A polypeptide targeting human cancer cells and its application
A cancer cell and targeting technology, applied in the preparation of anticancer drugs or imaging preparations, polypeptides and their application fields, can solve the problems of high cost, cumbersome preparation, weak penetrating power, etc., and achieve small molecular weight and high purity High, selective effect
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Embodiment 1
[0041] Example 1 Construction of polypeptide library and screening of APN targeting polypeptide
[0042] 1. Experimental instruments and materials
[0043] 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, peptide synthesis tubes, shaking Bed, vacuum water pump, rotary evaporator, above reagents and materials were obtained from commercial sources.
[0044] 2. Synthesis of "one bead, one object" polypeptide library
[0045] The peptide library was synthesized by Fmoc solid-phase peptide synthesis method, and the chemical formula constructed by screening the peptide library of APN target peptide was as follows: figure 1 shown. The specific method is to randomly couple amino acids one by one to the ...
experiment example 1
[0051] Experimental Example 1 Detection of affinity between polypeptide and human aminopeptidase N (APN) protein by surface plasmon resonance imaging (SPRi) method
[0052] Spot 1mg / mL AP-1, AP-2, AP-3, AP-4 and 1×PBS (phosphate buffer) onto the chip, incubate overnight at 4°C under humid conditions, then wash with 10×PBS for 10min , then wash with 1×PBS for 10min, and finally wash twice with deionized water, 10min each time, immerse in 1×PBS containing 5% milk, incubate overnight at 4°C, then wash with 10×PBS for 10min, 1× Wash with PBS for 10 min, and finally wash twice with deionized water for 10 min each time, blow dry with nitrogen, and install the chip on the machine (Plexera HT surface plasmon resonance imaging system).
[0053] The mobile phase was sequentially passed through 1×PBS, 2×PBS, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL and 20 μg / mL of APN purified protein, and the SPRi signal was recorded and analyzed.
[0054] binding effect as figure 2 As shown, the b...
experiment example 2AP-1
[0055] Experimental Example 2 Interaction of AP-1, AP-2, AP-3, AP-4 with APN-positive cells SKOV-3 and APN-negative cells 293A
[0056] AP-1, AP-2, AP-3, fluorescein isothiocyanate (FITC) conjugates of AP-4 are obtained by solid-phase synthesis, AP-1 obtained in Example 1, AP-2, Continue to couple ε-aminocaproic acid to the AP-3 and AP-4 polypeptide microbeads. In a solution of pyridine / N,N dimethylformamide / dichloromethane at a ratio of 1:5:7, FITC was mixed with peptide beads and reacted overnight.
[0057] The FITC conjugates of AP-1, AP-2, AP-3 and AP-4 were obtained after trifluoroacetic acid cleavage. Polypeptide SP was coupled with FITC according to the above method, and the SP-FITC conjugate was obtained as a control. MALDI-TOF identification and HPLC purification were used for subsequent experiments.
[0058] The human ovarian cancer cell line SKOV-3 was cultured in McCoy’s 5A medium containing 100 mL / L fetal bovine serum at 1×10 5 / mL cell concentration into a ro...
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