Ngf aptamer and application thereof
An aptamer, N42-N48 technology, applied in recombinant DNA technology, DNA/RNA fragments, medical preparations containing active ingredients, etc., can solve the problem of no tyrosine kinase domain, etc., achieve good NGF inhibitory activity, Effect of high neurite outgrowth inhibitory activity
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Embodiment 1
[0168] Embodiment 1: the production of NGF aptamer-1
[0169] RNA aptamers specifically binding to NGF were prepared using the SELEX method. SELEX was performed by referring to the method of Ellington et al. (Ellington and Szostak, Nature 346, 818-822, 1990) and the method of Tuerk et al. (Tuerk and Gold, Science 249, 505-510, 1990). Human NGF (manufactured by R&D Systems) was used as a target substance.
[0170] RNA (40N) used for the first round was transcribed from chemically synthesized DNA using DuraScribe TM T7 Transcription Kit (manufactured by Epicentre) was obtained. In the NTPs contained in the kit, 2'-OH ATP was replaced by 2'-deoxyadenosine 5'-triphosphate (2'-H ATP or dATP, manufactured by GE Healthcare) and other substances contained in the kit were used . The RNA obtained by this method has the fluorinated 2' position of the ribose of pyrimidine nucleotide, and G (purine nucleotide) is of RNA type, and A is of DNA type. The 83-nucleotide DNA of the primer ...
Embodiment 2
[0182] Example 2: Aptamers that inhibit the binding of NGF and NGF receptors
[0183] Whether or not the aptamer obtained in Example 1 inhibits the binding of NGF and NGF receptors (TrkA and p75) was determined using the surface plasmon resonance method.
[0184] Protein A (21181, PIERCE) was immobilized on a CM5 sensor chip according to the protocol guidelines at BIAcore. About 700 to 1200 RU of human TrkA (175-TK, R&D systems) or human P75 (367-NR, R&D systems) fused to the Fc portion of IgG was immobilized thereon. After allowing to stand for 30 minutes, a mixture of NGF (0.1 μM) and each aptamer (0.3 μM) was injected as analyte. If the aptamer inhibits the binding of NGF and TrkA or p75, the signal on the sensorgram is not expected to increase; if the aptamer does not inhibit binding, a three-part complex will form and the signal is expected to increase. When NGF binds to the receptor more strongly than the aptamer, the aptamer can be removed and NGF can bind to the rece...
Embodiment 3
[0186] Embodiment 3: the neurite outgrowth inhibition activity of aptamer
[0187] The neurite outgrowth inhibitory activity of the aptamer obtained in Example 1 was evaluated using Neuroscreen-1 cells, which are a subclone of PC-12 cells.
[0188] Cells (2500 cells per well) were cultured in RPMI-1640 medium containing 2.5% horse serum and 1.25% fetal bovine serum in 96-well flat bottom plates coated with type IV collagen for one day. Add a mixed solution of human NGF (final concentration 0.38nM or 1.14nM) and aptamer (final concentration 500–0.01nM) that have been pre-reacted in serum-free RPMI-1640 medium at room temperature or 37°C for 30 minutes to 1 hour . Two days later, the cytoplasm and nucleus were stained using Cellomics Neurite Outgrowth Kit (manufactured by Thermo Scientific), and the neurite length per cell was measured by Cellomics ArrayScan VTI (manufactured by Thermo Scientific). The neurite length per cell obtained by adding only NGF was taken as the inhibi...
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