Polypeptides specifically targeting epha8 protein and uses thereof

Abstract

The present application relates to a kind of polypeptide of specifically targeting EphA8 protein, its amino acid sequence is as shown in SEQ ID NO:1.The present application also provides the use of the polypeptide and the kit containing the polypeptide.The present application provides a kind of new EphA8 targeting polypeptide, is obtained by four rounds of strict phage display subtraction screening, has clear sequence information;The polypeptide has high specific targeting ability, and is used for the cell localization of EphA8 protein after being labeled with fluorescence, expression level detection, and also can be used as targeted head group for the targeted delivery of drug or nano carrier.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a polypeptide that specifically targets the EphA8 protein and its applications. Background Technology

[0002] EphA8 is a member of the Eph family of receptor tyrosine kinases and is abnormally highly expressed in various tumor tissues, including breast cancer. Its expression level is closely related to lymph node metastasis and poor prognosis, making it a potential tumor marker and drug target. Currently, there is a severe lack of molecules specifically binding to EphA8. Existing anti-EphA8 monoclonal antibodies are mainly used for basic research, and there are no commercially available or clinically applicable high-affinity, high-specificity targeting peptides. Peptide drugs / reagents have advantages such as low immunogenicity, good tissue penetration, and ease of chemical synthesis and labeling. Therefore, developing a peptide that can specifically bind to the EphA8 protein is of great value for EphA8-related detection, diagnosis, and targeted delivery. Summary of the Invention

[0003] The first objective of this invention is to provide a polypeptide that specifically targets the EphA8 protein, capable of specifically binding to the EphA8 protein, thus providing a tool molecule for the detection, cell labeling, and targeted delivery of the EphA8 protein.

[0004] A second object of the present invention is to provide the use of the above-mentioned polypeptide.

[0005] A third objective of this invention is to provide a kit containing the aforementioned polypeptide.

[0006] This invention is achieved through the following technical solution: I. A polypeptide that specifically targets the EphA8 protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Furthermore, the N-terminus or C-terminus of the polypeptide is attached with a marker or functional molecule.

[0008] Furthermore, the marker is selected from fluorescent markers, biotin tags, enzyme markers, or radioisotope markers.

[0009] Furthermore, the fluorescent label is Rhodamine B, FITC, or Cy5.

[0010] Furthermore, the functional molecules are selected from cell-penetrating peptides, polyethylene glycol, or nanocarriers.

[0011] II. Application of the above-mentioned peptides in the preparation of EphA8 protein-targeted diagnostic reagents or imaging agents.

[0012] III. An EphA8 protein detection kit, wherein the kit comprises the polypeptide described in any one of claims 1-5.

[0013] The positive effects of adopting the above technical solution: 1. This invention provides a novel EphA8-targeting polypeptide, obtained through four rounds of rigorous phage display attenuation screening, and possesses clearly defined sequence information; 2. High specificity targeting ability: Immunofluorescence co-localization assays confirmed that the fluorescently labeled EphA8-T1 peptide could co-localize with eukaryotically expressed EGFP-EphA8 protein on HEK 293T cells, showing yellow fluorescence in the merge plot, while the control peptide showed no binding signal; flow cytometry quantitative analysis showed that the binding rate of EphA8-T1 peptide to EphA8 protein was approximately 80%, while the binding rate of the control peptide was almost zero, indicating that the peptide has excellent targeting specificity. 3. Excellent tool properties: This peptide can be used for cell localization and expression level detection of EphA8 protein after fluorescent labeling, and can also be used as a targeting head for targeted delivery of drugs or nanocarriers. Attached Figure Description

[0014] Figure 1 Figure A shows the results of EphA8 protein purification and Western blot validation. A: Coomassie Brilliant Blue staining results for EphA8 protein purification: M: protein marker, 1: supernatant before induction with pET-28a(+) / EphA8 bacterial culture, 2: precipitate before induction with pET-28a(+) / EphA8 bacterial culture, 3: supernatant after induction with pET-28a(+) / EphA8 bacterial culture, 4: precipitate after induction with pET-28a(+) / EphA8 bacterial culture, 5: whole protein after purification with pET-28a(+) / EphA8 bacterial culture; B: Western blot results for EphA8 protein purification: M: protein marker, 1: whole protein before induction with pET-28a(+) / EphA8 bacterial culture, 2: whole protein after purification with pET-28a(+) / EphA8 bacterial culture. Figure 2 The image shows the enrichment results of four rounds of phage screening. In the image, there are plates for measuring the titer of phage eluted after the first round of screening, plates for measuring the titer of phage eluted after the second round of screening, plates for measuring the titer of phage eluted after the third round of screening, and plates for measuring the titer of phage eluted after the fourth round of screening. The titer measurements were all performed with serial dilutions, and each dilution was repeated three times. Figure 3 The figure shows the ELISA results of positive phage clones. In the figure, the phage binding to the target protein was detected by ELISA. The blank group is the well that is not coated with any protein. "*" indicates that there is a significant difference between the target protein EphA8 group and the BSA protein control group, P<0.05. Figure 4 This is a diagram of the phage DNA sequencing results.

[0015] Figure 5 The figures show the cell viability of MDA-MB-231 cells after treatment with different concentrations of EphA8-T1 using the CCK-8 assay. In the figures, Figure A shows the cell viability after 12 h of EphA8-T1 peptide solution, Figure B shows the cell viability after 24 h of EphA8-T1 peptide solution, and Figure C shows the cell viability after 36 h of EphA8-T1 peptide solution. Figure 6 The image shows the immunofluorescence results of EphA8-T1 peptide binding to EphA8 protein. In the image, HEK293T(NP) represents the immunofluorescence results of HEK293T cells transfected with the expression vector plasmid and Rhodamine B-labeled meaningless 12-peptide NP, while HEK293T(EphA8-T1) represents the immunofluorescence results of HEK293T cells transfected with the expression vector plasmid and Rhodamine B-labeled targeted EphA8 protein. Figure 7 Figures show the results of flow cytometry detection of EphA8-T1 peptide targeting. Figure A shows the blank control group without plasmid transfection; the FITC fluorescence intensity is almost zero in the FITC channel due to the lack of plasmid transfection. Figure B shows the negative control group without peptide after plasmid transfection; the fluorescence intensity in the FITC channel is 90%, indicating successful EphA8 protein expression. Figure C shows the group transfected with EphA8-T1 peptide; the fluorescence intensity is 90% in the FITC channel and 80% in the PC channel, indicating successful EphA8 protein expression and binding to the EphA8-T1 peptide. Figure D shows the group transfected with NP peptide; the fluorescence intensity is 89% in the FITC channel and almost zero in the PC channel, indicating that the NP peptide failed to bind to the EphA8 protein. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to specific embodiments, but it should not be construed as a limitation of the present invention. Example

[0017] This example illustrates the prokaryotic expression and purification of the EphA8 protein.

[0018] 1. Construction of expression carrier The gene encoding the extracellular domain of the human EphA8 receptor protein (82-1620 bp) was extracted, codon-optimized, fused with an HIS tag, and cloned into the pET-28a(+) vector to construct the pET-28a(+) / EphA8 recombinant plasmid. Double enzyme digestion was used for identification. Hin dⅢ and EcoThe R I results showed the target band at 1539 bp, proving that the recombinant plasmid was successfully constructed.

[0019] 2. Protein induction expression and purification The recombinant plasmid was transformed into E. coli BL21(DE3), and after induction with 0.1 mM / L IPTG for 4 hours, the bacterial cells were collected and sonicated. The target protein (approximately 58 kDa) existed in the form of inclusion bodies. The inclusion bodies were dissolved in 6 mol / L urea, dialyzed, and refolded to obtain purified protein. Western blot validation showed that the purified product was an EphA8 protein fused with the HIS tag. Figure 1 ). Example

[0020] This example illustrates the use of phage display technology to screen for EphA8-targeting peptides.

[0021] 1. Four rounds of subtractive screening Using the purified EphA8 protein from Example 1 as the target, four rounds of attenuated screening were performed using a phage display library of random 12 peptides. The screening conditions were progressively tightened: the target protein concentration was decreased from 100 μg / mL to 25 μg / mL, the binding time was decreased from 2 hours to 1 hour, and the Tween-20 concentration in TBST was increased from 0.1% to 0.25%. Phage titers were measured after each round of screening.

[0022] The results are shown in Table 1. With the increase of screening rounds, the phage recovery rate increased from 0.007% to 0.428%, and the enrichment increased to 611.43 times. Figure 2 This indicates that phages that specifically bind to EphA8 are efficiently enriched.

[0023]

[0024] 2. ELISA identification of positive clones After the final round of screening, 12 monoclonal phages were selected for ELISA testing. The results are as follows: Figure 3 As shown, five clones showed significantly higher binding affinity to the EphA8 protein than the BSA control group (P<0.05).

[0025] 3. DNA sequencing and peptide sequence determination DNA from positive bacteriophages was extracted and sequenced, and the results were as follows: Figure 4 As shown in the figure. Sequencing results showed that most positive clones carried the same random 12-peptide coding sequence, and the amino acid sequence obtained after translation was: SQHQNTRRTRKT (SEQ ID NO:1). This polypeptide was named EphA8-T1.

[0026] This example illustrates the targeting verification of the EphA8-T1 peptide.

[0027] 1. Construction of eukaryotic expression vector and expression of EphA8 protein Construct a pcDNA-EGFP / EphA8 eukaryotic expression vector (EphA8 and EGFP fusion expression), and then... Hin dⅢ and Bam After successful identification via HⅠ double digestion, HEK 293T cells were transfected. After 48 hours of culture, green fluorescence was observed under a fluorescence microscope, confirming successful expression of the EphA8 protein.

[0028] 2. CCK-8 assay for the cytotoxicity of EphA8-T1 peptide To assess the biosafety of the EphA8-T1 peptide, its effect on the survival rate of triple-negative breast cancer MDA-MB-231 cells was detected using the CCK-8 assay.

[0029] MDA-MB-231 cells were cultured at 5.0 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured until approximately 70% confluence. EphA8-T1 peptide solution was then added at final concentrations of 0 (PBS control group), 5, 25, 50, and 100 μM, with three replicates for each concentration. After 12, 24, and 36 hours of treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The results showed no significant change in cell viability, indicating that the peptide had no significant cytotoxicity.

[0030] 3. Immunofluorescence detection Rhodamine B-labeled EphA8-T1 peptide (final concentration 100 mg / mL) was incubated with transfected HEK 293T cells for 2 hours. Results are as follows: Figure 5 As shown: EphA8-T1 peptide (red fluorescence) and EGFP-EphA8 (green fluorescence) co-localize on cells, and the merge image shows yellow fluorescence; while the control group of insignificant peptides (NP group) does not show binding signals.

[0031] 4. Flow cytometry detection Flow cytometry results as follows Figure 6 As shown, in HEK 293T cells expressing EGFP-EphA8, approximately 80% simultaneously detected red fluorescence signals of EphA8-T1; while the red fluorescence signal of the control peptide NP group was almost zero. These results demonstrate that the EphA8-T1 peptide possesses good targeting specificity.

[0032] 5. Immunofluorescence detection The N-terminus of the EphA8-T1 peptide is linked to rhodamine B fluorescent dye. After fixing and permeabilizing the test cells (MDA-MB-231 cells), they are incubated with 1-100 mg / mL of the fluorescently labeled EphA8-T1 peptide for 1-2 hours. After washing, the cells are observed under a fluorescence microscope, clearly showing the cellular localization and expression level of the EphA8 protein. This method can replace or supplement traditional antibody immunofluorescence staining.

[0033] This invention provides a novel EphA8-targeting peptide, obtained through four rounds of rigorous phage display attenuation screening, possessing clear sequence information; it exhibits high specificity targeting capability: immunofluorescence co-localization experiments confirmed that the fluorescently labeled EphA8-T1 peptide can co-localize with eukaryotically expressed EGFP-EphA8 protein on HEK 293T cells, showing yellow fluorescence in the merge plot, while the control peptide showed no binding signal; flow cytometry quantitative detection showed that the binding rate of the EphA8-T1 peptide to EphA8 protein was approximately 80%, while the binding rate of the control peptide was almost zero, indicating that this peptide has excellent targeting specificity; it also possesses good tool properties: this peptide can be used for cellular localization and expression level detection of EphA8 protein after fluorescent labeling, and can also be used as a targeting head for targeted delivery of drugs or nanocarriers.

Claims

1. A polypeptide that specifically targets the EphA8 protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that: The peptide has a marker or functional molecule attached to its N-terminus or C-terminus.

3. The polypeptide according to claim 2, characterized in that: The marker is selected from fluorescent markers, biotin tags, enzyme markers, or radioisotope markers.

4. The polypeptide according to claim 3, characterized in that: The fluorescent label is Rhodamine B, FITC, or Cy5.

5. The polypeptide according to claim 2, characterized in that: The functional molecules are selected from cell-penetrating peptides, polyethylene glycol, or nanocarriers.

6. The use of the polypeptide according to any one of claims 1-5 in the preparation of EphA8 protein-targeting diagnostic reagents or imaging agents.

7. An EphA8 protein detection kit, characterized in that: The kit contains the polypeptide according to any one of claims 1-5.

Images