CN115246876A - Scyrepsin as antibacterial polypeptide of scylla paramamosain 34-55 And uses thereof - Google Patents
Scyrepsin as antibacterial polypeptide of scylla paramamosain 34-55 And uses thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A—HUMAN NECESSITIES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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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
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
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- 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
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Abstract
The invention discloses an Scyrepsin as an antibacterial polypeptide of Scyrepsin 34‑55 And the use thereof, of formula C 123 H 194 N 38 O 25 The amino acid sequence is shown as SEQ ID NO. 01. The invention has broad-spectrum antibacterial and antifungal activity, strong antibacterial effect, high sterilization efficiency, no cytotoxicity and high safety, is derived from crustacean, can be applied to aquatic feed additives, and can also be developed into antibacterial combinationsThe compound, antifungal composition and the like, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of marine molecular biology, and particularly relates to Scyrepsin as an antibacterial polypeptide of Scyrepsin of scylla paramamosain 34-55 And applications thereof.
Background
The scylla paramamosain is an important economic breeding variety in the southeast coast of China. With the annual increase of the cultivation scale of the blue crabs, the cultivation density is continuously increased, so that the disease outbreak is frequent, and the healthy development of the cultivation industry is seriously hindered. In order to control the disease problem in cultivation, a large amount of antibiotics are put into use, but a series of problems such as drug resistance, drug residue, environmental pollution and the like are brought.
Antimicrobial peptides (AMPs) are a class of small polypeptides that are widely found in nature and are an important component of the innate immune system. Blue crabs, as invertebrates, must fight various pathogens through rapid cellular and humoral responses due to the lack of an adaptive immune system, establishing a more diverse innate immune system. The antibacterial peptide is one of the important innate immune factors of the blue crabs and plays an important role in the process of resisting the invasion of foreign pathogens. Therefore, the novel antibacterial polypeptide of the blue crab is further developed, so that scientific basis can be provided for aquatic disease control strategies, and a foundation is laid for developing antibacterial drugs, feed additives, immunomodulators and the like.
Disclosure of Invention
The invention aims to provide an antibacterial polypeptide Scyrepsin of scylla paramamosain 34-55 。
The invention also aims to provide the Scyrepsin as the Scyrepsin polypeptide for inhibiting the bacterial infection of scylla paramamosain 34-55 The use of (1).
The technical scheme of the invention is as follows:
scyrepsin as antibacterial polypeptide of scylla paramamosain 34-55 Of the formula C 123 H 194 N 38 O 25 The amino acid sequence is shown as SEQ ID NO. 01.
The Scyrepsin serving as the antibacterial polypeptide of Scyrepsin 34-55 Use in the preparation of an antibacterial composition.
In a preferred embodiment of the present invention, the antibacterial composition has an inhibiting and killing effect on staphylococcus aureus, corynebacterium glutamicum, micrococcus muralis, enterococcus faecium, enterococcus faecalis, escherichia coli, acinetobacter baumannii, shigella flexneri, pseudomonas aeruginosa, pseudomonas stutzeri, aeromonas hydrophila, and vibrio alginolyticus.
An antibacterial composition, the active ingredient of which comprises the Scyrepsin of the Scyrepsin 34-55 。
The Scyrepsin serving as the antibacterial polypeptide of Scyrepsin 34-55 Use in the preparation of an antifungal composition.
In a preferred embodiment of the present invention, it has inhibitory and bactericidal effects on candida albicans, cryptococcus neoformans, aspergillus niger, aspergillus fumigatus and aspergillus ochraceus.
An antifungal composition, the active ingredient of which comprises the Scyrepsin of the Scyrepsin 34-55 。
The Scyrepsin serving as the antibacterial polypeptide of Scyrepsin 34-55 Application in preparing aquatic feed additive.
In a preferred embodiment of the present invention, it has an inhibitory and bactericidal effect against Aeromonas hydrophila and Vibrio alginolyticus.
An aquatic feed additive, the effective component of which comprises the Scyrepsin as the antibacterial polypeptide of Scyrepsin 34-55 。
The beneficial effects of the invention are:
1. the invention consists of 22 amino acids, and the molecular formula is C 123 H 194 N 38 O 25 The cationic polypeptide has a molecular weight of 2605.13 daltons, a theoretical isoelectric point of 12.31 and a hydrophilic average coefficient of-0.114, is a cationic polypeptide with positive charges, is derived from crustaceans, has no cytotoxicity and high safety, can be applied to aquatic feed additives, can be ground into antibacterial compositions, antifungal compositions and the like, and has wide applicationAnd (4) foreground.
2. The invention has obvious antibacterial effect on bacteria and fungi, and simultaneously has no cytotoxicity to normal mammal cells, human kidney epithelial cells and scylla paramamosain blood cells under high concentration.
3. The invention has broad-spectrum antibacterial and antifungal activity, strong antibacterial effect, quick sterilization efficiency and good antibacterial effect on common aquatic pathogenic bacteria.
Drawings
FIG. 1 shows Scyrepsin, an antibacterial polypeptide of Scyrepsin from Scyreopsis pseudonana in example 3 of the present invention 34-55 Graph of bactericidal kinetics for staphylococcus aureus and acinetobacter baumannii with time (min) on the abscissa and bactericidal index (%) on the ordinate.
FIG. 2 shows Scyrepsin, an antibacterial polypeptide of scylla paramamosain in example 4 of the present invention 34-55 Thermal stability plots of antibacterial activity against Staphylococcus aureus and Acinetobacter baumannii, where the abscissa is time (h) and the ordinate is OD 600 The value is obtained.
FIG. 3 shows Scyrepsin, an antibacterial polypeptide of scylla paramamosain in example 5 of the present invention 34-55 Scanning electron microscope observation images of treated staphylococcus aureus and acinetobacter baumannii. Wherein A is Staphylococcus aureus, and B is Staphylococcus aureus +48 μ M Scyrepsin 34-55 C is Acinetobacter baumannii, D is Acinetobacter baumannii +24 mu M Scyrepsin 34-55 。
FIG. 4 shows Scyrepsin, an antibacterial polypeptide of Scyrepsin from Scyreopsis pseudonana in example 6 of the present invention 34-55 Experimental diagram for inhibiting spore germination of Aspergillus niger, aspergillus fumigatus and Aspergillus ochraceus (from left to right), wherein Scyrepsin 34-55 The final concentrations from top to bottom were: 0. Mu.M, 3. Mu.M, 6. Mu.M, 12. Mu.M and 24. Mu.M.
FIG. 5 shows the MTS-PMS method for detecting Scyrepsin, an antibacterial polypeptide of Scyrepsin, from scylla paramamosain in example 7 of the present invention 34-55 Cytotoxicity test chart of (a); wherein the abscissa is antibacterial polypeptide Scyrepsin 34-55 The concentration (. Mu.M) of (C) in the ordinate represents the cell growth rate (%).
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1 Scyrepsin, an antibacterial polypeptide of Scyrepsin, from Sclerotinia 34-55 Preparation of (2)
The Scyrepsin of the antibacterial polypeptide of Scyrepsin of Scyreopsis pseudonana 34-55 The amino acid sequence of (a) is:
Gly-Val-Val-Trp-Lys-Lys-Gly-Pro-Trp-Arg-Leu-Val-Thr-Phe-Arg-Arg-Ala-Asn-His-Leu-L eu-Ala(SEQ ID NO.01)
the Scyrepsin with the Scyrepsin antibacterial polypeptide of Scyrepsin with the purity of more than 95 percent can be obtained by adopting the existing solid-phase chemical synthesis method 34-55 . The Scyrepsin of the Scyrepsin for antibacterial polypeptide of Scyrepsin in the embodiment 34-55 The molecular weight and HPLC detection information of the polypeptide are obtained and provided by entrusting Kingsry Biotechnology Co., ltd through solid phase synthesis. Scyrepsin 34-55 The physicochemical parameters are shown in table 1:
TABLE 1 Scyrepsin 34-55 Physical and chemical parameters of
From Table 1, scyrepsin is known 34-55 The cationic polypeptide has small molecular weight and good stability, and is a cationic polypeptide with positive charges.
Example 2 Scyrepsin, an antibacterial polypeptide of Scyrepsin, from Sclerotinia 34-55 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
The strains referred to in this example were: staphylococcus aureus (Staphylococcus aureus), micrococcus lysodeiikicus (Micrococcus lysodeiikicus), enterococcus faecium (Enterococcus faecium), enterococcus faecalis (Enterococcus faecium), corynebacterium glutamicum (Corynebacterium glutamicum), escherichia coli (Escherichia coli), shigella flexneri (Shigella flexneri), acinetobacter baumannii (Acinetobacter baumannii), pseudomonas Aeruginosa (Pseudomonas Aeruginosa), pseudomonas stutzeri (Pseudomonas stutzeri), aeromonas hydrophila (Aeromonas hydrophila), pseudomonas fluorescens (Pseudomonas fluorescens), vibrio harveyi (Vibrio harveyi), vibrio bacteriolyticus (Vibrio bacteriolyticus), candida albicans (Candida albicans), aspergillus niger (Aspergillus niger) and Aspergillus niger (Aspergillus niger). The strains are purchased from the strain preservation center of the institute of microbiology, china academy of sciences, and are preserved and stored in the laboratory.
The specific method comprises the following steps:
(1) Coating preserved staphylococcus aureus, micrococcus muralis, enterococcus faecium, enterococcus faecalis, corynebacterium glutamicum, escherichia coli, shigella flexneri, acinetobacter baumannii, pseudomonas aeruginosa, pseudomonas stutzeri, aeromonas hydrophila and pseudomonas fluorescens on a nutrient broth plate, and performing inverted culture at appropriate temperatures for 18-24h; spreading Vibrio harveyi and Vibrio alginolyticus on 2216 plate, and performing inverted culture at 28 deg.C for 18-24h; coating Candida albicans and Cryptococcus neoformans on YPD plate, and performing inversion culture at 28 deg.C for 1-3d; aspergillus niger, aspergillus fumigatus and Aspergillus ochraceus were spread on potato dextrose plates and cultured in an inverted culture at 28 ℃ for 1-7 days.
(2) Colonies from each plate were picked and inoculated on the corresponding media slant and the bacteria were cultured for a further 18-24h. Bacteria were washed off the slant with 10mM sodium phosphate buffer (pH = 7.4) and the suspension concentration was adjusted. The bacteria were diluted with MH liquid medium and the Vibrio was diluted with seawater medium so that the final concentration of the cells was 5X 10 5 CFU/mL. Washing fungal spores from the slant with 10mM sodium phosphate buffer (pH = 7.4), diluting yeast fungi with 1640 medium, diluting filamentous fungal spores with potato dextrose broth and sodium phosphate buffer mixture, counting spores with a light microscope using a hemocytometer, adjusting spore concentration to give a final fungal spore concentration of 5X 10 4 One per mL.
(3) Synthesized Scyrepsin 34-55 The powders were dissolved in sterile MilliQ water, filtered through 0.22. Mu.M filter, and then diluted to 3. Mu.M, 6. Mu.M, 12. Mu.M, 24. Mu.M, 48. Mu.M, and 96. Mu.M protein concentrations by fold, and placed on ice for use.
(4) On a 96-well cell culture plate, each bacterium to be tested is provided with a blank control group, a negative control group and an experimental group to be tested, and each group is provided with three parallels:
a blank control group: 50 mu L of protein sample to be detected and 50 mu L of culture medium;
b negative control group: 50 μ L of sterile MilliQ water and 50 μ L of bacterial suspension;
c test group: 50 μ L of the protein sample to be tested and 50 μ L of the bacterial suspension.
(5) Placing the 96-hole cell culture plate in an incubator at 28 ℃, culturing for 1-2d, and observing the MIC result in the experimental group to be tested; and (3) blowing and uniformly mixing the experimental group to be detected, sucking a proper amount of bacterial liquid, dripping the bacterial liquid on a corresponding solid culture medium flat plate, performing inverted culture at a proper temperature for 1-2d, and observing an MBC result.
Scyrepsin, an antibacterial polypeptide of scylla paramamosain, used in example 1 34-55 Has broad-spectrum antibacterial activity, and has strong antibacterial and bactericidal effects on Corynebacterium glutamicum, micrococcus muralyticus, acinetobacter baumannii, pseudomonas stutzeri, shigella flexneri and Cryptococcus neoformans, and the minimum bactericidal concentration is less than 12 μ M; the bactericidal composition also has good inhibition and killing effects on enterococcus faecium, staphylococcus aureus, escherichia coli, aspergillus fumigatus and aspergillus ochraceus, and the minimum bactericidal concentration is 12-24 mu M; has certain inhibiting and killing effects on enterococcus faecalis, pseudomonas aeruginosa, aeromonas hydrophila, vibrio alginolyticus and candida albicans, and the minimum bactericidal concentration is 24-48 mu M. The results are shown in table 2:
TABLE 2 Scyrepsin, an antibacterial polypeptide of Scyrepsin from Sclerotinia punctata 34-55 Antibacterial activity of
Note: MIC: the minimum inhibitory concentration (. Mu.M), indicated as a-b. a: the highest protein concentration of the thallus growth can be seen by naked eyes; b: the lowest protein concentration for the growth of the cells was not seen visually. MBC: minimum bactericidal concentration (. Mu.M), indicated as a-b. a: the highest protein concentration for the growth of the visible colony on the plate; b: the lowest protein concentration at which colonies grew was not seen in the plates.
Example 3 Scyrepsin, an antibacterial polypeptide of Scyrepsin, scyrepsin 34-55 Sterilization kinetics curve
In the embodiment, staphylococcus aureus and acinetobacter baumannii are selected as bacteria to be detected to carry out Scyrepsin on the antibacterial polypeptide of Scyrepsin of Sclerotinia punctata 34-55 The bactericidal kinetics of (a) were determined.
The specific method was similar to the antimicrobial activity assay described in example 2. Regulation of Scyrepsin 34-55 Final concentration 1-fold MBC (Staphylococcus aureus: 24. Mu.M; acinetobacter baumannii: 12. Mu.M), scyrepsin 34-55 After incubating with the tested bacteria for a certain period of time, diluting 6 μ L of bacterial suspension, coating a proper amount of diluted bacterial suspension on a nutrient broth plate, and culturing at 37 deg.C for 1-2d for clone counting. The samples incubated with sterile MilliQ water and bacterial suspension for 0h were used as positive control, 6. Mu.L of clones plated on nutrient broth plates at the same dilution was defined as 100%, and the bactericidal index was expressed as the percentage of the number of clones in the experimental group incubated for a certain period of time relative to the number of clones in the positive control, as shown in FIG. 1, and Scyrepsin 34-55 Can kill staphylococcus aureus within 10min, and kill acinetobacter baumannii within 30 min.
Example 4 Scyrepsin, an antibacterial polypeptide of Scyrepsin, from Sclerotinia 34-55 Antimicrobial active thermal stability
In the embodiment, staphylococcus aureus and acinetobacter baumannii are selected as bacteria to be detected to carry out Scyrepsin on the antibacterial polypeptide of Scyrepsin of Sclerotinia punctata 34-55 The heat stability of the antibacterial activity was measured.
The specific method was similar to the antimicrobial activity assay described in example 2. Regulation of Scyrepsin 34-55 To a final concentration of 1-fold MBC (Staphylococcus aureus: 24. Mu.M; acinetobacter baumannii: 12. Mu.M), the antimicrobial polypeptide Scyrepsin was added 34-55 Heating in boiling water at 100 deg.C for 10min, 20min, and 30min, and placing on ice for use. Scyrepsin (a) 34-55 Incubating with bacteria to be tested, and measuring OD with enzyme labeling instrument at 0h, 12h, 24h, 36h, and 48h 600 Value of (c), as shown in FIG. 2, scyrepsin 34-55 Through boiling water bathGood antibacterial activity is still kept after 30 min.
Example 5 SEM Observation of Scyrepsin 34-55 Morphological structural change of bacteria after treatment
In the embodiment, staphylococcus aureus and acinetobacter baumannii are selected as strains to be detected, and the preparation of the scanning electron microscope sample is carried out according to the following steps:
(1) Activating strains, randomly selecting 3-5 clones to corresponding nutrient broth liquid culture medium when the clones grow to a proper size, shaking to logarithmic growth phase, measuring OD, centrifuging to remove supernatant, resuspending the strains by using MH culture medium, adjusting OD to 0.1, and placing on ice for later use.
(2) The polypeptide powder was solubilized with sterile MilliQ and filtered through a 0.22 μ M filter to adjust the final polypeptide concentration to 2-fold MBC. Respectively setting an experimental group and a control group, uniformly mixing, and then incubating for 1h at 37 ℃. The supernatant was centrifuged off, and the cells were collected after washing once with PBS.
(2) The collected cells were resuspended with 2.5% glutaraldehyde and fixed in a freezer at 4 ℃ for 1.5h or more. After the fixed thalli is washed for three times by PBS, high-concentration suspension is prepared, the suspension is dripped on a cut glass slide, the adhesion is carried out for 30min, and ethanol gradient dehydration is carried out after the filtration is carried out by using filter paper.
(3) 30% -50% -70% -80% -90% -95% -100% (v/v) ethanol is dehydrated step by step, and each step of dehydration lasts for 10-15min.
(4) Drying the critical point, then spraying gold for 60s at 10mA current; observing by a scanning electron microscope and shooting and recording.
The results are shown in FIG. 3, where the control group had a normal bacterial morphology, intact structure, smooth surface and no shrinkage. Antimicrobial polypeptide Scyrepsin 34-55 The treated bacteria have obvious change of the shape, collapse and fold on the surface, cell rupture, leakage of intracellular substances and bubble-shaped bulge on the surface of part of the bacteria.
Example 6 Scyrepsin, an antibacterial polypeptide of Scyrepsin, from Sclerotinia 34-55 Optical microscope observation of post-operative fungal spore germination
In the embodiment, aspergillus fumigatus, aspergillus ochraceus and aspergillus niger are selected as bacteria to be detected, and Scyrepsin, an antibacterial polypeptide of Scyrepsin, of scylla paramamosain, is observed 34-55 Against germination of fungal sporesThe influence of hair.
The specific procedure was similar to the antimicrobial activity assay described in example 2. Regulation of Scyrepsin 34-55 The concentration is 6 muM, 12 muM, 24 muM and 48 muM, and the mixture is put on ice for standby; adjusting the final concentration of each fungal spore to 5 × 10 4 One per mL. Equal volumes of each concentration of Scyrepsin 34-55 Mixing with each fungal spore in 96-well cell culture plate, setting blank control group, placing in 28 deg.C incubator, standing and culturing for 24 hr, and observing fungal spore germination under optical microscope. As shown in fig. 4, scyrepsin 34-55 At a concentration of 12. Mu.M, germination of Aspergillus niger, aspergillus fumigatus and Aspergillus ochraceus was inhibited.
Example 7 Scyrepsin, an antibacterial polypeptide of Scyrepsin, scyrepsin 34-55 Cytotoxicity assays
In the embodiment, human kidney epithelial cells (HEK-293T) and Scyrepsin, which is an antibacterial peptide for Scyrepsin, are selected from normal blood cells of scylla paramamosain 34-55 Cytotoxicity was measured.
(1) Collecting Eriocheir sinensis blood cells and human renal epithelial cells with good growth state, counting with cell counting plate, and adjusting cell concentration to 1 × 10 with culture medium 5 And (4) mixing the cell suspensions per mL, adding 100 mu L of cell suspension into each hole of a 96-hole cell culture plate, and placing the cell suspension in an incubator at a proper temperature to culture the cells until more than 80% of the cells adhere to the wall.
(2) Carefully aspirate the medium, add 100 μ L of the antimicrobial polypeptide diluted with medium gradient to concentrations of 3, 6, 12, 24, 48, 96 μ M, and incubate at the appropriate temperature for 24h.
(3) Adding 20 mu L of MTS-PMS solution into each well, incubating for 2h in dark place, and measuring OD by using an enzyme-linked immunosorbent assay 492 Value, evaluation of Scyrepsin 34-55 The cytotoxicity of (4).
The results are shown in FIG. 5, scyrepsin at concentrations up to 96 μ M 34-55 Has no toxic effect on human kidney epithelial cells and scylla paramamosain blood cells.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Sequence listing
<110> university of mansion
Xiamen Jiacheng Biotechnology Co.,Ltd.
<120>Scyrepsin as antibacterial polypeptide of scylla paramamosain 34-55 And applications thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213> Scylla Paramosain)
<400> 1
Gly Val Val Thr Leu Leu Gly Pro Thr Ala Leu Val Thr Pro Ala Ala
1 5 10 15
Ala Ala His Leu Leu Ala
20
Claims (10)
1. Scyrepsin as antibacterial polypeptide of scylla paramamosain 34-55 The method is characterized in that: having a molecular formula of C 123 H 194 N 38 O 25 The amino acid sequence is shown in SEQ ID NO. 01.
2. The Scyrepsin as defined in claim 1, wherein the Scyrepsin is an antibacterial polypeptide of Scyrepsin 34-55 Use in the preparation of an antibacterial composition.
3. An antibacterial composition characterized by: the effective component of the Scyrepsin comprises the Scyrepsin as the antibacterial polypeptide of scylla paramamosain of claim 1 34-55 。
4. An antibacterial composition according to claim 3, wherein: it has inhibiting and killing effects on Staphylococcus aureus, corynebacterium glutamicum, micrococcus muralyticus, enterococcus faecium, enterococcus faecalis, escherichia coli, acinetobacter baumannii, shigella flexneri, pseudomonas aeruginosa, pseudomonas stutzeri, aeromonas hydrophila and Vibrio alginolyticus.
5. The Scyrepsin as defined in claim 1, wherein the Scyrepsin is an antibacterial polypeptide of Scyrepsin 34-55 Use in the preparation of an antifungal composition.
6. An antifungal composition, characterized by: the effective component of the Scyrepsin comprises the Scyrepsin as the antibacterial polypeptide of scylla paramamosain of claim 1 34-55 。
7. An antifungal composition according to claim 6, wherein: it has inhibiting and killing effects on Candida albicans, cryptococcus neoformans, aspergillus niger, aspergillus fumigatus and Aspergillus ochraceus.
8. The Scyrepsin as an antibacterial polypeptide of scylla paramamosain of claim 1 34-55 Application in aquatic feed additive.
9. An aquatic feed additive, which is characterized in that: the effective component of the Scyrepsin comprises the Scyrepsin as the antibacterial polypeptide of Scyrepsin as claimed in claim 1 34-55 。
10. An aquaculture feed additive according to claim 8 wherein: it has inhibiting and killing effects on Aeromonas hydrophila and Vibrio alginolyticus.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110028568A (en) * | 2019-03-11 | 2019-07-19 | 厦门大学 | A kind of Scylla paramamosain antibacterial polypeptide Sp-NPFin and its application |
| WO2019241938A1 (en) * | 2018-06-20 | 2019-12-26 | 厦门大学 | Scylla paramamosain antibacterial peptide sparamosin and application thereof |
| CN112159460A (en) * | 2020-09-29 | 2021-01-01 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamprin4-23And uses thereof |
| CN113307855A (en) * | 2021-06-25 | 2021-08-27 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Spampcin56-86And uses thereof |
| CN114014923A (en) * | 2020-10-27 | 2022-02-08 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Sp-LECin and application thereof |
-
2022
- 2022-06-09 CN CN202210649799.1A patent/CN115246876A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019241938A1 (en) * | 2018-06-20 | 2019-12-26 | 厦门大学 | Scylla paramamosain antibacterial peptide sparamosin and application thereof |
| CN110028568A (en) * | 2019-03-11 | 2019-07-19 | 厦门大学 | A kind of Scylla paramamosain antibacterial polypeptide Sp-NPFin and its application |
| CN112159460A (en) * | 2020-09-29 | 2021-01-01 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamprin4-23And uses thereof |
| CN114014923A (en) * | 2020-10-27 | 2022-02-08 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Sp-LECin and application thereof |
| CN113307855A (en) * | 2021-06-25 | 2021-08-27 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Spampcin56-86And uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王晓飞;彭会;陈芳奕;张财亮;黄文树;: "拟穴青蟹抗菌肽Crustin新变体的表达特性与抗菌功能", 厦门大学学报(自然科学版), no. 03, 14 November 2018 (2018-11-14), pages 68 - 75 * |
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