Tumor stem cell marker CD133-targeting polypeptide and application thereof
A technology of tumor stem cells and markers, which is applied in the direction of anti-tumor drugs, medical preparations of non-active ingredients, peptides, etc., can solve the problems of difficult labeling, weak penetration, large molecules, etc., to improve the ability of targeted penetration, No immunogenicity, small molecular weight effect
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experiment example 1
[0056] Experimental example 1 Construction and screening of CD133 targeting polypeptide screening system of the present invention
[0057] 1) Experimental instruments and materials
[0058] 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, tetraphenylethylene ( TPE), PE-anti-CD133 antibody, MB-Streptavidin (streptavidin magnetic beads), 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.
[0059] 2) Synthesis of CD133 "one bead one object" polypeptide library
[0060]Use the Fmoc solid-phase peptide synthesis method to synthesize the peptide library, the specific met...
experiment example 2
[0077] Experimental example 2 Detection of affinity between CS133-P2 polypeptide and CD133 protein by surface plasmon resonance (SPRi) method
[0078] Spot the 1mg / mL CS133-P2 polypeptide and 1×PBS on the chip, incubate overnight at 4°C under humid conditions, then wash with 10×PBS for 10 minutes, then wash with 1×PBS for 10 minutes, and finally wash with deionized water for 2 minutes. Once, 10min each time, immersed in 1×PBS containing 5% milk, incubated overnight at 4°C, then washed with 10×PBS for 10min, 1×PBS for 10min, and finally washed twice with deionized water, 10min each time , blown dry with nitrogen, and put the chip on the machine (Plexera HT surface plasmon resonance imaging system).
[0079] The mobile phase was sequentially passed through 1×PBS, 2×PBS, 0.78μg / mL, 1.56μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL and 25μg / mL human CD133 purified protein, and the SPRi signal was recorded and analyzed .
[0080] Depend on figure 2 It can be seen that the SPRi sign...
experiment example 3
[0081] Experimental Example 3 CS133-P2 and CD133 isolated from leukemia + Specific staining of tumor stem cells and tissue sections of colorectal cancer patients
[0082] CD133 was sorted by CD133 antibody flow cytometry + Tumor stem cells were cultured in complete stem cell medium containing cell growth factors for 24 hours at 37°C, and then mixed with 1×10 3 / mL cell concentration into a round glass-bottomed culture dish (35mm), add 1μmol / L Hoechst 33342 and 5μL PE-anti-CD133 antibody to the three kinds of cells respectively, incubate at 4°C for 60min in the dark, then use pre-cooled 1 Wash twice with ×PBS, add 50 μmol / L FITC-labeled CS133-P2 polypeptide respectively, incubate at 4°C in the dark for 20 min, and wash three times with pre-cooled 1×PBS. Fluorescence distribution in stem cells was detected with a laser scanning confocal microscope (ZEISS LSM 710). Tumor tissue samples were fixed with paraformaldehyde for more than 48 hours (paraformaldehyde was replaced once ...
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