A kind of nogo-a receptor binding peptide and its derivatives and application
A receptor binding and drug technology, applied in the field of biomedicine, can solve problems such as the recovery of nerve function of patients, achieve huge social and economic benefits, promote nerve regeneration, and promote recovery.
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Embodiment 1
[0047] Example 1 Organic Synthesis and Detection of Nogo-A Receptor Binding Peptides
[0048] Organically synthesized No. 2 polypeptide (His-Ile-Tyr-Thr-Ala-Leu-Val) and No. 5 polypeptide (Gly-Phe-Ala-Thr-Ile-Thr-Gly) (synthesized by Shanghai Qiangyao Biological Company), called Take 1g of KP resin and place it in a reactor, add DMF to swell for 30min, filter out DMF, dissolve Fmoc amino acid, HBTU, HOBT 0.4mmol and NMM 0.6mmol in an appropriate amount of DMF, add it into the reactor, Shake for 2 hours, filter the reaction solution, wash the resin twice with methanol and DMF alternately, and wash once with anhydrous ether; elute the Fmoc group with 20% piperidine / DMF solution by volume fraction; react for 10 minutes, filter the reaction solution methanol, Wash the resin twice with DMF alternately; carry out the coupling of the next amino acid residue, so that the first amino acid at the C-terminal is extended to the N-terminal until the short peptide is completed. The synthes...
Embodiment 2
[0050] Example 2 ELISA detection of phage display: NgR specificity, specificity binding positive monoclonal
[0051] A 96-well ELISA plate (Corning, Inc.) was coated with sNgR protein diluted in PBS at a concentration of 2.5 μg / mL (R&D Company, USA), 150 μL per well, and the wells not coated with sNgR were used as a control. Shake slowly overnight at 4°C, pour off the coating solution, add a blocking solution with a mass fraction of 1% BSA diluted in PBS, and block for 1 hour; add the monoclonal 1×10 10100 μL of pfu / mL phage (New England Biolabs, Beverly, MA), combined with slow shaking at room temperature for 1 h; then washed 6 times with 0.1% PBST, and added 100 μL of HRP-anti-M13 antibody (NEB Company) (1:5000 diluted), incubated at 37°C for 40 min, washed 6 times with PBST, and then added 50 μL of the display substrate TMB (Beiyuntian Company) for 10 min, 1M H 2 SO 4 Stop the color development, and detect the OD value with a microplate reader at 450nm. According to the ...
Embodiment 3
[0052] Example 3 Confocal binding of Nogo-A receptor-binding peptides to NgR on the cell membrane surface of PC-12 and CGCs cells with high NgR expression, and colocalization of Nogo-A receptor-binding peptides with NgR on the surface of PC-12 cells
[0053] Confocal plates were inoculated with PC-12 and CGCs cells, among which PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (ATCC CRL-1721), and CGCs cells were obtained from Sprague-Dawley (SD) suckling mice (SPF grade, Guangdong Province, 6-8 days after birth) Experiment Center (SCXK Guangdong 2013-0002)); 10,000 cells per dish, cultured for 24 hours, washed three times with PBS, fixed with paraformaldehyde for 15 minutes, washed three times with PBS, blocked with BSA for 1.5 hours, and then labeled with 1 μM FITC No. 2 Peptide or peptide No. 5 (the FITC markers of peptide No. 2 or peptide No. 5 were prepared by Shanghai Qiangyao Biological Co., Ltd.) and incubated at room temperature for 2 hour...
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