mentioned TPEs is shown in formula (II);

[0031]

[0032] The present invention also protects the application of any of the above-described couplings, which may be at least one of the following (c1)-(c12):

[0033] (c1) Prepare a kit for detecting CD81 protein;

[0034] (c2) Prepare a kit for detecting peptide EL1;

[0035] (c3) Prepare a kit for detecting cells expressing CD81 protein;

[0036] (c4) Prepare a kit for inhibiting the migration and / or invasion of cells expressing CD81 protein;

[0037] (c5) Prepare a kit for inhibiting the migration and / or invasion of cancer tissues expressing CD81 protein;

[0038] (c6) Prepare a kit for inhibiting CD81 protein function;

[0039] (c7) Detection of CD81 protein;

[0040] (c8) Detection of peptide EL1;

[0041] (c9) Detection of cells expressing CD81 protein;

[0042] (c10) Inhibits the migration and / or invasion of cells expressing CD81 protein;

[0043] (c11) Inhibits the migration and / or invasion of cancer tissues expressing CD81 protein;

[0044] (c12) acts as an inhibitor of CD81 protein function;

[0045] The amino acid sequence of polypeptide EL1 is shown in SEQ ID NO: 10.

[0046] In the above applications, the cells expressing CD81 protein can be human breast cancer cells MCF-7, human liver cancer cells Huh-7, or human glioma cells U251.

[0047] This invention also protects reagent kit A, which contains any of the polypeptides described above; the use of reagent kit A may be at least one of (c7)-(c12):

[0048] (c7) Detection of CD81 protein;

[0049] (c8) Detection of peptide EL1;

[0050] (c9) Detection of cells expressing CD81 protein;

[0051] (c10) Inhibits the migration and / or invasion of cells expressing CD81 protein;

[0052] (c11) Inhibits the migration and / or invasion of cancer tissues expressing CD81 protein;

[0053] (c12) acts as an inhibitor of CD81 protein function;

[0054] The amino acid sequence of polypeptide EL1 is shown in SEQ ID NO: 10.

[0055] In the kit A, the cells expressing CD81 protein can be human breast cancer cells MCF-7, human liver cancer cells Huh-7, or human glioma cells U251.

[0056] The present invention also protects reagent kit B, which contains any of the conjugates described above; the use of reagent kit B may be at least one of (c7)-(c12):

[0057] (c7) Detection of CD81 protein;

[0058] (c8) Detection of peptide EL1;

[0059] (c9) Detection of cells expressing CD81 protein;

[0060] (c10) Inhibits the migration and / or invasion of cells expressing CD81 protein;

[0061] (c11) Inhibits the migration and / or invasion of cancer tissues expressing CD81 protein;

[0062] (c12) acts as an inhibitor of CD81 protein function;

[0063] The amino acid sequence of polypeptide EL1 is shown in SEQ ID NO: 10.

[0064] In the kit B, the cells expressing CD81 protein can be human breast cancer cells MCF-7, human liver cancer cells Huh-7, or human glioma cells U251.

[0065] CD81 is a 25kD, four-transmembrane protein with four hydrophobic transmembrane regions and two extracellular regions. Compared to the extracellular macrocycle, the extracellular microcycle SEL (amino acids 34-63 at the N-terminus) has a more extended conformation, making it suitable for ligand docking. The extracellular microcycle of CD81 contains the hydrophilic polypeptide EL1 (amino acids 44-54 at the N-terminus), which can serve as a target for recognizing CD81.

[0066] Experiments have shown that the peptides and peptide conjugates provided by this invention can not only bind to CD81 protein on the cell surface with high selectivity and high affinity, enabling highly selective analysis and detection of CD81, but also selectively inhibit cell migration by targeting and binding to CD81 protein, thus serving as a novel functional inhibitor of human CD81 protein.

[0067] Therefore, the peptide (i.e., affinity peptide) provided by this invention exhibits high affinity and selectivity for the extracellular small loop fragment of CD81 protein, enabling it to recognize and bind to CD81 protein in both free and living cells. This allows for highly sensitive detection and in-situ imaging of CD81 protein, and further, it can be used as a selective inhibitor of the migration process of tumor cells expressing CD81 protein. Simultaneously, the peptide provided by this invention has a short amino acid sequence, facilitating large-scale production, and is non-toxic to cells. This overcomes the shortcomings of antibody and other biological agents, such as cumbersome preparation, poor stability, high cost, susceptibility to immune responses, and weak penetration. It can serve as a novel small-molecule recognition tool targeting CD81 protein. The affinity peptide provided by this invention can specifically target and recognize CD81 protein, making it a specific probe for CD81 protein analysis, detection, and functional studies, and it shows promise in the detection of malignant tumors and the development of targeted drugs. This invention has significant application value. Attached Figure Description

[0068] Figure 1 This is a schematic diagram of the chemical structure of the screening probe TPE-EL1.

[0069] Figure 2 The statistical results of the relative fluorescence intensity of 9 peptides after incubation with TPE-EL1 are presented.

[0070] Figure 3 The results show the statistical results of the relative fluorescence intensity of the peptide APQQ after incubation with CD81 protein and other peptides and proteins.

[0071] Figure 4 The results show the affinity and detection limit of the peptide APQQ for CD81 protein.

[0072] Figure 5 Immunofluorescence analysis and FITC-APQQ staining-based cell imaging analysis of CD81 expression levels in Hep3B, Huh-7, MCF-7, and U251 human hepatocellular carcinoma cells.

[0073] Figure 6 The peptide APQQ inhibits cell migration by targeting and binding to the CD81 protein. Detailed Implementation

[0074] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0076] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0077] Example 1: Design and screening of affinity peptides targeting human CD81 protein

[0078] I. Design of affinity peptides targeting human CD81 protein

[0079] Using the undecapeptide (EL1 or EL1) in the extracellular small ring of human CD81 protein as the target, based on the principle of positive-antisense peptide interaction and the degeneracy of the leucine genetic codon, a group of functionally guided candidate peptides that specifically bind to EL1 were designed and named as follows: peptide APEQ (APEQ), peptide APEK (APEK), peptide APEE (APEE), peptide APKQ (APKQ), peptide APKK (APKK), peptide APKE (APKE), peptide APQQ (APQQ), peptide APQK (APQK), and peptide APQE (APQE).

[0080] The amino acid sequences of EL1 and the above 9 candidate peptides are shown in Table 1; where each letter represents an amino acid as follows: A for alanine, D for aspartic acid, P for proline, Q for glutamine, Y for tyrosine, S for serine, E for glutamic acid, L for leucine, G for glycine, I for isoleucine, R for arginine, V for valine, and K for lysine.

[0081] Table 1

[0082]

[0083]

[0084] II. Screening of Affinity Peptides

[0085] Aggregation-induced emission fluorophores (AIEs) are increasingly used in intermolecular interaction studies due to their fluorescence activation in aggregated states or when intramolecular rotation is restricted. TPE is a typical AIE; when TPE is coupled to a polypeptide, the presence of the hydrophilic polypeptide significantly weakens its fluorescence in physiological solutions.

[0086] When a peptide interacts with other molecules, the TPE exhibits fluorescence activation, indicating the strength of the interaction in the system. Based on this principle, the fluorescence intensity of the TPE-EL1 probe can be used to examine the strength of the specific interaction between nine candidate peptides and EL1, thereby obtaining the preferred peptide with the strongest interaction with EL1.

[0087] 1. Preparation of fluorescent probes for peptide screening

[0088] A peptide screening probe (TPE-EL1) was synthesized on Wang resin starting from the C-terminus using the FMOC solid-phase peptide synthesis method. Specifically, the amino acids of peptide EL1 were coupled one by one to the solid-phase resin. A glycine residue was attached to the N-terminus of EL1 as a spacer arm. Finally, the N-terminal amino group of the peptide was coupled to a fluorescently open TPE molecule with a carboxyl group. The peptide chain was then cleaved from the resin using a lysis buffer (composed of 95% (v / v) trifluoroacetic acid, 2.5% (v / v) triisopropylsilane, and 2.5% (v / v) water), simultaneously removing the side-chain protecting groups.

[0089] A schematic diagram of the chemical structure of TPE-EL1 is shown below. Figure 1 As shown.

[0090] 2. Screening of affinity peptides

[0091] To investigate the strength of the interaction between TPE-EL1 and candidate peptides (APEQ, APEK, APEE, APKQ, APKK, APKE, APQQ, APQK, or APQE), the following experiments were conducted:

[0092] (1) Using dimethyl sulfoxide (DMSO) as a solvent, TPE-EL1 stock solution and candidate peptide stock solution were prepared.

[0093] (2) Mix 10 mmol / L phosphate buffer (pH 7.4) containing 0.05% Tween-20, TPE-EL1 stock solution and candidate peptide stock solution to obtain mixed system 1; in mixed system 1, the concentration of TPE-EL1 is 10 μmol / L, the concentration of candidate peptide is 20 μmol / L and the content of DMSO is 0.3%.

[0094] (3) Take the mixed system 1 obtained in step (2) and incubate it in the dark for 1 hour.

[0095] (4) After completing step (3), use a microplate reader (USA) to quickly scan the solution, with an excitation wavelength of 320 nm and record the fluorescence intensity data at an emission wavelength of 457 nm, which is the fluorescence intensity of the candidate peptide.

[0096] (5) Following steps (2)-(4) above, replace mixing system 1 with mixing system 2, keeping all other steps unchanged, to obtain the fluorescence intensity of the blank control. Mixing system 2 consists of 10 mmol / L phosphate buffer (pH 7.4) containing 0.05% Tween-20 and TPE-EL1 stock solution; in mixing system 2, the concentration of TPE-EL1 is 10 μmol / L, and the content of DMSO is 0.3%.

[0097] (6) Calculate the relative fluorescence intensity of the candidate peptide. Relative fluorescence intensity of the candidate peptide = (Fluorescence intensity of the candidate peptide - Fluorescence intensity of the blank control) / Fluorescence intensity of the blank control

[0098] The results are as follows Figure 2 As shown in the figure. The results indicated that the nine candidate peptides exhibited different binding abilities to EL1. In phosphate buffer at pH 7.4 and 10 mmol / L, peptide APQQ showed the strongest targeting binding ability to EL1, with a 2.98-fold increase in fluorescence intensity at 457 nm. Using peptide APQQ as the preferred peptide, its specific recognition performance for CD81 protein was further investigated.

[0099] Example 2: Specific recognition and high-sensitivity detection of CD81 protein using the peptide APQQ

[0100] Using the screened peptide APQQ as the recognition molecule, a TPE molecule was modified at its N-terminus to investigate the specific recognition of the peptide APQQ with the CD81 protein. When the peptide APQQ interacts with the CD81 protein, the TPE fluorescence is activated, thus indicating the strength of the interaction.

[0101] 1. Preparation of TPE-labeled polypeptide fluorescent probe TPE-APQQ

[0102] The peptide was synthesized directly on Wang resin starting from the C-terminus using the FMOC solid-phase peptide synthesis method, and Fmoc-Lys(Dde)-OH was coupled to the N-terminus, retaining the N-terminal FMOC protecting group. Dde deprotection solution (composed of imidazole, hydroxylamine hydrochloride, 5 mL N-methylpyrrolidone and 1 mL dichloromethane; the amount of imidazole in the Dde deprotection solution was 1.35 mmol and the amount of hydroxylamine hydrochloride was 1.80 mmol) was added, and the peptide was removed at room temperature for 3 h. Weigh a certain amount of the above resin, dissolve 3 times the amount of TPE-COOH, 3 times the amount of HATU and 3 times the amount of HOBt in a DMF solution containing 0.4 mol / L N-methylmorpholine, couple at room temperature in the dark for 2.5 h, wash with DMF, add a DMF solution containing 20% ​​piperidine to remove the N-terminal FMOC protecting group, and then use a lysis buffer (composed of 95% (v / v) trifluoroacetic acid, 2.5% (v / v) triisopropylsilane and 2.5% (v / v) water) to cleave the TPE-modified peptide chain from the resin, while removing all side chain protecting groups to obtain the cleavage product.

[0103] The pyrolysis products were purified by HPLC. The HPLC purification parameters were as follows: the column was a Diamonsil C18(2) 250×4.6 mm; mobile phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. Elution was performed by increasing the percentage of mobile phase B from 47% to 67% within 25 min. The corresponding target fraction was collected, lyophilized, and TPE-APQQ with high purity and correct structure was obtained.

[0104] 2. Recognition selectivity of peptide APQQ with CD81 protein

[0105] (1) Mix 10 mmol / L phosphate buffer (pH 7.4) containing 0.05% Tween-20, TPE-APQQ and the analyte to obtain mixture system A; in mixture system A, the concentration of TPE-APQQ is 1 μmol / L and the concentration of the analyte is 2 μmol / L.

[0106] The substances to be tested are CD81 protein, transferrin, human serum albumin (HSA), cytochrome C, trypsin, horseradish peroxidase (HRP), lysozyme, oxytocin, C-type atrial natriuretic peptide (CNP), vasopressin, Substance P, or L-glutathione (L-GSH).

[0107] (2) Take the mixed system obtained in step (1) and incubate it in the dark for 1 hour.

[0108] (3) Use a fluorescence spectrometer (Hitachi F-4600, Japan) to perform fluorescence scanning on the solution after completing step (2). The excitation wavelength is 320 nm, and the fluorescence intensity data of the emission wavelength is 457 nm, which is the fluorescence intensity of the substance to be tested.

[0109] (4) Following steps (1)-(3) above, replace mixture system A with mixture system B, keeping all other steps unchanged, to obtain the fluorescence intensity of the blank control. Mixture system B consists of 10 mmol / L phosphate buffer (pH 7.4) containing 0.05% Tween-20 and TPE-APQQ; the concentration of TPE-APQQ in mixture system B is 1 μmol / L.

[0110] (5) Calculate the relative fluorescence intensity of the analyte. The relative fluorescence intensity of the analyte = (fluorescence intensity of the analyte - fluorescence intensity of the blank control) / fluorescence intensity of the blank control.

[0111] The results are as follows Figure 3 As shown in the figure. The results indicate that, except for CD81 protein, other peptides or proteins do not specifically bind to APQQ, and their fluorescence signals are significantly weaker than those of CD81 protein, indicating that the binding of peptide APQQ to CD81 protein is highly selective.

[0112] 3. Affinity and detection limit of peptide APQQ to CD81 protein

[0113] (1) Mix 10 mmol / L phosphate buffer (pH 7.4) containing 0.05% Tween-20, TPE-APQQ and CD81 protein to obtain mixture A; in mixture A, the concentration of TPE-APQQ is 1 μmol / L, and the concentration of CD81 protein is 0.2 μmol / L, 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L, 1 μmol / L, 1.5 μmol / L or 2.0 μmol / L.

[0114] (2) Take the mixed system obtained in step (1) and incubate it in the dark for 1 hour.

[0115] (3) Use a fluorescence analyzer to perform fluorescence scanning on the solution after completing step (2), with an excitation wavelength of 320 nm and record the fluorescence intensity data at an emission wavelength of 457 nm, which is the fluorescence intensity of the experimental group.

[0116] (4) Following steps (1)-(3) above, replace mixture A with mixture B, keeping all other steps unchanged, to obtain the fluorescence intensity of the blank control. Mixture B consists of 10 mmol / L phosphate buffer (pH 7.4) containing 0.05% Tween-20 and TPE-APQQ; the concentration of TPE-APQQ in mixture B is 1 μmol / L.

[0117] (5) Calculate the relative fluorescence intensity of the experimental group (relative fluorescence intensity of the experimental group = (fluorescence intensity of the experimental group - fluorescence intensity of the blank control) / fluorescence intensity of the blank control), and use SigmaPlot software to calculate the equilibrium dissociation constant K. D The CD81 protein concentration was linearly fitted using relative fluorescence intensity, and the detection limit was calculated according to the formula LOD = 3N / S (where N is the blank standard deviation and S is the slope of the standard curve).

[0118] The results are as follows Figure 4 As shown. The results indicate that the peptide APQQ interacts with the K-cell receptor of the CD81 protein. D The limit of detection (LOD) was 514.1 ± 81.0 nM, indicating that APQQ has a high affinity for CD81 protein. The limit of detection was 61.1 nM, suggesting that the peptide APQQ can be used as a recognition probe for detecting CD81 protein.

[0119] Example 3: Application of the peptide APQQ in CD81 protein imaging analysis in living cells

[0120] 1. Preparation of FTIC-APQQ conjugates

[0121] The peptide was synthesized directly on Wang resin starting from the C-terminus using the FMOC solid-phase peptide synthesis method, and Fmoc-Lys(Dde)-OH was coupled to the N-terminus, retaining the N-terminal FMOC protecting group. Dde deprotection solution (composed of imidazole, hydroxylamine hydrochloride, 5 mL N-methylpyrrolidone and 1 mL dichloromethane; the amount of imidazole in the Dde deprotection solution was 1.35 mmol and the amount of hydroxylamine hydrochloride was 1.80 mmol) was added, and the peptide was removed at room temperature for 3 h. Weigh a certain amount of the above resin, dissolve 2 times the amount of FITC and 4 times the amount of N,N-diisopropylethylamine in DMF solution, add the solution to the swollen resin, and couple at room temperature in the dark for 3 hours. After washing with DMF, add DMF solution containing 20% ​​piperidine to remove the N-terminal FMOC protecting group. Then use lysis buffer (composed of 95% (v / v) trifluoroacetic acid, 2.5% (v / v) triisopropylsilane and 2.5% (v / v) water) to cleave the FITC-modified peptide chain from the resin, while removing all side chain protecting groups to obtain the cleavage product.

[0122] The pyrolysis products were purified by HPLC. The HPLC purification parameters were as follows: the column was a Diamonsil C18(2) 250×4.6 mm; mobile phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. Elution was performed by increasing the percentage of mobile phase B from 28% to 35% within 25 min. The corresponding target fraction was collected, lyophilized, and then FITC-APQQ with high purity and correct structure was obtained.

[0123] 2. Immunofluorescence staining combined with flow cytometry was used to analyze the expression level of CD81 protein in cells.

[0124] (1) Human breast cancer cells MCF-7, human liver cancer cells Huh-7, and human glioma cells U251 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin (composed of 10,000 Units / mL penicillin and 10,000 μg / mL streptomycin), respectively. After reaching the desired density, they were digested with 0.25% trypsin solution to obtain MCF-7 suspension cells, Huh-7 suspension cells, and U251 suspension cells, respectively. Human liver cancer cells Hep3B were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin (composed of 10,000 Units / mL penicillin and 10,000 μg / mL streptomycin), respectively. After reaching the desired density, they were digested with 0.25% trypsin solution to obtain Hep3B suspension cells.

[0125] (2) 1×10 6 One suspension of cells (MCF-7 suspension cells, Huh-7 suspension cells, U251 suspension cells, or Hep3B suspension cells) was placed in a centrifuge tube, and blocking solution (composed of bovine serum albumin and PBS, with a bovine serum albumin concentration of 5%) was added. The cells were then blocked at room temperature for 1 hour.

[0126] (3) After completing step (2), discard the solution, wash once with PBS, add CD81 antibody solution, and incubate at room temperature for 1 hour; the CD81 antibody solution consists of CD81 antibody (Abcam, catalog number ab219209, UK), bovine serum albumin and PBS; the CD81 antibody dilution ratio is 1:500, and the mass concentration of bovine serum albumin is 0.5%;

[0127] (4) After completing step (3), discard the solution, wash once with PBS, add secondary antibody solution, and incubate at room temperature for 1 hour; the secondary antibody solution consists of Alexa Fluor 488 labeled goat anti-rabbit IgG antibody (Abcam, catalog number ab150077, UK), bovine serum albumin and PBS; the dilution ratio of the secondary antibody is 1:2000, and the mass concentration of bovine serum albumin is 0.5%.

[0128] (5) After completing step (4), discard the solution, add PBS to wash, redisperse the cells in PBS, analyze using a flow cytometer (BD, USA), and process the data using FlowJo software.

[0129] The results are as follows Figure 5 As shown in a) of the diagram. The results showed that CD81 protein was expressed in human breast cancer cells MCF-7, human liver cancer cells Huh-7, and human glioma cells U251, making them CD81-positive cells; however, CD81 protein was not expressed in human liver cancer cells Hep3B, making them CD81-negative cells.

[0130] 3. In situ imaging analysis of CD81 protein in live cells

[0131] (1) with approximately 1×10 6 Cancer cells (human breast cancer cells MCF-7, human liver cancer cells Huh-7, human glioma cells U251, or human liver cancer cells Hep3B) were seeded at a density of cells / dish in confocal dishes (Φ=15mm) and cultured overnight at 37℃ and 5% CO2 to allow them to adhere.

[0132] (2) After completing step (1), take the confocal dish containing cancer cells and divide it into group I (FITC-APQQ group) and group II (EL1+FITC-APQQ group), and perform the following operations:

[0133] Group I: Discard the original culture medium in the confocal dish, then add FITC-APQQ solution (diluted with DMEM medium) to make the concentration of FITC-APQQ in the system 25 μmol / L, then incubate at 37℃ and 5% CO2 in the dark for 1 h, discard the solution, and wash once with PBS.

[0134] Group II: Discard the original culture medium in the confocal dish, then add the FITC-APQQ+EL1 mixture (made by mixing FITC-APQQ solution (diluted with DMEM medium) and EL1 solution (diluted with DMEM medium)) and make the concentration of FITC-APQQ and EL1 in the system 25 μmol / L. Then incubate at 37℃ and 5% CO2 in the dark for 1 h, discard the solution, and wash once with PBS.

[0135] (3) The fluorescence distribution in the cells was detected using a laser scanning confocal microscope (Olympus FV1000-IX81, Japan).

[0136] The results are as follows Figure 5 As shown in b). For CD81-positive human breast cancer cells MCF-7, human liver cancer cells Huh-7, and human glioma cells U251, the addition of FITC-APQQ resulted in significant green fluorescence on the cell membrane, consistent with the membrane localization distribution of CD81 protein. For CD81-negative human liver cancer cells Hep3B, no significant green fluorescence was observed in the field of view. This indicates that FITC-APQQ specifically recognizes CD81 protein, and the signal response intensity is positively correlated with the expression level of CD81 protein. However, when FITC-APQQ was premixed with EL1 and incubated in each group of cells for 1 hour, almost no green fluorescence was observed in the cells. This is because FITC-APQQ preferentially binds to free EL1 fragments in solution, blocking binding to the EL1 site on the CD81 protein in the cells. This competitive relationship between FITC-APQQ and EL1 also verifies the targeted recognition of the EL1 site by APQQ in cells.

[0137] Example 4: The peptide APQQ inhibits cell migration by targeting and binding to CD81 protein.

[0138] 1. Wound healing experiment

[0139] (1) In a six-hole plate, use 5×10 4 Cancer cells (human breast cancer cells MCF-7, human liver cancer cells Huh-7, human glioma cells U251, or human liver cancer cells Hep3B) were implanted into wound healing inserts (Ibidi, Germany) at a density of cells / well and cultured at 37°C and 5% CO2 until 100% confluence.

[0140] (2) After completing step (1), carefully remove the plug-in with tweezers, leaving a scratch area with a width of 500μm.

[0141] (3) After completing step (2), discard the culture medium, carefully wash the detached cells with PBS buffer, and then add the test solution to a total volume of 1 mL.

[0142] The test solutions were 10 μmol / L APQQ solution (diluted with DMEM medium) (as the experimental group), 10 μmol / L Apamin solution (diluted with DMEM medium) (as the positive group), or DMEM medium (as the blank group).

[0143] (4) After completing step (3), continue incubation at 37℃ and 5% CO2 for 24 hours. Take pictures of the scratched area at 0h and 24h, respectively, and use ImageJ software to calculate the healing area of ​​the scratched area. The cell migration ability is measured by the ratio of the healing area of ​​the experimental group to the healing area of ​​the control group.

[0144] The experimental results are shown in Figure 6 (a) and b) (Control group: blank). The results showed that APQQ, by targeting and binding to the CD81 protein, selectively inhibited the migration of human breast cancer cells MCF-7, human liver cancer cells Huh-7, and human glioma cells U251, reducing the migration rates of the three cell types to 47.4%, 31.1%, and 37.1%, respectively, comparable to the inhibitory level of the cell migration inhibitor melittin. However, APQQ was ineffective in inhibiting the migration of CD81-negative human liver cancer cells Hep3B.

[0145] 2. Competitive binding experiment

[0146] (1) In a six-hole plate, use 5×10 4Cancer cells (human breast cancer cells MCF-7, human liver cancer cells Huh-7, or human glioma cells U251) were implanted into wound healing inserts (Ibidi, Germany) at a density of cells / well and cultured at 37°C and 5% CO2 until 100% confluence.

[0147] (2) After completing step (1), carefully remove the plug-in with tweezers, leaving a scratch area with a width of 500μm.

[0148] (3) After completing step (2), discard the culture medium, carefully wash the detached cells with PBS buffer, and then add the test solution to a total volume of 1 mL.

[0149] The test solutions were 10 μmol / L APQQ solution (diluted with DMEM medium) (as the APQQ experimental group), mixed solutions (as the APQQ+EL1 experimental group), or DMEM medium (as the blank group). The mixed solutions consisted of APQQ, EL1, and DMEM medium; the concentration of APQQ in the mixed solutions was 10 μmol / L, and the concentration of EL1 was 20 μmol / L.

[0150] (4) After completing step (3), continue incubation at 37℃ and 5% CO2 for 24 hours. Take pictures of the scratched area at 0h and 24h, respectively, and use ImageJ software to calculate the healing area of ​​the scratched area. The cell migration ability is measured by the ratio of the healing area of ​​the experimental group to the healing area of ​​the control group.

[0151] The experimental results are shown in Figure 6 (c) and d) (Control group was the blank group). The results showed that when the target EL1 was added, APQQ preferentially bound to EL1 in the solution, blocking the binding to the EL1 site on the CD81 protein in the cell, and the cell migration ability was restored.

[0152] Therefore, APQQ can be used as an inhibitor of CD81 protein by selectively inhibiting its role in cell migration by targeting and binding to the extracellular small loop fragment EL1 of CD81 protein.

[0153] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A polypeptide having the amino acid sequence shown in SEQ ID NO:

7.

2. The application of the polypeptide of claim 1 is at least one of the following (c1)-(c4): (c1) Prepare a kit for detecting CD81 protein; (c2) Prepare a kit for detecting peptide EL1; the amino acid sequence of peptide EL1 is shown in SEQ ID NO: 10; (c3) Prepare a kit for detecting cells expressing CD81 protein; (c4) Prepare a kit for inhibiting cell migration; said cells are human breast cancer cells MCF-7, human liver cancer cells Huh-7, or human glioma cells U251.

3. A conjugate obtained by conjugating the polypeptide of claim 1 with a substance, wherein the substance is FITC or TPE; The structural formula of the FITC is shown in formula (Ⅰ); ； Equation (I); The structural formula of the TPE is shown in formula (Ⅱ); ； Formula (II).

4. The application of the coupling compound according to claim 3 is at least one of the following (c1)-(c4): (c1) Prepare a kit for detecting CD81 protein; (c2) Prepare a kit for detecting peptide EL1; the amino acid sequence of peptide EL1 is shown in SEQ ID NO: 10; (c3) Prepare a kit for detecting cells expressing CD81 protein; (c4) Prepare a kit for inhibiting cell migration; said cells are human breast cancer cells MCF-7, human liver cancer cells Huh-7, or human glioma cells U251.

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