CN116925204A - Scylla paramamosain antibacterial polypeptide Spgilstin 10-33 And applications thereof - Google Patents
Scylla paramamosain antibacterial polypeptide Spgilstin 10-33 And applications thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- 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
- A23K—FODDER
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- 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
- 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 discloses a scylla paramamosain antibacterial polypeptide Spgilstin 10‑33 And uses thereof, of formula C 118 H 206 N 42 O 30 S, the amino acid sequence of which is shown as SEQ ID NO. 01. The invention has broad-spectrum antibacterial and antifungal activity, high sterilization efficiency, no cytotoxicity and high safety, can be used as an active ingredient to be applied to antibacterial agents, mildew inhibitors and aquatic feed additives, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of marine molecular biology, and in particular relates to scylla paramamosain antibacterial polypeptide Spgilstin 10-33 And applications thereof.
Background
The antibacterial peptide is used as an important immune factor, has good antibacterial effect on various pathogenic bacteria, is different from antibiotics in antibacterial mechanism, is not easy to induce the pathogenic bacteria to generate drug resistance, and becomes an effective substitute of the antibiotics. Marine animals live in complex aquatic environments, such as those rich in environmental microorganisms, harmful pollutants, and water eutrophication pressures, and have evolved unique and efficient immune systems to cope with environmental pressures.
Scylla paramamosain (Scylla paramamosain), abbreviated as blue crab, is an important marine invertebrate crustacean species in China, only has an innate immune system, and is susceptible to pathogen infection due to the fragile stage of the blue crab after multiple molting in the growth and development process, and the antibacterial peptide is an important defense factor in the innate immune system, plays an important role in anti-infective immunity of the blue crab, and is an important source for separating and identifying novel antibacterial peptide.
Disclosure of Invention
The invention aims to provide a scylla paramamosain antibacterial polypeptide Spgilstin 10-33 。
Another object of the present invention is to provide the scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Is used in the application of (a).
The technical scheme of the invention is as follows:
scylla paramamosain antibacterial polypeptide S [ mu ] gilstin 10-33 Its molecular formula is C 118 H 206 N 42 O 30 S, the amino acid sequence of which is shown as SEQ ID NO. 01.
The scylla paramamosain antibacterial polypeptide Spgilstin 10-33 Use in the preparation of an antibacterial composition.
In a preferred embodiment of the present invention, the antibacterial composition has inhibitory and killing effects on staphylococcus epidermidis, micrococcus luteus, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, pseudomonas stutzeri and shigella flexneri.
An antibacterial composition comprises the above scylla paramamosain antibacterial polypeptide Spgilstin as effective component 10-33 。
In a preferred embodiment of the invention, the effective component is the scylla paramamosain antibacterial polypeptide Spgilstin 10-33 。
The scylla paramamosain antibacterial polypeptide Spgilstin 10-33 Use in the preparation of an antimycotic composition.
In a preferred embodiment of the present invention, the mildew-resistant composition has inhibitory and killing effects on Aspergillus niger, fusarium putrescence, fusarium graminearum, and Fusarium oxysporum.
An antifungal composition comprises the above scylla paramamosain antibacterial polypeptide Spgilstin as effective component 10-33 。
In a preferred embodiment of the invention, the effective component is the scylla paramamosain antibacterial polypeptide Spgilstin 10-33 。
An aquatic feed additive comprises the above scylla paramamosain antibacterial polypeptide Spgilstin as effective component 10-33 。
The beneficial effects of the invention are as follows:
1. the scylla paramamosain antibacterial polypeptide Spgilstin 10-33 Consists of 24 amino acids, and has a molecular formula of C 118 H 206 N 42 O 30 S, the molecular weight is 2725.26 daltons, the theoretical isoelectric point is 11.57, the hydrophobicity is 22.1%, and the S is a positive-charge cationic polypeptide.
2. The scylla paramamosain antibacterial polypeptide Spgilstin 10-33 Has broad-spectrum antibacterial activity, and has no cytotoxicity to normal mammalian cells such as human renal epithelial cells and zebra fish embryo cells at high concentration.
3. The scylla paramamosain antibacterial polypeptide Spgilstin 10-33 Has strong antibacterial effect and high sterilization efficiency, and is derived from the first partThe shell animal has no cytotoxicity and high safety, can be used as an active ingredient for antibacterial agents, mildew preventive agents and aquatic feed additives, and has wide application prospect.
Drawings
FIG. 1 shows an antibacterial polypeptide Spgilstin of scylla paramamosain in example 3 of the present invention 10-33 The sterilization kinetics diagram for Acinetobacter baumannii and Pseudomonas stutzeri, wherein the abscissa is time (min) and the ordinate is sterilization index (%).
FIG. 2 shows an antibacterial polypeptide Spgilstin of scylla paramamosain in example 4 of the present invention 10-33 Thermal stability diagram of antibacterial activity against Acinetobacter baumannii and Pseudomonas stutzeri, wherein the abscissa is time (h) and the ordinate is OD 600 Values.
FIG. 3 shows an antibacterial polypeptide Spgilstin of scylla paramamosain in example 5 of the present invention 10-33 Scanning electron microscope observation images of Acinetobacter baumannii and pseudomonas aeruginosa are processed. Wherein A is Pseudomonas aeruginosa, B is Pseudomonas aeruginosa+24 mu M Spgilstin 10-33 C is Acinetobacter baumannii, D is Acinetobacter baumannii+24 mu M Spgilstin 10-33 。
FIG. 4 shows an antibacterial polypeptide Spgilstin of scylla paramamosain in example 6 of the present invention 10-33 Experimental figures for inhibiting germination of spores of Aspergillus niger, fusarium graminearum, fusarium oxysporum, and Fusarium solani (left to right), wherein Spgilstin 10-33 The final concentrations from top to bottom were respectively: 0. Mu.M, 3. Mu.M, 6. Mu.M, 12. Mu.M, 24. Mu.M and 48. Mu.M.
FIG. 5 shows the detection of scylla paramamosain antimicrobial polypeptide Spgilstin by MTS-PMS method in example 7 of the present invention 10-33 Is a cytotoxicity test chart of (2); wherein the abscissa is antibacterial polypeptide Spgilstin 10-33 Is the concentration (. Mu.M), and the ordinate is the cell proliferation rate (%).
Detailed Description
The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.
Example 1 scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Is prepared from
The scylla paramamosain antimicrobial polypeptide Spgilstin of this embodiment 10-33 The amino acid sequence of (2) is:
Lys-Val-Ser-Ile-Ala-Lys-Gln-Asp-Gly-Arg-Leu-Gly-Arg-Val-Ile-Asn-Ala-Cys-Trp-Arg-Arg-Lys-Gly-Ile(SEQ ID NO.01)
the scylla paramamosain antibacterial polypeptide Spgilstin with the purity of more than 95 percent can be obtained by adopting the existing solid-phase chemical synthesis method 10-33 . The scylla paramamosain antimicrobial polypeptide Spgilstin in this embodiment 10-33 The entrusted gold srey biotechnology limited company is synthesized by a solid phase synthesis method and provides detection information such as polypeptide molecular weight, HPLC and the like. Spgilstin 10-33 The physicochemical parameters are shown in Table 1:
TABLE 1 Spgilstin 10-33 Physicochemical parameters of (2)
From Table 1, spgilstin 10-33 Is a cationic polypeptide with positive charges.
Example 2 scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Determination of minimum inhibitory concentration (mInimum inhibition concentration, MIC) and minimum bactericidal concentration (minimum bactericidal concentration, MBC)
The strains involved in this example are: staphylococcus epidermidis (Staphylococcus epidermidis), micrococcus luteus (Micrococcus luteus), acinetobacter baumannii (Acinetobacter baumannii), escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas stutzeri (Pseudomonas stutzeri), shigella flexneri (Shigella flexneri), aspergillus niger (Aspergillus niger), fusarium solani (Fusarium solani), fusarium graminearum (Fusarium graminearum), and Fusarium oxysporum (Fusarium oxysporum). All the strains are purchased from the culture collection of the microbiological institute of China academy of sciences and are preserved and stored in the laboratory.
The specific method comprises the following steps:
(1) Coating the bacteria with seed protection on a nutrient broth plate, and culturing in an inverted mode for 18-24 hours in each incubator with proper temperature; mould is coated on a potato dextrose plate, and the mould is cultured for 1-7d in an incubator at 28 ℃.
(2) Colonies were picked from each plate and inoculated into the corresponding liquid medium and placed in a constant temperature shaking incubator overnight. The bacterial suspension concentration was adjusted with 10mM sodium phosphate buffer (ph=7.4). The bacteria were diluted with MH broth to give a final concentration of 5X 10 cells 5 CFU/mL. Washing fungal spores from the slant with 10mM sodium phosphate buffer (pH=7.4), diluting with a mixture of potato dextrose broth and sodium phosphate buffer, counting the spores under an optical microscope with a hemocytometer, and adjusting the concentration of the fungal spores to give a final concentration of 5×10 fungal spores 4 And each mL.
(3) To synthesize Spgilstin 10-33 The powder was dissolved in sterile MilliQ water, and filtered through a 0.22. Mu.M filter, and the protein concentration was diluted to 1.5. Mu.M, 3. Mu.M, 6. Mu.M, 12. Mu.M, 24. Mu.M, 48. Mu.M, 96. Mu.M by a double ratio, and placed on ice for further use.
(4) On a 96-well cell culture plate, each test bacterium is provided with a blank control group, a negative control group and a test experiment group, and each group is provided with three parallels:
a blank control group: 50. Mu.L of protein sample to be tested and 50. Mu.L of culture medium;
b negative control group: 50. Mu.L of sterile MilliQ water and 50. Mu.L of the bacterial suspension;
c test group: 50. Mu.L of the protein sample to be tested and 50. Mu.L of the bacterial suspension.
(5) Placing the 96-well cell culture plate in a 28 ℃ incubator, culturing for 1-2d, and observing MIC results in the experimental group to be tested; blowing and uniformly mixing the experimental group to be tested, sucking a proper amount of fungus liquid drops on a corresponding solid culture medium plate, inversely culturing for 1-2d at a proper temperature, and observing an MBC result.
Scylla paramamosain antibacterial polypeptide Spgilstin 10-33 Has broad-spectrum antibacterial activity, and has strong bactericidal effect on Pseudomonas stutzeri, shigella flexneri and Fusarium putrescens, and minimum bactericidal concentration<12. Mu.M; has good inhibiting and killing effect on micrococcus luteus, acinetobacter baumannii, pseudomonas aeruginosa, fusarium oxysporum and fusarium graminearum, and the minimum sterilizing concentration is 12-24 mu M; for staphylococcus epidermidis,Escherichia coli and Aspergillus niger also have bactericidal effect, and the minimum bactericidal concentration is<48. Mu.M. The results are shown in Table 2:
table 2 scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Is used as an antimicrobial agent
Note that: MIC: minimum inhibitory concentration (μM), indicated as a-b. a: the highest protein concentration at which the cells grow is visible to the naked eye; b: minimal protein concentration at which the cells grew was not seen with the naked eye. MBC: minimum bactericidal concentration (μm), indicated as a-b. a: the highest protein concentration for visible colony growth on the plate; b: the plates did not see the lowest protein concentration for colony growth.
Example 3 scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Sterilization kinetics curve
In this example, pseudomonas stutzeri and Acinetobacter baumannii are selected as bacteria to be tested, and the scylla paramamosain antibacterial polypeptide Spgilstin is selected 10-33 Is determined by the sterilization kinetics of (2).
The specific procedure is similar to the antibacterial activity assay described in example 2. Adjusting Spgilstin 10-33 The final concentration reaches the sterilization concentration (Pseudomonas stutzeri: 6 mu M; acinetobacter baumannii: 24 mu M), after a certain period of co-incubation with the bacteria to be tested, a proper amount of co-incubation bacterial suspension is diluted, a proper amount of the suspension is coated on a nutrient broth plate, and the culture is carried out for 24 hours at 37 ℃ for clone counting.
As shown in FIG. 1, spgilstin 10-33 The sterilization effect can reach 100% after 60min of incubation with pseudomonas stutzeri, and the sterilization effect can reach 100% after 30min of incubation with acinetobacter baumannii.
Example 4 scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Antimicrobial active thermal stability
In the embodiment, pseudomonas stutzeri and Acinetobacter baumannii are selected as bacteria to be detected,anti-bacterial polypeptide Spgilstin for scylla paramamosain 10-33 The thermal stability of the antibacterial activity was determined.
The specific procedure is similar to the antibacterial activity assay described in example 2. Adjusting Spgilstin 10-33 Final concentration to sterilizing concentration (Pseudomonas stutzeri: 6. Mu.M; acinetobacter schbaumannii: 24. Mu.M), and mixing the antibacterial polypeptide Spgilstin 10-33 Heating in 100deg.C boiling water for 10min, 20min, and 30min, and placing on ice for use. Spgilstin 10-33 Incubating with the bacteria to be detected, and measuring OD (optical density) at 0h, 6h, 12h, 18h and 24h by using an enzyme-labeled instrument 600 As shown in FIG. 2, spgilstin 10-33 The antibacterial activity is still good after the water is subjected to boiling water bath for 30 min.
Example 5 scanning electron microscope observation of Spgilstin 10-33 Morphological structure changes of treated bacteria
In the embodiment, pseudomonas aeruginosa and acinetobacter baumannii are selected as strains to be detected, and the preparation of a scanning electron microscope sample is carried out according to the following steps:
(1) Activating strain, randomly picking 3-5 clones to corresponding nutrient broth liquid culture medium, shaking to logarithmic phase, measuring OD, centrifuging to remove supernatant, re-suspending thallus with MH culture medium, and adjusting to 5×10 7 cfu/mL, placed on ice for use.
(2) Spgilstin 10-33 The powders were dissolved in sterile MilliQ water and filtered through a 0.22. Mu.M filter to adjust the final concentration of polypeptide to 1-fold MBC. An experimental group and a control group are respectively arranged, and after uniform mixing, the mixture is incubated for 1h at 37 ℃. The supernatant was centrifuged off, and after washing with PBS once, the cells were collected.
(2) The collected cells were resuspended with 2.5% glutaraldehyde and fixed at 4℃for 1.5h or more in a refrigerator. Washing the fixed thalli with PBS for three times, preparing high-concentration suspension, dripping the suspension onto a glass slide, adhering for 30min, and carrying out ethanol gradient dehydration after the thalli are adhered.
(3) And (5) performing metal spraying treatment after the critical point is dried, and finally observing and shooting record by using a scanning electron microscope.
As shown in FIG. 3, the bacteria of the control group are normal in morphology, complete in structure and smooth in surface without shrinkage. Antibacterial polypeptide Spgilstin 10-33 Folds appear on the surface of the treated bacteria, the bacterial membrane breaks, and cytoplasmic contents flow out.
Example 6 scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Optical microscopic observation of fungal spore germination after action
In this example, aspergillus niger, fusarium graminearum, fusarium oxysporum and Fusarium solani are selected as the bacteria to be tested, and the scylla paramamosain antimicrobial polypeptide Spgilstin is observed 10-33 Effects on fungal spore germination.
The specific procedure is similar to the antibacterial activity assay described in example 2. Adjusting Spgilstin 10-33 The concentrations were 6. Mu.M, 12. Mu.M, 24. Mu.M and 48. Mu.M, and placed on ice for further use; adjusting the final concentration of each fungal spore to 5×10 4 And each mL. Equal volumes of Spgilstin at various concentrations 10-33 Mixing with each fungus spore in 96-well cell culture plate, setting blank control group, placing in 28 deg.C incubator, standing for 24 hr, and observing fungus spore germination condition by using optical microscope. As shown in FIG. 4, spgilstin 10-33 Can inhibit the germination of Fusarium solani at 6 mu M; can inhibit the germination of Fusarium graminearum and Fusarium oxysporum at 12 μm; aspergillus niger germination was inhibited at 48. Mu.M.
EXAMPLE 7 scylla paramamosain antimicrobial polypeptide Spgilstin 10-33 Cytotoxicity assays
In this example, human kidney epithelial cells (HEK-293T) and zebra fish embryo cells (ZF 4) were selected, and scylla paramamosain antibacterial peptide Spgilstin was selected 10-33 Cytotoxicity was measured.
(1) Collecting embryo cells of Zebra fish and epithelial cells of human kidney in good growth state, counting by cell counting plate, and regulating cell concentration to 1×10 with culture medium 5 The cell suspension is uniformly mixed per mL, 100 mu L of the cell suspension is added into each hole of a 96-hole cell culture plate, and the mixture is placed in a proper temperature incubator for culturing until more than 80% of cells are attached to the wall.
(2) Sucking out the culture medium, adding Spgilstin with different concentration 10-33 Is subjected to stationary culture for 24 hours.
(3) After adding 20 mu LMTS-PMS solution to each well and incubating for 4 hours in dark place, OD is measured by an enzyme-labeled instrument 492 Value, evaluation of Spgilstin 10-33 Is a cell cytotoxicity of (a).
The results are shown in FIG. 5, which shows Spgilstin 10-33 Has no toxic effect on human kidney epithelial cells and zebra fish embryo cells at 96 mu M.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, i.e., the invention is not to be limited to the details of the invention.
Claims (10)
1. Scylla paramamosain antibacterial polypeptide Spgilstin 10-33 The method is characterized in that: its molecular formula is C 118 H 206 N 42 O 30 S, the amino acid sequence of which is shown as SEQ ID NO. 01.
2. The scylla paramamosain antimicrobial polypeptide Spgilstin of claim 1 10-33 Use in the preparation of an antibacterial composition.
3. The use according to claim 2, wherein: the antibacterial composition has inhibiting and killing effects on staphylococcus epidermidis, micrococcus luteus, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, pseudomonas stutzeri and shigella flexneri.
4. An antibacterial composition characterized in that: the effective components of the composition comprise the scylla paramamosain antibacterial polypeptide Spgilstin of claim 1 10-33 。
5. The antibacterial composition of claim 4, wherein: the effective component is the scylla paramamosain antibacterial polypeptide Spgilstin 10-33 。
6. The scylla paramamosain antimicrobial polypeptide Spgilstin of claim 1 10-33 Use in the preparation of an antimycotic composition.
7. The use according to claim 6, wherein: the mildew-proof composition has inhibiting and killing effects on aspergillus niger, fusarium solani, fusarium graminearum and fusarium oxysporum.
8. A mildew-resistant composition, characterized in that: the effective components of the composition comprise the scylla paramamosain antibacterial polypeptide Spgilstin of claim 1 10-33 。
9. A mildew resistant composition according to claim 8, wherein: the effective component is the scylla paramamosain antibacterial polypeptide Spgilstin 10-33 。
10. An aquatic feed additive, characterized in that: the effective components of the composition comprise the scylla paramamosain antibacterial polypeptide Spgilstin of claim 1 10-33 。
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