Specific HER2 protein targeted polypeptide and application thereof
A specific and targeted technology, applied in applications, specific peptides, immunoglobulins, etc., can solve problems such as large toxic and side effects, and achieve the effects of small molecular weight, strong selectivity 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 SEQIDNO: 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, suck the medium in the small dish, and then add respectively 200 μL of medium containing SEQIDNO:1 and SEQIDNO:2 polypeptides labeled with fluorescein isothiocyanate (FITC) (FITC). The culture medium of PBS (phosphate buffer pH 7.4) equal to the amount of the polypeptide, and the nuclei were stained with hoechst reagent, and used in a 1:200 dilution. 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 fluorescent signal was observed usi...
Embodiment 3
[0055] Embodiment 3 Immunofluorescence method detects the binding effect of SEQIDNO: 1 and 2 polypeptides 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, suck the medium in the small dish, and then add respectively 200 μL of medium containing SEQIDNO:1 and SEQIDNO:2 polypeptides labeled with fluorescein isothiocyanate (FITC) (FITC). The culture medium of PBS (phosphate buffer pH 7.4) equal to the amount of the polypeptide, and the nuclei were stained with hoechst reagent, and used in a 1:200 dilution. 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 fluorescent signal was observed using a confocal laser micro...
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