CN115819515B - Antibacterial peptide and preparation method and application thereof - Google Patents
Antibacterial peptide and preparation method and application thereof Download PDFInfo
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- CN115819515B CN115819515B CN202211526393.0A CN202211526393A CN115819515B CN 115819515 B CN115819515 B CN 115819515B CN 202211526393 A CN202211526393 A CN 202211526393A CN 115819515 B CN115819515 B CN 115819515B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention relates to an antibacterial peptide, a preparation method and application thereof, wherein the antibacterial peptide is antibacterial peptide AC-1, and the amino acid sequence is as follows: f (F) 1 F 2 L 3 K 4 F 5 L 6 N 7 S 8 F 9 G 10 K 11 V 12 V 13 K 14 A 15 I 16 G 17 V 18 A 19 P 20 The method comprises the steps of carrying out a first treatment on the surface of the The antibacterial peptide AC-1 can specifically have good antibacterial effect on candida albicans, and the minimum antibacterial concentration is as low as 7.8 mug/ml, so that the growth of other strains is not affected; the antibacterial peptide has strong potential as an anti-candida albicans drug.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide and a preparation method and application thereof.
Background
In recent years, due to the evolution of bacteria and the abuse of antibiotics, multi-drug resistant bacteria are growing, so that the difficulty of clinical anti-infection treatment is increased, more and more patients die continuously because proper antibacterial agents cannot be found, and the patients become the second leading cause of death worldwide, and great trouble and challenges are caused to first-line clinicians. Existing antibiotic therapies have failed to meet the clinical needs of treatment, and thus there is an urgent need to find new alternative drugs or therapeutic strategies.
Currently, invasive candidiasis is a serious threat to human health, and Candida albicans (Candida albicans) is the most common type of pathogenic microorganism causing this type of disease. The pathogenic bacteria are conditional pathogenic fungi, are often parasitic on human skin, oral cavity, upper respiratory tract, intestinal tract, vaginal mucosa and the like, and are clinically common in oral candidiasis and candidal vaginitis. The current clinical drugs against candida albicans are polyenes (such as amphotericin B), azoles (such as fluconazole, voriconazole, etc.), echinocandins (such as caspofungin, micafungin, etc.), pyrimidine analogues (such as 5-fluorocytosine, etc.), propionamides (such as terbinafine, etc.). With the continuous emergence of multi-drug resistant candida albicans strains, the development of novel antifungal drugs is urgent. The antibacterial peptide (antimicrobial peptide) has been attracting more attention as a novel drug having many advantages such as low toxicity, high activity, high synthesis efficiency, small side effects, and difficulty in drug resistance.
Most of the existing antibacterial peptides have broad-spectrum antibacterial effects, and are more likely to cause negative effects in clinical application, such as damage to the oral cavity and the intestinal flora.
For this reason, this patent is filed.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an antibacterial peptide and application thereof, and the antibacterial peptide AC-1 is screened, so that the growth of candida albicans can be specifically inhibited without affecting the growth of other strains.
The invention aims to provide a preparation method of antibacterial peptide.
It is another object of the present invention to provide an antibacterial peptide obtained by the above preparation method.
It is a further object of the present invention to provide the use of the above-mentioned antibacterial peptide AC-1.
A method for preparing an antibacterial peptide according to an embodiment of the present invention is: leaching the skin secretion collected from giant salamander with acetic acid for 20-28h; centrifuging to collect supernatant, adjusting pH to 7.2 with phosphate buffer solution, centrifuging, and lyophilizing the supernatant to obtain crude extract; dissolving the crude extract with sterilized distilled water, ultrafiltering and intercepting a filtrate with molecular weight less than 5000Da, screening a solution with antibacterial activity, and purifying to obtain the antibacterial peptide.
According to the antibacterial peptide of the specific embodiment of the invention, the antibacterial peptide is antibacterial peptide AC-1, and the amino acid sequence of the antibacterial peptide AC-1 is shown as SEQ ID NO. 1: F1F2L3K4F5L6N7S8F9G10K11V12V13K14A15I16G17V18A19P20. (the key amino acid positions are 3 rd, 8 th, 9 th, 10 th, 11 th, 14 th and 16 th)
The antibacterial peptide is an antibacterial peptide AC-263, and the amino acid sequence of the antibacterial peptide AC-263 is shown as SEQ ID NO. 2; the antibacterial peptide is antibacterial peptide AC-265, and the amino acid sequence of the antibacterial peptide AC-265 is shown as SEQ ID NO. 3; the antibacterial peptide is antibacterial peptide AC-278, and the amino acid sequence of the antibacterial peptide AC-278 is shown as SEQ ID NO. 4.
The AC-1 antibacterial peptide sequence (SEQ ID NO. 1) is as follows:
Phe-Phe-Leu-Lys-Phe-Leu-Asn-Ser-Phe-Gly-Lys-Val-Val-Lys-Ala-Ile-
Gly-Val-Ala-Pro
the AC-263 antimicrobial peptide sequence (SEQ ID NO. 2) is as follows:
Phe-Lys-Leu-Asn-Phe-Leu-Asn-Ser-Phe-Gly-Lys-Val-Val-Lys-Ala-Ile-
Gly-Val-Ala-Pro
the AC-265 antibacterial peptide sequence (SEQ ID NO. 3) is as follows:
Phe-Lys-Leu-Lys-Phe-Leu-Asn-Ser-Phe-Gly-Lys-Val-Val-Lys-Ala-Ile-
Gly-Val-Ala-Pro
the AC-278 antibacterial peptide sequence (SEQ ID NO. 4) was as follows:
Phe-Lys-Leu-Lys-Phe-Leu-Asn-Ser-Phe-Gly-Lys-Val-Val-Lys-Ala-Ile-
Gly-Val-Ala-Lys
the polypeptide is AC-263 antibacterial peptide (F1K 2L3N4F5L6N7S8F9G10K11V12V13K14A15I16G 17A 19P 20), AC-265 antibacterial peptide (F1K 2L3K4F5L 6S 8F9G10K11V12V13K14A15I16G 17A 19P 20), AC-278 antibacterial peptide (F1K 2L3K4F5L6N7S8F9G10K11V12V 13G 14A15I16G 18A19K 20).
According to the antibacterial peptide AC-1 of the specific embodiment of the invention, the antibacterial peptide AC-1 has good antibacterial effect on candida albicans, and the minimum antibacterial concentration is as low as 7.8 mug/ml.
Further, the centrifugation was 12000rpm/min for 30min.
Further, leaching the skin secretion collected from giant salamander with 5% acetic acid according to a volume ratio of 1:1, and stirring for 24 hours at 4 ℃; centrifuging at 12000rpm/min for 30min, collecting supernatant, adjusting pH to 7.2 with phosphate buffer, centrifuging at 12000rpm/min for 30min, and lyophilizing the supernatant to obtain crude extract; dissolving the crude extract with sterilized distilled water, ultrafiltering and separating with ultrafiltration membrane with molecular weight cut-off of 5000Da at 4deg.C, collecting filtrate with molecular weight less than 5000Da, screening solution with antibacterial activity by antibacterial zone method, and purifying to obtain antibacterial peptide AC-1.
Use of an antimicrobial peptide AC-1 according to a specific embodiment of the present invention for the manufacture of an antimicrobial medicament for the prevention and/or treatment of candida albicans infection.
Compared with the prior art, the invention has the following beneficial effects:
(1) The antibacterial peptide AC-1 screened by the invention has good antibacterial effect on candida albicans, and the minimum antibacterial concentration is as low as 7.8 mug/ml;
(2) The antibacterial peptide AC-1 provided by the invention can specifically inhibit the growth of candida albicans, and has weak or no antibacterial effect on other candidas (candida glabrata, candida krusei and candida tropicalis);
(3) The antibacterial peptide AC-1 has the highest antibacterial performance on candida albicans, and has no antibacterial effect on the growth of other strains except candida albicans, candida glabrata and candida tropicalis Ctr 3450.
Drawings
FIG. 1 shows a schematic diagram of an antibacterial test procedure of the antibacterial peptide AC-1 of the present invention;
FIG. 2 shows concentration profiles of antibacterial peptides.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
The reagents used in the invention are all commercially available unless specified.
Example 1 preparation of antibacterial peptide AC-1
The present example provides an antimicrobial peptide AC-1, which is prepared as follows:
leaching the skin secretion collected from giant salamander with 5% acetic acid according to the volume ratio of 1:1, and stirring at 4deg.C for 24 hr; centrifuging at 12000rpm/min for 30min, collecting supernatant, adjusting pH to 7.2 with phosphate buffer, centrifuging at 12000rpm/min for 30min, and lyophilizing the supernatant to obtain crude extract; dissolving the crude extract with sterilized distilled water, ultrafiltering and separating with ultrafiltration membrane with molecular weight cut-off of 5000Da at 4deg.C, collecting filtrate with molecular weight less than 5000Da, screening solution with antibacterial activity by antibacterial zone method, and purifying to obtain antibacterial peptide.
And (3) carrying out sequence identification on antibacterial peptides with antibacterial activity in giant salamander skin secretion by using a mass spectrometry method. The conserved regions of the candidate polypeptides are determined by sequence alignment of the candidate polypeptides. The polypeptide containing the protecting sequence is named as No.1, then the physical and chemical properties of the polypeptide are predicted by means of an antibacterial peptide analysis website, and the polypeptide No.1 is subjected to sequence modification and optimization, namely the addition and deletion of amino acids. Finally screening to obtain the antibacterial peptide named as AC-1.
Providing amino acid sequence of antibacterial peptide AC-1, subjecting to chemical synthesis by polypeptide synthesis company, desalting to obtain dry powder, storing at-20deg.C, and dissolving with sterilized distilled water to 5mg/mL.
Example 2 bacteriostasis test
In order to detect the antimicrobial spectrum of the antimicrobial peptide AC-1, we selected a plurality of gram-positive bacteria (staphylococcus aureus, enterococcus faecalis), gram-negative bacteria (escherichia coli, pseudomonas aeruginosa), and fungi (candida albicans, candida tropicalis, candida glabrata, candida krusei) strains, and then examined the bacteriostatic effect of the antimicrobial peptide AC-1 on these strains. The main technical approach used is to find the minimum inhibitory concentration (MIC, minimum inhibitory concentration) of the antimicrobial peptide against the strain tested, in order to consider whether it can be used as a drug.
The test flow is shown in FIG. 1, and the concentration distribution of the antibacterial peptide is shown in FIG. 2. The specific experimental details are as follows:
(1) Preparation of test strains, in particular table 1 below:
table 1 strains for testing
(2) Preparing antibacterial peptide, namely diluting the antibacterial peptide AC-1 to 5mg/mL with sterilized distilled water for later use;
(3) The experimental method comprises the following steps:
(1) preparation of the culture medium: the glucose liquid medium (SDB)/solid medium (SDA), mueller-Hinton broth liquid Medium (MHB) and solid Medium (MHA) were prepared. Both SDB and MHB media are commercially available.
(2) And (3) strain recovery: the frozen candida fungi at-80 ℃ are streaked on SDA solid culture dishes and are cultured for 48 hours at 30 ℃ in an inverted manner. The frozen bacteria at the temperature of minus 80 ℃ are streaked on an MHA solid culture dish, and are inversely cultured for 16 to 24 hours at the temperature of 37 ℃.
(3) Preparing bacterial liquid: the monoclonal colonies in the plates were picked up in 15 ml tubes and incubated for 12 hours at the optimum temperature with MHB broth (bacteria) or SDB broth (fungi). Diluting the bacterial solution with fresh MHA liquid culture medium (bacteria) or SDB liquid culture medium (fungi) to OD 600=0.12, and diluting 20 times (bacteria) or 200 times (fungi) to final concentration of 5×10 6 CFU/mL (bacteria) or 0.5X10 4 CFU/mL～1×10 4 CFU/mL (fungi) for use;
(4) preparation of antibacterial peptide: 5mg/ml was diluted in a gradient with sterile distilled water. The concentrations of the antimicrobial peptides diluted to the following were: 500ug/ml, 250ug/ml, 125ug/ml, 62.5ug/ml, 31.2ug/ml, 15.6ug/ml, 7.8ug/ml, 3.9ug/ml, 1.95ug/ml, 0.975ug/ml, 0.48ug/ml, 0.24ug/ml, 0.121ug/ml; each concentration is repeated three times to ensure the reliability of the data;
(5) and (3) result judgment: the bacteria are cultivated for 16 to 20 hours at the constant temperature of 37 ℃ and the fungi are cultivated for 48 hours at the constant temperature of 28 ℃, and then the growth of the bacterial liquid in each hole is detected by an enzyme-labeled instrument.
(4) Test results
The in vitro antibacterial effect of the antibacterial peptide AC-1 on candida albicans and the like is shown in the following table 2:
TABLE 2 antibacterial results
Remarks: N/A indicates that the highest concentration of 500ug/ml has no inhibitory activity.
From the experimental results in the table, compared with other strains, the antibacterial peptide AC-1 has the strongest antibacterial performance on candida albicans, and can reach 7.8ug/ml. The antibacterial peptide N8 has strong potential as an anti-candida albicans drug, and has no antibacterial effect on gram-positive bacteria (staphylococcus aureus and enterococcus faecalis), gram-negative bacteria (escherichia coli and pseudomonas aeruginosa) and candida krusei.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (1)
1. An application of antibacterial peptide in preparing antibacterial agent for treating candida albicans;
the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.
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| CN202211526393.0A CN115819515B (en) | 2022-11-30 | 2022-11-30 | Antibacterial peptide and preparation method and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000009553A2 (en) * | 1998-08-14 | 2000-02-24 | Bionebraska, Inc. | Antimicrobial peptides isolated from the skin of american frogs |
| WO2009149554A1 (en) * | 2008-06-11 | 2009-12-17 | National Research Council Of Canada | Antimicrobial components of the mucus and extruded slime of hagfish (myxine glutinosa) |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000009553A2 (en) * | 1998-08-14 | 2000-02-24 | Bionebraska, Inc. | Antimicrobial peptides isolated from the skin of american frogs |
| WO2009149554A1 (en) * | 2008-06-11 | 2009-12-17 | National Research Council Of Canada | Antimicrobial components of the mucus and extruded slime of hagfish (myxine glutinosa) |
Non-Patent Citations (3)
| Title |
|---|
| 中国大鲵主要病原菌的鉴定及皮肤分泌液抗菌活性成份的分离;袁瑞;中国优秀硕士学位论文全文数据库 农业科技辑(第2期);D052-36 * |
| 大鲵皮肤分泌液中抗菌肽对铜绿假单胞菌感染小鼠创面的抗菌作用;王利锋 等;华西药学杂志;第26卷(第4期);336-339 * |
| 大鲵皮肤分泌液中抗菌肽的鉴定及生物活性研究;王利锋 等;中国生化药物杂志;第32卷(第4期);269-272 * |
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