GPC-3 targeting binding peptide F3 and derivative, probe and application thereof
A targeted combination and derivative technology, applied in the field of biomedicine, can solve the problems of slow imaging background clearance, excessive accumulation of liver metabolism, poor tumor permeability, etc., achieve good tumor targeting and application prospects in diagnosis and treatment, and simple synthesis process , the effect of small molecular weight
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Embodiment 1
[0039] Example 1: Phage Display Random Peptide Library Biopanning for GPC-3-specific Binding Peptides.
[0040]1. Coating the target protein: irradiate the sterile polystyrene culture dish with ultraviolet light for 30 min in advance, then add 2 mL of recombinant human GPC-3 protein with a concentration of 100 μg / mL, then put it into an airtight container and place it at 4 Shake gently overnight at ℃, discard the excess coating solution the next day, add 0.5% BSA blocking solution, and incubate at 37 ℃ for 1 h.
[0041] 2. Biopanning of phage display random peptide library: remove the blocking solution and wash 6 times with PBS-T (containing 0.1% Tween-20). Take 10 μL of the original phage display random peptide library (original library titer 2 × 1013 pfu / mL), dilute and mix with 2 mL of 0.1% PBS-T, add to the coated protein culture dish, and incubate at room temperature for 2.5 h .
[0042] 3. Remove unbound phage display peptides: wash 10 times with 0.1% PBS-T (concentrat...
Embodiment 2
[0046] Example 2: ELISA of monoclonal phage displaying F3 peptide.
[0047] 1. Target coating: prepare 3 coated plates, including protein plate, HepG-2 plate and PC-3 plate. Protein plate: Add recombinant human GPC-3 protein at 100 μg / mL to 100 μL / well of a 96-well plate, overnight at 4 °C; HepG-2 plate: HepG-2 is a human liver cancer cell line positive for GPC-3 expression, Add 2 × 105 cells / well to a 96-well plate, culture overnight at 37 °C, 5% CO2; PC-3 plate: PC-3 is a human prostate cancer cell line negative for GPC-3 expression, add 2 × 10 cells to a 96-well plate 105 cells / well, cultured overnight at 37°C, 5% CO2.
[0048] 2. Block the target: discard the supernatant from the coated plate, add 200 μL / well of 0.5% BSA blocking solution, and block at 4 °C for 2 hours. Before sealing the cell-coated plate, the supernatant was discarded, 120 μL / well of 4% paraformaldehyde was added, left at room temperature for 15 min, washed twice with 0.5% PBS-T, and then blocked.
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Embodiment 3
[0055] Example 3: Verification of cell binding and target blocking verification.
[0056] 1. According to the polypeptide sequence F3 obtained in Example 1, carry out chemical synthesis and label the fluorescent dye Rhodamine B (RhodamineB, RhoB), and chemically synthesize the control polypeptide G7 (sequence is CGGGGGGGC, the disulfide bond at the first and last ends is a cyclic peptide) and Label RhoB to obtain fluorescent probes RhoB-F3 and RhoB-G7.
[0057] 2. Cell plating: HepG-2 and PC-3 cells were plated on 12-well cell culture plates at 1.5 × 104 cells / well, and cultured overnight at 37 °C and 5% CO2.
[0058] 3. Blocking: Discard the supernatant, wash with PBS, add 4% paraformaldehyde, let stand at room temperature for 20 min, then wash with PBS, add 1% BSA blocking solution, and incubate at 37 °C for 1 h.
[0059] 4. Fluorescent peptide probe binding: Discard the blocking solution, add RhoB-F3 fluorescent probe or control probe RhoB-G7 at a concentration of 10 μM, r...
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