CN111658761A - Application of natural host defense peptide Cm-CATH2 - Google Patents
Application of natural host defense peptide Cm-CATH2 Download PDFInfo
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- CN111658761A CN111658761A CN202010566319.6A CN202010566319A CN111658761A CN 111658761 A CN111658761 A CN 111658761A CN 202010566319 A CN202010566319 A CN 202010566319A CN 111658761 A CN111658761 A CN 111658761A
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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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
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- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A61P31/04—Antibacterial agents
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Abstract
The invention relates to application of a natural host defense peptide Cm-CATH2, in particular to application of the natural host defense peptide Cm-CATH2 in preparation of an anti-aquatic pathogenic bacterium medicament and an application in preparation of an aquaculture animal immunomodulator. The invention further discloses application of the natural host defense peptide Cm-CATH2 in preparation of aquaculture animal feed. The research result of taking the largemouth bass as an experimental animal shows that the Cm-CATH2 can kill various common aquatic pathogenic bacteria, regulate the immune function of the largemouth bass, improve the resistance of the largemouth bass to bacterial infection and improve the survival rate of the largemouth bass. Cm-CATH2 has wide application prospect in the field of aquaculture.
Description
Technical Field
The invention relates to the field of polypeptide application, in particular to application of a natural host defense peptide Cm-CATH 2.
Background
Antibiotics refer to chemical substances produced by microorganisms that are capable of inhibiting or killing other microorganisms at low concentrations. Antibiotics have played an important role in the development of the breeding industry because of their effects of promoting animal growth, increasing feed utilization, and preventing and treating animal epidemic diseases.
However, with the extensive use of antibiotics in the aquaculture industry, especially unscientific abuse, problems of bacterial resistance and drug residues are becoming more prominent. Due to the problems of overproof antibiotic residues and the like, the export quantity of the products of the breeding industry in China only accounts for 0.9 to 1.2 percent of the production quantity, and the development of the economy of the breeding industry in China is seriously influenced. Therefore, the development of novel antibiotic substitute drugs with high antibacterial and anti-infection effects and no residue is increasingly important.
CN 106188265A discloses an antimicrobial peptide Cm-CATH2 and a gene, a preparation method and application thereof, wherein Cm-CATH2 has strong antibacterial activity on gram-negative bacteria, gram-positive bacteria and fungi, comprises various clinical drug-resistant bacteria, and has good application prospects in daily chemicals such as cosmetics, health products, foods, feeds and the like. The value of the polypeptide can be more fully exerted by developing the application of Cm-CATH2 in other fields.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide application of a natural host defense peptide Cm-CATH2, and discloses an antibacterial effect of Cm-CATH2 on aquatic pathogenic bacteria and an immunoregulation effect on aquatic animals, which can be used as an aquatic animal feed in the field of aquaculture.
The invention discloses application of a natural host defense peptide Cm-CATH2 in preparing a medicament for resisting aquatic pathogenic bacteria, wherein the amino acid sequence of Cm-CATH2 is shown as SEQ ID No. 1.
Further, the aquatic pathogenic bacteria include one or more of Vibrio alginolyticus, Vibrio anguillarum, Vibrio Brazilian, Vibrio cholerae, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio lautus, Vibrio vulnificus, Aeromonas hydrophila, Aeromonas sobria and Aeromonas veronii.
Further, the minimum inhibitory concentration of Cm-CATH2 to aquatic pathogenic bacteria is 2.34-18.75 mug/mL.
The second purpose of the invention is to disclose the application of natural host defense peptide Cm-CATH2 in the preparation of an immunomodulator for aquaculture animals, wherein the amino acid sequence of Cm-CATH2 is shown as SEQ ID No. 1.
Further, the immunomodulator is used for treating inflammation caused by aquatic pathogenic bacteria.
Further, the immunomodulator is used for inhibiting the gene expression of proinflammatory factors TNF-alpha and IL-1 beta caused by aquatic pathogenic bacteria infection in an aquaculture animal body and improving the expression level of a cell chemotactic factor IL-8 gene.
Further, the dose of the immunomodulator is 1-100 mg/kg. Preferably, the dose of immunomodulator is 10-20 mg/kg.
Further, the aquaculture animal is a fish. Preferably, the aquaculture animal is largemouth bass, grass carp, silver carp, bighead carp, black carp, mandarin fish, flounder, turbot, sciaenops ocellatus or grouper.
Preferably, the inflammation is caused by one or more of the aquatic pathogenic bacteria Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio cholerae, Vibrio lautus and Aeromonas hydrophila.
The third purpose of the invention is to disclose the application of natural host defense peptide Cm-CATH2 in preparing aquaculture animal feed, wherein the amino acid sequence of Cm-CATH2 is shown as SEQ ID No. 1.
Further, the aquaculture animal feed is used for combating aquatic pathogenic bacteria and/or modulating immune function of the aquaculture animal.
Further, the aquaculture animal is a fish. Preferably, the aquaculture animal is Lateolabrax japonicus, grass carp, silver carp, bighead carp, black carp, mandarin fish, Paralichthys olivaceus, Scophthalmus maximus, sciaenops ocellatus and Epinephelus rockfish.
In the present invention, the natural host defense peptide Cm-CATH2 is derived from the reptile green turtle.
By the scheme, the invention at least has the following advantages:
the invention discloses a natural host defense peptide Cm-CATH2 which has broad-spectrum and efficient antibacterial action on common aquatic pathogenic bacteria, has rapid bactericidal action, can regulate the immune function of an aquaculture animal, improve the resistance of the aquaculture animal to bacterial infection, improve the survival rate of the aquaculture animal and has wide application prospect in the field of aquaculture.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
FIG. 1 is a graph showing the effect of Cm-CATH2 on spleen cytokine expression in micropterus salmoides;
FIG. 2 is a graph showing the effect of Cm-CATH2 on survival rate after bacterial infection of Lateolabrax japonicus.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 Cm-CATH2 preparation
(1) The complete sequence derived from reptile green turtle Cm-CATH2 was synthesized by an automated polypeptide synthesizer (433A, Applied Biosystems), and desalted and purified by HPLC reverse phase column chromatography.
(2) And the molecular weight is measured by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF).
(3) And the purity of the purified Cm-CATH2 is determined by a High Performance Liquid Chromatography (HPLC) method, the molecular weight is determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), isoelectric point is determined by isoelectric focusing electrophoresis, and the amino acid sequence structure is determined by an automatic amino acid sequencer.
The amino acid sequence of the natural host defense peptide Cm-CATH2 is shown in SEQ ID No. 1. It consists of 33 amino acids, has molecular weight of 4089.9Da and isoelectric point of 12.96, wherein all amino acids are L-type.
Example 2 detection of the antibacterial Activity of Cm-CATH2 against common aquatic pathogenic bacteria
(1) Respectively picking test strains (aquatic pathogenic bacteria) preserved on the inclined plane, uniformly coating the test strains on a nutrient broth solid culture medium (OXOID company, UK) plate, placing a sterilized filter paper sheet with the diameter of 0.5Cm on the surface of the culture medium, dropwise adding 10 mu L of Cm-CATH2 sample solution dissolved in 2mg/mL of sterilized deionized water, culturing at 37 ℃ for 18-20 hours in an inverted mode, and observing whether a bacteriostatic zone is formed or not. If the sample has antibacterial activity, clear and transparent inhibition zones can be formed around the filter paper sheet, and the larger the inhibition zone is, the stronger the antibacterial activity of the sample is.
(2) Cm-CATH2 Minimum Inhibitory Concentration (Minimum inhibition Concentration) assay (2-fold dilution method):
the test strains were inoculated into NB broth (OXOID, UK), cultured with shaking at 37 ℃ until logarithmic phase, and then the culture broth cultured to logarithmic phase was diluted to 2 × 10 with fresh NB broth5cfu/mL for standby.
According to the dilution method in Table 1, 100 μ L of liquid culture medium was added to each well of a sterile 96-well plate, 100 μ L of Cm-CATH2 sample solution diluted to a certain concentration with NB liquid culture medium and filtered through a 0.22 μm-well filter was added to the first well, 100 μ L of the mixed solution was added to the second well, and the mixed solution was diluted in duplicate (see Table 1), 100 μ L of the mixed solution was aspirated from the second well and discarded, and the 10 th well was a control tube containing a positive control neomycin sulfate.
TABLE 1 dilution method
The 96-well plate was incubated at 37 ℃ for 18 hours with slow shaking, and the light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration was the lowest sample concentration at which no bacterial growth was seen, and the results are shown in table 2.
TABLE 2 Cm-CATH2 antibacterial Activity against aquatic pathogenic bacteria
As can be seen from the table 2, Cm-CATH2 shows extremely strong antibacterial activity to aquatic pathogenic bacteria in the table 2, and the MIC value is in the range of 2.34-18.75 mug/mL, which is equivalent to the antibacterial activity of positive control neomycin sulfate.
EXAMPLE 3 Cm-CATH2 determination of Sterilization Rate
Vibrio parahaemolyticus was cultured in NB broth (OXOID, UK) at 37 ℃ for 12 hours, and then diluted to 10 with fresh NB broth6Adding a Cm-CATH2 sample dissolved in sterilized deionized water into the bacterial suspension to enable the final concentration to be 5 × MIC, placing bacterial liquid added with the Cm-CATH2 sample into an incubator at 37 ℃, carrying out shake culture, respectively taking 50 mu L of bacterial liquid to dilute 1000 times in 0, 15, 30, 60, 120 and 180 minutes, then taking 50 mu L of the diluted bacterial liquid to coat on an NB solid culture medium plate, and counting bacterial colonies after the incubator at 37 ℃ is cultured overnight.
As shown in Table 3, Cm-CATH2 showed rapid bactericidal action against Vibrio parahaemolyticus, killing all bacteria within 15 minutes, faster than that of the positive control neomycin sulfate.
TABLE 3 Cm-CATH2 Sterilization rates for Vibrio parahaemolyticus
Example 4 Cm-CATH2 assay for Micropterus salmoides immune System modulating Activity
The micropterus salmoides used in the following examples were purchased from Wujiang farms, Suzhou, and after 1 week of temporary rearing in the laboratory, healthy individuals (1-2 g/individual) of uniform size were randomly selected and group-bred in a sort box, with 20 individuals per group. The experiment was performed in 2 groups: bacterial infection group, 10mg/kg Cm-CATH2 group.
Vibrio parahaemolyticus was cultured in NB liquid medium (OXOID, UK) at 37 ℃ for 12 hours, washed 2 times with PBS and diluted to 1 × 108CFU/mL of bacterial suspension. Perch micropterus salmoides were intraperitoneally injected with the diluted bacterial suspension (20. mu.L/strip) and immediately after the injection of the bacteria, the polypeptide sample (10mg/kg) dissolved in PBS was injected. 3 Perciformes were randomly selected from each group at 0, 6, 12, and 24 hours, spleen tissue was taken after sacrifice, total RNA of tissue was extracted with TRIzol reagent (Life Technology, USA), cDNA single Strand was synthesized by reverse transcription with PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Japan), and SYBR Premix Ex Taq was usedTMII (TliRNaseH plus) two-step qRT-PCR kit (Takara, Japan) to detect the expression of the pro-inflammatory factors TNF- α, IL-1 β and the cell chemotactic factor IL-8. the β -actin gene was used as an internal reference, the primers used are shown in Table 4:
TABLE 4 qRT-PCR primers
The results are shown in figure 1, 10mg/kg Cm-CATH2 can obviously inhibit the gene expression of proinflammatory factors TNF-alpha and IL-1 beta caused by vibrio parahaemolyticus infection in a largemouth bass 6 and 12 hours after bacterial infection, and the Cm-CATH2 has better in-vivo anti-inflammatory activity. Meanwhile, compared with a bacterial infection group, the expression level of a cell chemotactic factor IL-8 gene in the largemouth bass is remarkably improved by 10mg/kg Cm-CATH2, which shows that the Cm-CATH2 has a better immunoregulation effect, and can regulate chemotaxis of immune cells to an infected part by inducing the expression of the cell chemotactic factor, thereby playing an anti-infection effect.
Example 5 Effect of Cm-CATH2 on survival rates following bacterial infection of Perch Perciformis
The micropterus salmoides used in the following examples were purchased from Wujiang farms, Suzhou, and after 1 week of temporary rearing in the laboratory, healthy individuals (1-2 g/individual) of uniform size were randomly selected and group-bred in a sort box, with 20 individuals per group. The experiment was performed in 3 groups: blank group, bacterial infection group, 10mg/kg Cm-CATH2 group.
Vibrio parahaemolyticus was cultured in NB liquid medium (OXOID, UK) at 37 ℃ for 12 hours, washed twice with PBS buffer and resuspended to 1 × 108CFU/mL of bacterial suspension is ready for use. Perch micropterus salmoides were intraperitoneally injected with the diluted bacterial suspension (40. mu.L/strip) and immediately after the injection of the bacteria, the polypeptide sample (10mg/kg) dissolved in PBS was injected. After the injection is finished, the largemouth black bass is kept for 96 hours temporarily, the vitality is observed, and the final survival rate is calculated.
The result is shown in figure 2, Cm-CATH2 can provide effective immunoprotection for micropterus salmoides. The survival rate of the largemouth bass after bacterial infection can be obviously improved by injecting 10mg/kg of Cm-CATH 2.
In conclusion, the research result of the invention using the micropterus salmoides as experimental animals shows that the Cm-CATH2 can kill various common aquatic pathogenic bacteria, regulate the immune function of the micropterus salmoides, improve the resistance of the micropterus salmoides to bacterial infection and improve the survival rate of the micropterus salmoides. Cm-CATH2 has wide application prospect in the field of aquaculture.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Suzhou university
<120> use of native host defense peptide Cm-CATH2
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>33
<212>PRT
<213> (Artificial sequence)
<400>1
Arg Arg Ser Arg Phe Gly Arg Phe Phe Lys Lys Val Arg Lys Gln Leu
1 5 10 15
Gly Arg Val Leu Arg His Ser Arg Ile Thr Val Gly Gly Arg Met Arg
20 25 30
Phe
Claims (10)
1. The application of the natural host defense peptide Cm-CATH2 in preparing the medicine for resisting aquatic pathogenic bacteria is disclosed, wherein the amino acid sequence of Cm-CATH2 is shown as SEQ ID No. 1.
2. Use according to claim 1, characterized in that: the aquatic pathogenic bacteria include one or more of Vibrio alginolyticus, Vibrio anguillarum, Vibrio Brazilian, Vibrio cholerae, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio splendidus, Vibrio vulnificus, Aeromonas hydrophila, Aeromonas sobria and Aeromonas veronii.
3. Use according to claim 1, characterized in that: the minimum inhibitory concentration of Cm-CATH2 to aquatic pathogenic bacteria is 2.34-18.75 mug/mL.
4. The application of a natural host defense peptide Cm-CATH2 in preparing an immunomodulator for an aquaculture animal, wherein the amino acid sequence of the Cm-CATH2 is shown as SEQ ID No. 1.
5. Use according to claim 4, characterized in that: the immunomodulator is used for treating inflammation caused by aquatic pathogenic bacteria.
6. Use according to claim 4, characterized in that: the dose of the immunomodulator is 1-100 mg/kg.
7. Use according to claim 4, characterized in that: the aquaculture animal is fish.
8. The application of natural host defense peptide Cm-CATH2 in preparing aquaculture animal feed, wherein the amino acid sequence of Cm-CATH2 is shown as SEQ ID No. 1.
9. Use according to claim 8, characterized in that: the aquaculture animal feed is used for resisting aquatic pathogenic bacteria and/or regulating the immune function of aquaculture animals.
10. Use according to claim 8, characterized in that: the aquaculture animal is fish.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112450355A (en) * | 2020-11-23 | 2021-03-09 | 大连工业大学 | Application and application of antibacterial peptide Cm-CATH2 in inhibition of vibrio parahaemolyticus in marine products |
| CN112493312A (en) * | 2020-11-23 | 2021-03-16 | 大连工业大学 | Application and application of antibacterial peptide Cm-CATH2 in prevention and control of Fusarium meyeri in grains |
| CN112625107A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM8 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN112625108A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM5 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN112625109A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM6 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN113735956A (en) * | 2021-07-20 | 2021-12-03 | 苏州市第九人民医院 | Antibacterial peptide CCM7WC, and preparation method and application thereof |
| CN113999297A (en) * | 2021-07-20 | 2022-02-01 | 苏州市第九人民医院 | Antibacterial peptide hrNCM and preparation method and application thereof |
| CN114853865A (en) * | 2022-04-29 | 2022-08-05 | 苏州大学 | Modified antibacterial peptide dsNCM1 and application thereof |
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| CN112450355A (en) * | 2020-11-23 | 2021-03-09 | 大连工业大学 | Application and application of antibacterial peptide Cm-CATH2 in inhibition of vibrio parahaemolyticus in marine products |
| CN112493312A (en) * | 2020-11-23 | 2021-03-16 | 大连工业大学 | Application and application of antibacterial peptide Cm-CATH2 in prevention and control of Fusarium meyeri in grains |
| CN112625107A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM8 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN112625108A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM5 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN112625109A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM6 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN113735956A (en) * | 2021-07-20 | 2021-12-03 | 苏州市第九人民医院 | Antibacterial peptide CCM7WC, and preparation method and application thereof |
| CN113999297A (en) * | 2021-07-20 | 2022-02-01 | 苏州市第九人民医院 | Antibacterial peptide hrNCM and preparation method and application thereof |
| CN113999297B (en) * | 2021-07-20 | 2023-06-06 | 苏州市第九人民医院 | Antibacterial peptide hrNCM and preparation method and application thereof |
| CN114853865A (en) * | 2022-04-29 | 2022-08-05 | 苏州大学 | Modified antibacterial peptide dsNCM1 and application thereof |
| CN114853865B (en) * | 2022-04-29 | 2024-03-15 | 苏州大学 | Modified antibacterial peptide dsNCM1 and application thereof |
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