TRIM24-specific binding peptide

Abstract

The present invention discloses a TRIM24 binding peptide and a drug containing the same. The sequence of the TRIM24 binding peptide is: VLFX 1 X 2 YYTX 3 X 4 TM. The present invention screened 4 binding peptides that specifically bind to TRIM24 protein through D12 phage library, laying a foundation for treating glioma diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a TRIM24 specific binding peptide and a drug containing the same. Background Art

[0002] Gliomas are common malignant tumors of the central nervous system, among which glioblastoma multiforme (GBM) is the most malignant primary brain tumor. There are some cells in malignant gliomas that have self-renewal ability, multidirectional differentiation ability, and extremely strong tumorigenic ability, namely glioma stem cells (GSCs). GSCs are considered to be the main reason for the recurrence and poor prognosis of malignant gliomas. GSCs have been shown to have self-renewal ability in vitro, can differentiate into multiple cell lineages, form neurospheres, and express specific neural stem cell markers. For patients diagnosed with GBM, the current conventional treatment is surgical resection of the tumor, postoperative radiotherapy and adjuvant temozolomide. Surgery can relieve symptoms, reduce the burden of tumors, and provide sufficient tissue support for diagnosis and molecular level analysis.

[0003] TRIM24 protein has an amino-terminal RBCC domain (Ring, B-Box and Coiled-Coil) characteristic of the TRIM family, and a PHD–bromo domain that defines the TIF1 subfamily. TRIM24 has been shown to act as an oncogene or tumor suppressor. Although genetic deletion of TRIM24 in mice promotes hepatocellular carcinoma, abnormal overexpression of TRIM24 in humans is positively correlated with multiple cancers, cancer progression in patients, and patient survival. TRIM24 is highly expressed in gliomas, and expression increases significantly with increasing grade.

[0004] EGFR is a receptor that recognizes epidermal growth factor, which regulates cell proliferation and growth, and conducts signal transduction. It plays an important role in cell functions such as cell proliferation, differentiation, and migration. EGFR signal transduction promotes cell proliferation, inhibits cell apoptosis, and increases tumor cell motility and angiogenesis. The expression and strong expression of EGFR have been reported in various tumor types. It is known that EGFR expression levels are high in one-third of epithelial cancers, and strong expression of EGFR is often associated with poor prognosis. In addition, since EGFR is known to be related to the occurrence and development of cancer, the development of chemotherapeutic drugs targeting EGFR has been carried out. EGFR is also highly expressed in glioblastoma multiforme.

[0005] Inhibiting the expression levels of TRIM24 protein and EGFR protein in vivo can alleviate glioma or treat glioma disease. Summary of the invention

[0006] The object of the present invention is to provide a TRIM24-binding peptide and a drug containing the same.

[0007] A TRIM24 binding peptide, which is a short peptide having the following amino acid sequence: VLFX 1 X 2 YYTX 3 X 4 TM;

[0008] Among them, X 1 is Y, W or C; X 2 L, Y or T; X 3 is G, P, A or W; X 4 It is S or T.

[0009] A label for the binding peptide.

[0010] The label is a radioactive isotope, biotin, horseradish peroxidase, alkaline phosphatase, FITC, rhodamine or positron nuclide.

[0011] The binding peptide or the labeled body of the binding peptide is used for treating glioma.

[0012] The binding peptide or the binding peptide marker is used for the diagnosis of glioma.

[0013] The application of the binding peptide or the labeled body of the binding peptide in preparing medicine for treating glioma.

[0014] The application of the binding peptide or the binding peptide marker in the preparation of reagents for diagnosing glioma.

[0015] A medicine for treating glioma, comprising the binding peptide and a pharmaceutically acceptable carrier.

[0016] Beneficial effects of the present invention: The present invention screened 4 binding peptides that specifically bind to TRIM24 protein through the D12 phage library, laying a foundation for treating cellular glioma diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the ratio of output titer to input titer for experiments using phage libraries.

[0018] Figure 2 These are the results of the binding test of M13KE, 1-101, and 1-102 phages to the immobilized proteins.

[0019] Figure 3 These are the results of the binding test of 1-105 and 1-106 phages to the immobilized protein.

[0020] Figure 4The results of the competition experiment between 1-101 phage and 1-101 phage displayed peptide or unrelated peptide. DETAILED DESCRIPTION

[0021] In order to facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0022] Example 1

[0023] Phage display technology was used to screen short peptides that specifically bind to TRIM24. The steps are as follows:

[0024] (1) Add 200uL of recombinant human IgG1Fc (5ug / mL) and recombinant human TRIM24 / Fc chimera (1.5ug / well) to a 96-well plate and incubate at 4°C for 24h.

[0025] (2) Warm to room temperature, add 300uL blocking buffer (5mg / mL BSA, 50mM Tris-HCl, 150mM NaCl), incubate for 1h, and then wash three times with 0.1% TBST (200uL / well) solution.

[0026] (3) Add 100uL of 0.1% TBST to the wells with IgG1Fc fixed, and add D12 phage library (NEWENGLAND Biolabs Inc. 1x10 13 PFU / mL) 10uL.

[0027] (4) Incubate with shaking at room temperature for 10 min, wash 8 times with 200 uL / well of 0.1% TBST, add 100 uL of 1 mg / mL BSA / 0.2 M glycine-HCl (pH 2.2), incubate with shaking at room temperature for 10 min to elute the phage, recover the eluate, and neutralize it by adding 16 uL of 1 M Tris-HCl (pH 9.0) to the recovery solution. Use a portion of the recovered eluate to measure the phage titer.

[0028] (5) Infect 20 mL of LB with ER2738 bacteria in logarithmic growth and incubate the phage eluate for 5 h under vigorous stirring in a shaking incubator.

[0029] (6) Transfer the phage-infected culture medium to a 50-mL centrifuge tube and centrifuge at 4°C for 10 min using a high-speed refrigerated centrifuge.

[0030] (7) Pipette the supernatant into a new centrifuge tube, add 3.5 mL of PEG / NaCl (20% polyethylene glycol 6000, 2.5 M NaCl) solution, stir, and incubate at 4 °C for 18 h.

[0031] (8) Centrifuge in a high-speed centrifuge at 4°C and 9000×g for 10 min, remove the supernatant, add 1 mL of TBS solution to the precipitated phage, resuspend, transfer to a microcentrifuge tube and centrifuge at 4°C and 15000×g for 5 min. Recover the supernatant into another centrifuge tube, add 200uL PEG / NaCl, stir with a mixer, and incubate on ice for 1 h to precipitate the bacteria.

[0032] (9) Centrifuge at 4°C and 15,000 × g for 10 min to precipitate the phage, remove the supernatant, add 200 μL of 0.02% NaN3 / TBS to the phage precipitate, resuspend, and centrifuge at 4°C and 15,000 × g for 5 min. Recover the supernatant and measure the titer of the phage concentrate.

[0033] (10) Use the concentrated phage solution to perform the second or third screening.

[0034] Titer determination: ER2738 bacteria were cultured in 3 mL LB until the logarithmic growth phase, phage solution was added to the bacterial culture solution, stirred with a mixer, incubated at room temperature for 5 min, mixed with 4 mL of dissolved top agar solution, spread on LB / IPTG / X gal plates, incubated at 37°C for 16 h, and the number of blue plaques obtained was counted. The number of phages was calculated using the phage dilution factor (number of counted plaques × dilution factor / 10uL).

[0035] Figure 1 The changes in the ratio of output titer (the titer of phage eluted from the target molecule after washing) to input titer (the titer of phage added to the target molecule) of the experiments using the phage library are shown, showing a large increase in round 3, indicating that phages displaying specific binding to TRIM24 / Fc were selected.

[0036] Example 2

[0037] Sequencing analysis:

[0038] The phage obtained in the third round of the screening experiment of the D12 library was cloned according to a conventional method, and the base sequence of the phage-displayed peptide was determined, thereby determining the displayed peptide sequence.

[0039] The results are shown in Table 1:

[0040] Table 1

[0041]

[0042] Example 3

[0043] TRIM24 / Fc as the target protein, IgG1Fc as the Fc site recognized by TRIM24 / Fc, and BSA as the standard protein were added to a 96-well microplate at 1 μg / well and fixed by placing at 4°C for 12 hours. The protein solution was removed, 300 μl of blocking buffer was added, and the wells were blocked by incubating at 37°C for 1 hour. After removing the blocking buffer, the wells were washed 3 times with 200 μl of washing solution 0.5% TBST, and finally 100 μl of 0.5% TBST was added. The amplified phage solution (1-101, 1-102, 1-105, 1-106 phage, and M13KE (non-peptide display phage)) was added to the above wells to make it 1×10 10 PFU, mix by pipetting with a pipette. The reaction was shaken steadily at room temperature for 1. The reaction solution was removed, and the wells were washed 10 times with 200μl of 0.5% TBST. Then, 100μl of 0.2M glycine-HCl (pH 2.2) was added to the wells, and after stirring with a pipette, it was shaken at room temperature for 10 minutes. The eluate was recovered from the wells into a microcentrifuge tube, and neutralized by adding 15μl of 1M Tris-HCl (pH9.1) to obtain a phage solution bound to the target. The binding ability of the recovered phage was determined by titer determination.

[0044] The results are as follows Figure 2-3 As shown, the binding of 1-101, 1-102, 1-105, and 1-106 phages to TRIM24 / Fc is significantly higher than that of IgG1Fc as the Fc part and BSA as the standard protein. For M13KE phage, no difference in binding to the immobilized protein was found, and the values ​​of each were very low. From the above results, it can be seen that 1-101, 1-102, 1-105, and 1-106 phages specifically bind to TRIM24 / Fc under protein immobilization conditions. Such results indicate that the peptide sequences displayed by 1-101, 1-102, 1-105, and 1-106 phages are involved in binding to TRIM24 / Fc.

[0045] Example 4

[0046] TRIM24 / Fc, the target protein, was added to a 96-well microplate at 1 μg / well and fixed by placing at 4°C for 12 hours. The protein solution was removed, 300 μl of blocking buffer was added, and the wells were blocked by incubating at 37°C for 1 hour. After removing the blocking buffer, the wells were washed three times with 200 μl of 0.5% TBST, and finally 100 μl of 0.5% TBST was added. The amplified 1-101 phage solution was added to the above wells to make 1×10 10PFU, then add the artificially synthesized 1-101 phage display peptide (purity 90%, HPLC grade, AnyGen, Korea) and the synthetic peptide unrelated to TRIM24 / Fc binding (GTTSRHYLHRRI: purity 90%, HPLC grade, AnyGen, Korea), mix with a pipette, and shake steadily at room temperature for 1h. Remove the reaction solution and wash the wells 10 times with 0.5% TBST 200μl. Then, add 100μl 0.2M glycine-HCl (pH 2.2) to the wells, stir with a pipette, and shake steadily at room temperature for 10min. The eluate is recovered from the wells into a microcentrifuge tube and neutralized by adding 15μl 1M Tris-HCl (pH9.1) to obtain a phage solution bound to the target. The binding ability of the recovered phage is determined by titer determination.

[0047] The results are as follows Figure 4 As shown, the ratio of input titer / output titer of 1-101 phage decreased when 1 μL of 1-101 phage displayed peptide was added, indicating that the binding of 1-101 phage to TRIM24 / Fc was inhibited in a concentration-dependent manner. However, when an irrelevant synthetic peptide was added, the ratio of input titer / output titer of 1-101 phage remained high when an irrelevant synthetic peptide was added at a concentration of 100 μM. Since the irrelevant synthetic peptide does not participate in the binding of 1-101 phage to TRIM24 / Fc, even at high concentrations, no decrease in the ratio of input titer / output titer of 1-101 phage was found. However, it can be seen that the 1-101 phage displayed peptide inhibited the binding of 1-101 phage to TRIM24 / Fc in a concentration-dependent manner. Such results confirm that the 1-101 phage displayed peptide, even as a peptide monomer, competes with the 1-101 phage for binding to the same site of TRIM24 / Fc. Similarly, 1-102, 1-105, and 1-106 phage-displayed peptides also inhibited the binding of the respective phages to TRIM24 / Fc; it was confirmed that even as peptide monomers, 1-102, 1-105, and 1-106 phage-displayed peptides competed with the respective phages for binding to the same site of TRIM24 / Fc.

[0048] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A TRIM24 binding peptide, characterized in that It is a short peptide with the following amino acid sequence: VLFYLYYTGT™, VLFYLYYTPS™, VLFCTYYTAT™ or VLFCYYYTWT™.

2. The peptide-binding label according to claim 1, characterized in that The label is a radioactive isotope, biotin, horseradish peroxidase, alkaline phosphatase, FITC, rhodamine or positron nuclide.

3. Use of the binding peptide according to claim 1 or the labeled form of the binding peptide according to claim 2 in the preparation of a drug for treating glioma.

4. Use of the binding peptide according to claim 1 or a labeled form of the binding peptide according to claim 2 in the preparation of a reagent for diagnosing glioma.

5. A drug for treating glioma, characterized in that: It comprises the binding peptide according to claim 1 and a pharmaceutically acceptable carrier.

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