Polypeptide for restraining corneal epithelium adhered by pseudomonas aeruginosa
A technology of Pseudomonas aeruginosa and cornea, which is applied in the field of polypeptides that inhibit Pseudomonas aeruginosa from adhering to the corneal epithelium, and can solve the problem of less polypeptides
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Embodiment 1
[0025] Example 1: Screening and determination of polypeptides that inhibit Pseudomonas aeruginosa from adhering to corneal epithelium
[0026] 1) Human corneal epithelial cells (HCEC) cultured to a monolayer were mixed with 10 10 The pfu phage-displayed 12-peptide library (product of New England Biolabs, a 12-amino-acid-long random peptide embedded in the pIII protein of M13 phage) was incubated at 37°C for 1 hr, unbound phages were washed away with PBS, and the glycine eluent was eluted. The phage bound to the surface of HCEC was amplified and titrated, and then subjected to the same two rounds of panning to finally obtain phage polypeptides capable of binding to HCEC. Picked a single phage clone for sequence determination, and used the BLAST online software provided by NCBI to perform homology analysis on the obtained nucleotide sequence, and found that one of the polypeptides was highly homologous to a cell membrane protein of unknown function of Pseudomonas aeruginosa. Thr...
Embodiment 2
[0044] Embodiment 2: the preparation of complete antigen:
[0045] (1) The polypeptide of the present invention is dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.1 μg / μL polypeptide mother solution; water-soluble carbodiimide (EDC), N-hydroxysuccinimide ( NHS) and ovalbumin (OVA) were prepared into 1 mg / mL working solution with 0.01M phosphate buffered saline (PBS, pH7.5);
[0046] (2) Take 100 μL of polypeptide mother solution, add 20 μL of EDC working solution and 12 μL of NHS working solution, the molar ratio of STX, EDC and NHS reaction is 1:2-3:1-2, shake and react at room temperature for 3 hours, and obtain the initial reaction solution;
[0047] (3) Prepare the protein carrier with 0.01M phosphate buffered saline (PBS, pH7.5) to make a 1mg / mL working solution.
[0048] (4) The optimum molar ratio of STX and carrier protein reaction is 10:1; add the reactants in step (2) to 100 μL of carrier protein working solution, shake and react at 4°C for 12 hours, and obtai...
Embodiment 3
[0052] Example 3: Preparation of Composite Polypeptide Eye Drops and Detection of Its Effect on Prevention and Treatment of Pseudomonas aeruginosa Infection
[0053] Dissolve a certain amount of Pc-EP polypeptide freeze-dried powder in borate buffer, a common solvent for eye drops, to prepare eye drops with a polypeptide concentration of 0.1%, and use sodium bicarbonate to adjust the pH value to 5.5-7.5, and use glucose Adjust the osmotic pressure to be 270-310mOsm / kg, so that it meets the standard of conventional eye drops.
[0054] 1 hr before infection, healthy BALB / c mice were instilled with compound polypeptide eye drops, 15 min / time, for 5 consecutive times, and then Pseudomonas aeruginosa was established by corneal scratch method (10 7 cfu) infected mouse model, continue to use compound polypeptide eye drops for prevention and treatment after infection, 15min / time, 5 times in a row, remove the cornea after 8hr, homogenate and lyse, then gradient dilution plate counting,...
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