Polypeptide specifically targeting her2 protein and application thereof
A specific and targeted technology, applied in the direction of application, specific peptide, immunoglobulin, etc., can solve the problems of high toxicity and side effects, and achieve the effect of small molecular weight, non-immunogenicity and high purity
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Embodiment 1
[0036] Example 1 Synthesis of a polypeptide specifically targeting HER2 protein
[0037] 1. Experimental instruments and materials
[0038] Dimethylformamide (DMF), piperidine, resin, dichloromethane (DCM), ninhydrin reagent (ninhydrin, vitamin C, phenol), tetramethyluronium hexafluorophosphate (HBTU), six Hydropyridine (piperidine), triisopropylsilane (TIS), ethanedithiol (EDT), anhydrous ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methanol, various amino acids , Peptide solid-phase synthesis tube.
[0039] 2. Solution preparation
[0040] Deprotection solution - hexahydropyridine: DMF = 1:4
[0041] Reaction solution——NMM:DMF=1:24
[0042] Lysis solution - TFA (92.5%), TIS (2.5%), EDT (2.5%), H 2 O(2.5%)
[0043] Ninhydrin Test Solution—Ninhydrin: Vitamin C: Phenol = 1:1:1
[0044] 3. Experimental steps
[0045] Weigh the resin and put it into the peptide solid-phase synthesis tube (hereinafter referred to as the reactor), and add an appropriate amou...
Embodiment 2
[0050] Embodiment 2 Immunofluorescence method detects the binding effect of SEQ ID NO: 1 and 2 polypeptides to human HER2 positive breast cancer cells
[0051] 1. Experimental method
[0052] The human breast cancer HER2 high-expressing cell line SKBR3 was suspended in RPMI1640 culture medium containing 10% heat-inactivated fetal bovine serum, and seeded in 3 confocol small dishes at a density of 3000-5000 cells / dish. After cultivating for 24 hours, aspirate the medium in the small dish, and then add 200 μL of medium containing 50 μM mol / L of fluorescein isothiocyanate (FITC)-labeled SEQ ID NO:1 and SEQ ID NO:2 polypeptide respectively, and the control group Using the culture medium containing PBS (phosphate buffer pH 7.4) equal to the amount of the polypeptide, the nuclei were stained with the hoechst reagent and diluted 1:200. Incubate in an ice bath protected from light for 30 minutes. After washing with PBS and repeating the washing 3 times, 200 μL PBS was added, and the...
Embodiment 3
[0055] Example 3 Immunofluorescence detection of SEQ ID NO: 1 and 2 polypeptide binding to human HER2-negative breast cancer cells
[0056] 1. Experimental method
[0057] The HER2-negative breast cancer cell line 468 was suspended in H-DMEM medium containing 10% heat-inactivated fetal bovine serum, and seeded in 3 confocol small dishes at a density of 3000-5000 cells / dish. After cultivating for 24 hours, aspirate the medium in the small dish, and then add respectively 200 μL of medium containing SEQ ID NO:1 and SEQ ID NO:2 polypeptides labeled with fluorescein isothiocyanate (FITC) (FITC). The group used the medium containing PBS (phosphate buffer pH 7.4) equal to the amount of the polypeptide, and the nuclei were stained with hoechst reagent, which was diluted 1:200. Incubate in an ice bath protected from light for 30 minutes. After washing with PBS, repeat the washing 3 times, add 200 μL of PBS, and observe the fluorescent signal with a confocal laser microscope.
[0058...
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