Novel candida-killing polypeptide CFP1 and preparation method thereof

A new type of technology for Candida, which is applied in the field of new Candida-killing polypeptides and its preparation, and can solve the problems of unstable expression products, low product yields, and difficult purification

Active Publication Date: 2018-08-17
HENAN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Using genetic engineering technology to express polypeptides, the expression products are unstable, easily degraded, and difficult to purify
Chemically synthesize polypeptides with more than 30 amino acids, the product yield is low and the purity is low

Method used

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  • Novel candida-killing polypeptide CFP1 and preparation method thereof
  • Novel candida-killing polypeptide CFP1 and preparation method thereof
  • Novel candida-killing polypeptide CFP1 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Preparation of Example 1 FCP1

[0015] The target peptide was synthesized by the solid-phase Fmoc method from the C-terminus to the N-terminus. The carboxyl group of the first Fmoc-Gln(Trt)-OH at the C-terminus of the polypeptide is activated and combined with the resin, the Fmoc-protecting group is removed, combined with the second carboxyl-activated Fmoc-His(Trt)-OH, and then removed The Fmoc-protecting group was removed and the cycle repeated until all amino acids were added. Cut the peptide on the resin with the cutting solution, collect the cutting solution, dry it in a centrifuge tube, wash the precipitate with ice ether 2 to 3 times, centrifuge to remove the supernatant, and dry the precipitate. The obtained crude product was purified by HPLC.

Embodiment 2

[0016] Embodiment 2 MTT method measures FCP1 antimicrobial spectrum

[0017] 1) Weigh and dissolve protein. Prepare the FCP1 phosphoric acid solution, set the initial concentration in the first column to 250 μg / mL, and set the final concentration to 0.488 μg / mL. It is necessary to prepare 1mg / mL FCP1 stock solution. Take an appropriate amount (<10μl) of DMSO to dissolve 1mg FCP1, then add 20mmol / L pH6.0 sodium phosphate buffer to dissolve to 1ml, and filter the solution through a 0.22μm sterile filter membrane to sterilize.

[0018] 2) Using a pipette gun, add 100 μL of 20 mmol / L sodium phosphate buffer solution at pH 6.0 to each well of the 96-well plate to dilute FCP1.

[0019] 3) Use a pipette to pipette 100 μL of 1 mg / mL FCP1 stock solution into each well of the first row of the 96-well plate.

[0020] 4) Repeatedly blow and suck the solution in the first row in the board for 6-8 times, mix well without splashing.

[0021] 5) Draw 100 μL from the first row and add it t...

Embodiment 3

[0032] Example 3 FCP1 kills Candida tropicalis

[0033] 1) Weigh and dissolve protein. Prepare 1 mg / mL stock solution of FCP1. Take an appropriate amount (<10μl) of DMSO to dissolve 1mgFCP1, then add 20mmol / L pH6.0 sodium phosphate buffer to dissolve to 1ml, and filter the solution through a 0.22μm sterile filter membrane to sterilize.

[0034] 2) Using a pipette gun, add 100 μL of 20 mmol / L sodium phosphate buffer solution at pH 6.0 to each well of the 96-well plate to dilute FCP1.

[0035] 3) Use a pipette to pipette 100 μL of 1 mg / mL FCP1 stock solution into each well of the first row of the 96-well plate.

[0036] 4) Repeatedly blow and suck the solution in the first row in the board for 6-8 times, mix well without splashing.

[0037] 5) Draw 100 μL from the first row and add it to the second row, repeat blowing and mixing for 6 to 8 times, and then draw 100 μL to the third row. Repeat this step to the eighth row.

[0038] 6) Discard the 100 μL sucked out of the eight...

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Abstract

The invention discloses a candida-killing polypeptide sequence and a preparation method. An amino acid sequence of the candida-killing polypeptide is NH2-RILSILRHQ-COOH. A solid-phase polypeptide synthesis method is adopted for preparation. Defects of proneness to hemolysis and poor biological safety of common antibacterial peptides are overcome. The polypeptide is a novel antifungal polypeptide with a specific candida killing effect and without hemolysis and provides a novel primer for clinical antifungal drug research.

Description

technical field [0001] The invention belongs to biological preparations, and in particular relates to a novel candida-killing polypeptide and a preparation method thereof. Background technique [0002] Worldwide, as the number of immunocompromised patients increases, the incidence of fungal infections increases, and many clinical diseases caused by Candida spp are associated with significant mortality and morbidity in medical institutions. Studies have shown that among the invasive fungal infections, Candida infection is the main one. Due to the abuse of antibiotics, drug-resistant pathogenic Candida is increasing. Therefore, it is of great significance to research and develop new antifungal drugs with high efficiency, low toxicity and resistance to pathogenic fungi as soon as possible. [0003] Existing studies have shown that antimicrobial peptides (AMP) are considered as lead compounds in the development of new antibacterial drugs (LvY, et al.PLoS ONE, 2014, 9:e86364). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/04C07K1/06
CPCC07K7/06
Inventor 李瑞芳常俊朋陈晨
Owner HENAN UNIVERSITY OF TECHNOLOGY
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