A polypeptide inhibiting influenza virus and its application in the preparation of drugs for preventing and treating influenza virus infection
An influenza virus infection and influenza virus technology, applied in the field of biomedicine, can solve the problems of children's drug resistance mutation and frequent I38T mutation, and achieve the effect of inhibiting infection and inhibiting invasion
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0042] 1. Peptide acquisition
[0043] 1.1 Random peptide library screening for HA1-binding peptides
[0044] Influenza virus HA1 protein was used as a target molecule to screen a random 12-peptide library. Dilute the HA1 protein in the coating solution to a mass concentration of 20 micrograms per milliliter, take 100 microliters of the coated microtiter plate, place it in a humid box and incubate overnight at 4°C, streptavidin (0.2 milligrams per milliliter; 100 microliter) was also coated as a control. Discard the coating solution, fill up with blocking solution, act at 4°C for 1 h, wash 6 times with TBST (TBS+0.1% Tween-20) buffer, and take phage random 12-peptide library (2×10 11 PFU) were diluted in 200 μL TBST buffer and incubated at room temperature for 1 h. After washing 6 times with TBST, the bound phages were eluted with 0.2 mol / L Glycine-HCl (pH 2.2) and neutralized with 1 mol / L Tris-HCl (pH 9.1). Aspirate 1 μL of the eluate to determine the phage titer, and abs...
Embodiment 2
[0063] Detection of Influenza Virus Binding to Cells by Flow Cytometry
[0064] Influenza virus H1N1-PR8 strain was used to infect MDCK cells according to MOI=2. One day before infection, MDCK cells in the logarithmic growth phase were collected, the supernatant was discarded, washed twice with PBS, 3 ml each time, and the cells were digested with 0.25% trypsin. Briefly centrifuge for 5 minutes (1000 rpm), resuspend the cells in DMEM+FBS 10%, and distribute to 12-well plates at 100,000 cells per well. Incubate at 37°C, after 24 hours, discard the supernatant, wash twice with ice PBS, quickly add the mixed suspension of polypeptide IVA-P1 or IVA-P1-R4 and virus, set three replicates for each concentration, and incubate on ice for 1 hour , washed twice with ice-cold PBS, 1 ml each time. Cells were cultured for 48 hours.
[0065] Trypsinize the MDCK cells, blow down the cells, centrifuge at 2000 rpm, 4°C for 5 minutes. Discard the supernatant, resuspend in PBS, count the cell...
Embodiment 3
[0067] Elisa detection of binding of HA protein and peptide
[0068] The HA protein was coated onto a 96-well plate. The method is as follows, go to the polystyrene Elisa plate to equilibrate at room temperature. Dilute HA protein with coating solution to a final concentration of 0.1 microgram per microliter. Coating liquid composition: 0.1696 g of anhydrous sodium carbonate, 0.2856 g of sodium bicarbonate, dissolved in 100 ml of deionized water. Add 100 microliters of protein dilution to each well. Coating overnight at 4°C. Discard the coating solution, wash with PBST, add 300 microliters to each well, let it stand for 10 minutes, discard the washing solution, fill up the washing solution, repeat 5 times, and drain the washing solution. Washing solution PBST: 8 grams of sodium chloride, 2.9 grams of disodium hydrogen phosphate 12 hydrate, 0.2 grams of potassium chloride, 0.24 grams of potassium dihydrogen phosphate, 0.5 ml of Tween 20, and dilute to 1 liter. Blocking: EL...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More