A covalent polypeptide inhibitor targeting PD-L1 and its preparation method and application
A peptide inhibitor, PD-L1 technology, applied in the fields of molecular biology and medicine, can solve the problems of cumbersome preparation of antibody drugs, poor in vitro stability, weak penetration, etc., achieving good research prospects and clinical guiding significance, small molecular weight. , selective effect
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Embodiment 1
[0051] Example 1 Synthesis and Characterization of Covalent Peptide Inhibitor PD-P1
[0052] (1) Experimental instruments and materials
[0053] N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium hexafluorophosphate (HBTU), hexahydro Pyridine, triisopropylsilane (TIS), ethanedithiol (EDT), N,N dimethylformamide (DMF), anhydrous ether, resin, methanol, various Fmoc protected amino acids, carboxylated tetraphenyl Ethylene (TPE), peptide synthesis tube, shaker, vacuum water pump, rotary evaporator, laser confocal microscope (ZEISS LSM 710), the above reagents and materials were obtained from commercial sources.
[0054] (2) Solvent preparation
[0055]The preparation of the deprotection solvent is hexahydropyridine: N, N dimethylformamide = 1:4
[0056] The preparation of the reaction solution is N-methylmorpholine: N, N dimethylformamide = 1:24
[0057] The lysate was prepared as trifluoro...
Embodiment 2
[0064] Example 2 Self-assembled morphology of polypeptide inhibitor PD-P1 and analysis of its interaction with cells
[0065] (1) Analysis of the assembly morphology of peptide inhibitors by transmission electron microscopy
[0066] The purified PD-P1 polypeptide was placed in ultrapure water with a pH of 6.5, and reacted overnight at a constant temperature of 37°C, and a small amount was placed on a copper grid. After counterstaining with acetate glaze, it was imaged by a transmission electron microscope. The result is as image 3 As shown in a, the results showed that the peptide inhibitor PD-P1 formed nanofibrous structures under acidic conditions.
[0067] (2) Using scanning electron microscope imaging to analyze the interaction between polypeptide inhibitors and tumor cell MC38, the specific method is as follows:
[0068] ①Slide bubble acid (1% HCl, H 2 SO 4 )overnight.
[0069] ② Cleaning: Wash with tap water until colorless, replace tap water with triple distilled ...
Embodiment 3
[0078] Example 3 Analysis of the competition between PD-P1 and PD1 protein at the cellular level
[0079] The colon cancer cell line MC38 was used to verify the competition between PD-P1 and PD1 protein at the cellular level, the specific method is as follows:
[0080] Colon cancer cell line MC38 was cultured in MEM / EBSS medium containing 10% fetal bovine serum. Take 1×10 5 / mL cell concentration into a round glass-bottom Petri dish (35mm), 37 ° C, 5% CO 2 After culturing in the cell incubator for 24 hours, the culture medium was discarded, washed twice with 1×PBS, peptide inhibitors PD-P1 and PD1 protein were added to one dish, and PD1 protein was added to the other dish alone, and after incubation for 45 minutes, pre- Wash with cold 1×PBS for 3 times, block with BSA for 30min, then add streptavidin-labeled FITC dye, and incubate for 30min. ×PBS was washed 3 times, and the fluorescence distribution in the cells was detected with a laser confocal microscope (ZEISS LSM 710)....
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