Novel penetrating peptide and application thereof
A technology of penetrating peptides and residues, applied in the field of new penetrating peptides and their applications, can solve the problems of large cell stimulation, cytotoxicity, low import rate, etc., and achieve the effect of low immunogenicity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0030] Example 1: Synthesis of Penetrating Peptides
[0031] 1) Activation of the resin: Weigh 1000mg of Fmoc-Trp wang Resin or Fmoc-His wang Resin, add 10-15ml (immerse the entire resin) in DMF and soak for 30min to make it fully swell.
[0032] 2) Deprotection: soak the DMF of the resin to remove it by pressure filtration, add 10ml of DMF solution containing 20% piperidine, react with nitrogen blowing and boiling for 15min, then remove it by pressure filtration, remove the FMOC group of the amino group, and wash the resin with 10ml of isopropanol Three times, and then wash three times with 10ml DMF, and then use the ninhydrin method to detect that the resin should turn black or purple.
[0033] 3) Condensation reaction: connect the next amino acid, weigh the amount of FMOC-amino acid is 1.4mmol / g resin, 910mg TBTU, add 10ml DMF and 0.45g HOBt and mix evenly, add 0.52ml DIEA to make the reaction solution, nitrogen blowing at room temperature Boiling reaction 2h. After the...
Embodiment 2
[0038] Embodiment 2: Cytotoxicity experiment
[0039] 1) Seeding cells: Take a 96-well plate, add 7×10 cells to each well 3 A cell culture medium, 37 ℃, 5% carbon dioxide incubator for 24 hours, so that the cells adhere to the wall.
[0040] 2) Cultivate cells: prepare different concentrations of penetrating peptide SEQ ID NO: 1, and set three negative control wells at the same time, culture at 37°C and 5% carbon dioxide for 1-5h.
[0041] 3) Color development: Add 20 μl MTT to each well of adherent cells, discard the culture medium after continuing to incubate for 4-6 hours, add 150 μl DMSO (dimethyl sulfoxide) to each well, and shake for 10 minutes.
[0042] 4) Colorimetry: Select a wavelength of 490nm or 570nm, measure the light absorption value on a microplate reader immunodetector, and process the data to obtain the cell survival rate. see results figure 1.
Embodiment 3
[0043] Embodiment 3: cell membrane penetration experiment
[0044] 1) Add the medium containing a certain concentration of FITC-labeled penetrating peptide to incubate the cells, and use the medium containing FITC as a blank control. After incubation for 30min-4h, carefully aspirate the medium, and wash the adherent cells three times with PBS.
[0045] 2) Add fixative, 10min-20min, fix the cells and wash the cells 2-3 times with PBS.
[0046] 3) Add DAPI staining solution to stain for 5min-10min, then wash with PBS 2-3 times.
[0047] 4) Observe with a fluorescence microscope under the excitation light of 488nm and 543nm respectively, the results are shown in figure 2 .
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More