Peptides inhibiting adhesion of aspergillus fumigatus to cornea

A technology for Aspergillus fumigatus and cornea, which is applied in the field of polypeptides that inhibit the adhesion of Aspergillus fumigatus to cornea, can solve the problem that the specific mechanism of adhesion of corneal epithelial cells is not very clear.

Active Publication Date: 2012-10-17
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific mechanism by which Aspergillus fumigatus adheres to corneal epithelial cells is still unclear, so the research on how to block the infection of corneal cells by Aspergillus fumigatus during the adhesion stage is still in its infancy

Method used

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  • Peptides inhibiting adhesion of aspergillus fumigatus to cornea
  • Peptides inhibiting adhesion of aspergillus fumigatus to cornea
  • Peptides inhibiting adhesion of aspergillus fumigatus to cornea

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Screening for polypeptides that inhibit Aspergillus fumigatus from adhering to the cornea

[0032] 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 phages bound to the surface of HCECs were amplified and titrated, and then subjected to the same two rounds of panning, and finally 14 polypeptides of phages capable of binding to HCECs were obtained. Pick a single phage clone for sequence determination, translate the obtained nucleotide sequence into a polypeptide, and use pBLAST provided by NCBI to perform homology analysis on the polypeptides corresponding to the positive phage clones, and find that 10 of the polypeptides are similar to those of Aspergillus fum...

Embodiment 2

[0058] Embodiment 2: the preparation of complete antigen:

[0059] (1) Any one or several polypeptides described in the present invention are dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.1 μg / μL polypeptide mother solution; water-soluble carbodiimide (EDC), N-hydroxy Succinimide (NHS) and ovalbumin (OVA) were prepared with 0.01M phosphate buffer saline (PBS, pH7.5) to make 1mg / mL working solution respectively;

[0060] (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 is 1:2-3:1-2, shake and react at room temperature for 3 hours, and obtain the initial reaction solution;

[0061] (3) Prepare the protein carrier with 0.01M phosphate buffered saline (PBS, pH7.5) to make a 1mg / mL working solution.

[0062] (4) The optimal 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 reac...

Embodiment 3

[0067] Example 3: Preparation of Composite Polypeptide Eye Drops and Detection of Its Effect on Prevention and Treatment of Aspergillus Fumigatus Infection

[0068] Take a certain amount of two peptide freeze-dried powders, Pc-C and Pc-E, and dissolve them in borate buffer, a common solvent for eye drops, to prepare eye drops with a peptide concentration of 0.1%, and use sodium bicarbonate to adjust the pH value. To 5.5-7.5, use glucose to adjust the osmotic pressure to 270-310mOsm / kg, making it meet the standard of conventional eye drops.

[0069]1 hr before infection, the healthy BALB / c mice were instilled with compound polypeptide eye drops, 15 min / time, for 5 consecutive times, and then the Aspergillus fumigatus (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, only infection witho...

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Abstract

Provided are peptides inhibiting the adhesion of Aspergillus fumigatus to cornea, comprising: (1) peptide with the sequence of SEQ ID NOs: 1-10 respectively; (2) peptide having 70% or above homology to the peptide of (1) respectively, whose function is the same or similar with that of the peptide of (1). Also provided are polynucleotides. The polynucleotides are 1) a nucleotide of any peptide with the sequence of SEQ ID NOs: 1-10; 2) nucleotides of the peptides having 70 % or more homology with the peptide described in (1), and the encoded peptides have the same or the similar functions with the peptide described in (1). 10 peptides selected by the invention have competitive inhibitory effect on the interaction of the Aspergillus fumigatus and epithelial cells of the cornea in different degrees, can specifically block the adherence between ligands on the surface of the Aspergillus fumigatus and the epithelial cells of the cornea, and become new target spots for designing treatment drugs of fungal keretitis, thereby providing new ideas for clinical treatment thereof.

Description

technical field [0001] The invention belongs to the technical field of biological control of infectious eye diseases, and in particular relates to a polypeptide for inhibiting aspergillus fumigatus from adhering to the cornea. Background technique [0002] Keratitis is the most common frequently-occurring disease of the eye. The pathogenic microorganisms that cause keratitis include fungi, bacteria, viruses, and Acanthamoeba. In recent years, with the abuse of broad-spectrum antibiotics and immunosuppressants, and the widespread wearing of contact lenses, the incidence of keratitis has greatly increased. Fungal keratitis is more common in developing countries dominated by agricultural populations, and it has a tendency to increase year by year in my country. Relevant statistics show that fungal keratitis accounts for as high as 34.8% to 61.9% of infectious corneal diseases in my country. Some areas have become the leading cause of corneal blindness. Fungal keratitis is ofte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K19/00C07K16/14C12N15/31A61K38/10A61K39/395A61P31/10A61P27/02
CPCC07K7/06C07K2319/00C07K14/38A61K38/00A61P27/02A61P31/10
Inventor 王宜强赵格李思媛
Owner SHANDONG EYE INST
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