CN112759627A - Anti-candida albicans polypeptide and application thereof - Google Patents
Anti-candida albicans polypeptide and application thereof Download PDFInfo
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- CN112759627A CN112759627A CN202110269099.5A CN202110269099A CN112759627A CN 112759627 A CN112759627 A CN 112759627A CN 202110269099 A CN202110269099 A CN 202110269099A CN 112759627 A CN112759627 A CN 112759627A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention relates to the technical field of biological medicines, and particularly discloses an anti-candida albicans polypeptide and an application thereof, wherein the amino acid sequence of the anti-candida albicans polypeptide is shown as SEQ ID N0.1. The anti-candida albicans polypeptide can treat candida albicans related infection and inhibit the formation of a candida albicans biofilm.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to an anti-candida albicans polypeptide and an application thereof.
Background
Antimicrobial polypeptides, also known as peptides or peptide antibiotics, have a wide range of antibacterial, antifungal, antiviral, etc. activities. Since the antimicrobial polypeptide has good antimicrobial effect, low toxic and side effects and good killing activity on clinical drug-resistant strains, the antimicrobial polypeptide has become a hotspot in the research field of novel antimicrobial drugs in recent years.
Candida albicans as a conditional pathogen can cause oral mucosa infection, vaginal superficial infection and wound infection when the immunity of an individual is low or the immunity is deficient, and can also enter a blood system to cause deep tissue infection. The annual rise of the drug resistance rate of the candida albicans leads to great increase of treatment difficulty. In addition, the biofilm formed by candida albicans on host or non-biological surfaces is an important factor in causing infection and difficult to treat. Therefore, the compound can effectively kill candida albicans and inhibit the formation of a biological membrane, and has important significance in preventing and treating candida albicans related infection.
Disclosure of Invention
In order to solve the technical problems, the invention provides an anti-candida albicans polypeptide and an application thereof, wherein the polypeptide can treat candida albicans related infection and inhibit the formation of a candida albicans biofilm.
The first purpose of the invention is to provide an anti-candida albicans polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID N0.1.
The second purpose of the invention is to provide the application of the candida albicans resisting polypeptide in preparing antifungal medicines.
Further, the antifungal medicine comprises an antibacterial gel, an antibacterial spray and an antibacterial medicine coating.
The third purpose of the invention is to provide the application of the candida albicans resistant polypeptide in preparing a medicine for inhibiting the formation of a fungal biofilm.
The fourth purpose of the invention is to provide the application of the candida albicans resistant polypeptide in preparing polypeptide and antifungal medicines.
Further, the fungus is candida albicans.
Further, the polypeptide and antifungal drug combination comprises the Candida albicans resisting polypeptide and the antifungal drug Clotrimazole.
Compared with the prior art, the invention has the beneficial effects that:
1. the minimum inhibitory concentration of the anti-candida albicans polypeptide in the invention to candida albicans is 50-100 mug/mL, the minimum bactericidal concentration is 100 mug/mL, and the anti-candida albicans polypeptide has good antibacterial activity;
2. the anti-candida albicans polypeptide can kill 98.98% of candida albicans after being treated by 200 mu g/mL polypeptide for 15min, has good killing activity to the candida albicans, and can quickly and efficiently kill the candida albicans. .
3. The Candida albicans resistant polypeptide of the present invention has an inhibition rate of 49.17% for biofilm formation at an action concentration of 50. mu.g/mL, and has an inhibition rate of 88.82% for biofilm formation at an action concentration of 100. mu.g/mL. Therefore, the polypeptide can effectively inhibit the formation of candida albicans biofilms.
4. The anti-candida albicans polypeptide can efficiently kill candida albicans, inhibit the formation of candida albicans biomembranes, has a synergistic effect among antifungal medicaments, has the potential of being developed into anti-candida albicans medicaments, gel preparations, spray preparations and antibacterial medicament coatings, and is used for preventing and treating infectious diseases caused by candida albicans.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the effect of anti-Candida albicans polypeptides of the present invention on Candida albicans biofilm formation.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Obtaining of anti-Candida albicans polypeptide (hereinafter referred to as polypeptide)
The anti-candida albicans polypeptide provided by the invention is derived from a traditional Chinese medicine slug polypeptide sequence database constructed by people, and the amino acid sequence of the anti-candida albicans polypeptide is shown as SEQ ID N0.1;
SEQIDN0.1：Arg-Gly-Asp-Ile-Leu-Lys-Arg-Trp-Ala-Gly-His-Phe-Ser-Lys-Leu-Leu。
according to the shown amino acid sequence, the polypeptide is synthesized by Gill Biochemical (Shanghai) limited company by adopting a solid phase chemical synthesis method, and the polypeptide pure product with the purity of more than 95 percent is obtained by purification of reversed phase high performance liquid chromatography and identification of electrospray mass spectrometry.
Example 2
Antibacterial activity against Candida albicans polypeptide
The polypeptide preservation solution is prepared by using physiological salt as a solvent, and the polypeptide preservation solution is diluted by the physiological salt in a two-fold gradient manner. The bacterial liquid to be tested in logarithmic growth phase was adjusted to about 10 with fresh YPD medium3-104cfu/mAnd L. In a sterile 96-well cell culture plate, the diluted polypeptide solution and the diluted bacteria solution were added to each well, and the concentration of each polypeptide was 3 in parallel. Culturing at constant temperature of 35 deg.C for 24h, and taking the minimum polypeptide concentration at which Candida albicans can not be seen to grow as the Minimum Inhibitory Concentration (MIC) of the polypeptide to the detection bacteria. And simultaneously sampling the polypeptide concentration hole without the growth of the visible candida albicans, coating a YPD solid culture medium, culturing at the constant temperature of 35 ℃ for 24h, counting colonies, and taking the polypeptide concentration with the colony number as the initial colony number of 0.1% as the Minimum Bactericidal Concentration (MBC).
TABLE 1 bacteriostatic and bactericidal activity of anti-Candida albicans polypeptides
The antibacterial and bactericidal activity of the anti-candida albicans polypeptide is shown in table 1, and as can be seen from table 1, the polypeptide has good antibacterial activity on candida albicans polypeptide conventional strains and clinical drug-resistant strains, the minimum inhibitory concentration is 50-100 mu g/mL, and the minimum bactericidal concentration is 100 mu g/mL.
Example 3
Antibacterial effect against Candida albicans polypeptide
Candida albicans AY93025 strain concentration at logarithmic growth phase was adjusted to about 10 using fresh YPD medium4-105cfu/mL, the final concentrations of added antibacterial polypeptide are 100 mug/mL and 200 mug/mL respectively, and no antibacterial polypeptide is used as a control. Samples were taken after 15min incubation and were gradient diluted before plating YPD solid plates. Culturing at constant temperature of 35 ℃ and counting the number of colonies.
TABLE 2 Sterilization efficiency against Candida albicans polypeptide
The bactericidal effect against candida albicans polypeptides is shown in table 2. As can be seen from Table 2, 83.78% of Candida albicans can be killed after 100. mu.g/mL polypeptide treatment for 15min, and 98.98% of Candida albicans can be killed after 200. mu.g/mL polypeptide treatment for 15 min. Therefore, the polypeptide has good killing activity on candida albicans, and can quickly and efficiently kill the candida albicans.
Example 4
Effect of anti-Candida albicans Polypeptides on Candida albicans biofilm formation
Diluting Candida albicans AY93025 in logarithmic growth phase with YPD culture medium to (1-5) × 106cfu/mL, 100. mu.L of diluted bacterial solution was added to a sterile 96-well cell culture plate, and the mixture was subjected to static culture at 35 ℃ for 4 hours. Washing free thallus with PBS, adding new culture medium containing polypeptide, and performing static culture at 35 deg.C for 24 hr with polypeptide-free control group. Free cells were washed with PBS and stained with 0.1% crystal violet for 10 min. Washing off excessive dye solution with sterile water, adding 150 μ L of 33% acetic acid solution into each well, incubating at room temperature for 30min, taking out 100 μ L of the solution from each well, placing the solution in a new 96-well plate, measuring the light absorption value at 492nm with a microplate reader, and calculating the biofilm formation inhibition rate.
The effect of the anti-Candida albicans polypeptide on Candida albicans biofilm formation is shown in FIG. 1, the inhibition rate of the polypeptide on the biofilm formation is 49.17% at the action concentration of 50 μ g/mL, and the inhibition rate on the biofilm formation is 88.82% at the action concentration of 100 μ g/mL. Therefore, the polypeptide can effectively inhibit the formation of candida albicans biofilms.
Example 5
Interaction between anti-candida albicans polypeptide and antifungal drug
Using Candida albicans AY93025 as an example, a chessboard method is used to detect the interaction effect between the polypeptide and the antifungal drug on the activity of Candida albicans. The polypeptide preservation solution is diluted by gradient with physiological salt containing antibiotic with specific concentration. The minimal inhibitory concentration (cMIC) of the polypeptide in the mixed system was determined with reference to the method for detecting minimal inhibitory concentration in example 2, wherein the final concentration of the antibiotic was 1/4MIC, and the Fractional Inhibitory Concentration Index (FICI) was calculated. The calculation formula of the FICI is as follows: FICI ═ cICA/MICA + cMICB/MICB. FICI is less than or equal to 0.5, synergy is achieved, FICI <4 > 0.5 is no effect, and FICI >4 is antagonism.
TABLE 3 Effect of anti-Candida albicans Polypeptides in combination with antifungal Agents against Candida albicans AY93025
The mutual effect of the candida albicans resisting polypeptide and the antifungal drug is shown in the table 3, the polypeptide and the antifungal drug Clotrimazole have synergistic effect, and the polypeptide and the antifungal drug Amphiticin B, Nystatin and anidinlafugin have no synergistic effect or antagonistic effect. Therefore, the polypeptide can be used for preventing and treating candida albicans and can be used in combination with an antifungal drug Clotrimazole.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> university of Henan science and technology
<120> anti-candida albicans polypeptide and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> Artificial Synthesis
<400> 1
Ala Gly Asp Ile Leu Lys Arg Trp Ala Gly His Phe Ser Lys Leu Leu
1 5 10 15
Claims (7)
1. An anti-Candida albicans polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID N0.1.
2. Use of the anti-candida albicans polypeptide of claim 1 in the preparation of an antifungal medicament.
3. The use of the anti-candida albicans polypeptide of claim 2 for preparing an antifungal medicament, wherein said antifungal medicament comprises an antibacterial gel, an antibacterial spray and an antibacterial coating.
4. Use of the anti-candida albicans polypeptide of claim 1 for preparing a medicament for inhibiting fungal biofilm formation.
5. The use of the anti-candida albicans polypeptide of claim 1 in the preparation of a polypeptide combined antifungal medicament.
6. The use according to claim 2, 4 or 5, wherein the fungus is Candida albicans.
7. The use of the anti-candida albicans polypeptide of claim 5 for preparing a polypeptide and antifungal drug combination, wherein the polypeptide and antifungal drug combination comprises the anti-candida albicans polypeptide and the antifungal drug Clotrimazole.
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| CN202110269099.5A CN112759627B (en) | 2021-03-12 | 2021-03-12 | Anti-candida albicans polypeptide and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6199494A (en) * | 1993-05-17 | 1994-11-24 | Fujisawa Pharmaceutical Co., Ltd. | New polypeptide compound and a process for preparation thereof |
| CN107056893A (en) * | 2017-05-02 | 2017-08-18 | 东北农业大学 | A kind of antibacterial peptide RF3 of anti-Candida albicans of resistance to amphotericin B and application |
-
2021
- 2021-03-12 CN CN202110269099.5A patent/CN112759627B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6199494A (en) * | 1993-05-17 | 1994-11-24 | Fujisawa Pharmaceutical Co., Ltd. | New polypeptide compound and a process for preparation thereof |
| CN107056893A (en) * | 2017-05-02 | 2017-08-18 | 东北农业大学 | A kind of antibacterial peptide RF3 of anti-Candida albicans of resistance to amphotericin B and application |
Non-Patent Citations (3)
| Title |
|---|
| XIU-ZHEN WU ET AL.: "Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| 何晓玥，刘栋华: "临床常见真菌感染性皮肤病分类、致病菌生物学特征及发病机制", 《皮肤科学通报》 * |
| 韩亚君: "抗菌肽ZY4对白色念珠菌的杀菌作用研究", 《中国优秀硕士学位论文全文数据库》 * |
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