CN107252475B - The application of natural host defense peptide Alligatorin4 - Google Patents
The application of natural host defense peptide Alligatorin4 Download PDFInfo
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- CN107252475B CN107252475B CN201710383361.2A CN201710383361A CN107252475B CN 107252475 B CN107252475 B CN 107252475B CN 201710383361 A CN201710383361 A CN 201710383361A CN 107252475 B CN107252475 B CN 107252475B
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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
- 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
- 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
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The present invention relates to a kind of natural host defense peptide Alligatorin4 to prepare the application in anti-aquatic pathogenic bacteria drug.The invention also discloses natural host defense peptide Alligatorin4 to prepare the application in aquiculture animal immunoactivator.The present invention further discloses natural host defense peptide Alligatorin4 to prepare the application in aquiculture animal feed.Show that Alligatorin4 can enhance steamed crab immune function using steamed crab as the result of study of experimental animal, improves steamed crab to the resistance of bacterium infection, improve the survival rate of steamed crab.Alligatorin4 is with a wide range of applications in aquaculture field.
Description
Technical field
The present invention relates to answering for field of biomedicine technology more particularly to a kind of natural host defense peptide Alligatorin4
With.
Background technique
China is aquaculture big producer, and birds, beasts and eggs, meat and aquatic products total amount rank first in the world for years, and the gross output value reaches
To more than 10,000 hundred million yuan.Antibiotic refers to the chemistry for being able to suppress or killing other microorganisms at low concentrations generated by microorganism
Substance.Antibiotic is due to having the function of promoting growth of animal, improving efficiency of feed utilization, prevention and treatment animal epidemic, Zeng
Important function has been played in aquaculture development.
But a large amount of uses with antibiotic in aquaculture, especially unscientific abuse, bacterial drug resistance and medicine
The problems such as object remains becomes increasingly conspicuous.The problems such as exceeded due to antibiotic residue, aquaculture industry of China products export amount only account for output
0.9%-1.2%, seriously affect aquaculture industry of China expanding economy.Therefore new and effective antibacterial anti-infection, noresidue are researched and developed
New antibiotic alternative medicine become more and more important.
Host defense peptide (Host defense peptides, HDPs) be in animal body one kind encoded by gene it is natural
Micromolecule polypeptide has the effects that resist external microbe invasion and adjusts host immune response.
Summary of the invention
In order to solve the above technical problems, the object of the present invention is to provide a kind of natural host defense peptide Alligatorin4's
Using, the invention discloses antibacterial action of the Alligatorin4 to aquatic pathogenic bacteria, the immunological enhancement to aquatic livestock,
And its application in aquaculture field.
On the one hand, the present invention provides natural host defense peptide Alligatorin4 to prepare anti-aquatic pathogenic bacteria drug
In application.
Further, pathogenic bacteria are Aeromonas sobria, Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi, pair
One or more of hemolysis vibrion, Vibrio anguillarum, Vibrio vulnificus, Vibrio splindidus and comma bacillus.
On the other hand, the invention also discloses natural host defense peptide Alligatorin4 to prepare aquiculture animal
Application in immunoactivator.
It yet still another aspect, the invention also discloses natural host defense peptide Alligatorin4 to prepare aquiculture animal
Application in feed.
Further, the amino acid sequence of natural host defense peptide Alligatorin4 is as shown in SEQ ID No.1.
Further, natural host defense peptide Alligatorin4 derives from reptile Chinese alligator.
According to the above aspect of the present invention, the present invention has at least the following advantages:
Natural host defense peptide Alligatorin4 in the present invention there is broad-spectrum high efficacy to resist common aquatic pathogenic bacteria
Bacterium effect, bactericidal effect is rapid, can enhance aquiculture animal immune function, improves aquiculture animal to bacterium infection
Resistance improves the survival rate of aquiculture animal, is with a wide range of applications in aquaculture field.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
Influence Fig. 1 illustrates Alligatorin4 to steamed crab serum acid phosphatase and alkaline phosphatase activities；
Fig. 2 illustrates influence of the Alligatorin4 to survival rate after the infection of steamed crab vibrio parahaemolytious.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not intended to limit the scope of the invention for illustrating the present invention.
The preparation of 1 Alligatorin4 of embodiment
(1), with automatic Peptide synthesizer (433A, Applied Biosystems) synthesis source in reptile Yangtze
The complete sequence of crocodile Alligatorin4 passes through HPLC reversed phase column chromatography desalting and purifying.
(2), molecular weight determination uses Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF).
(3), the Alligatorin4 purified identifies its purity with high-efficient liquid phase chromatogram HPLC method, and molecular weight determination uses
Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF), isoelectric focusing electrophoresis measures isoelectric point, with automatic amino
Sour sequenator measures amino acid sequence structure.
The amino acid sequence of natural host defense peptide Alligatorin4 of the invention is as shown in SEQ ID No.1.Its by
38 amino acid compositions, molecular weight 4402.5Da, isoelectric point 12.78, wherein all amino acid are L-type.
2 Alligatorin4 of embodiment detects the antibacterial activity of common aquatic pathogenic bacteria
(1), it picks them separately the test strain being stored on inclined-plane and is spread evenly across nutrient broth solid medium
On (nutrient broth medium, OXOID company, Britain) plate, the filter paper of the 0.5cm diameter by sterilizing is placed in
The 10 μ l of Alligatorin4 sample solution for being dissolved in the 2mg/ml of sterile deionized water is added dropwise in media surface, falls in 37 DEG C
Culture 18-20 hours is set, whether observation inhibition zone is formed.If sample has antibacterial activity, can be formed around filter paper clear
Clear transparent inhibition zone, inhibition zone show that more greatly sample antibacterial activity is stronger.
(2), Alligatorin4 minimal inhibitory concentration (Minimum Inhibitory Concentration) measurement (2
Times dilution method):
The bacterial strain with inhibition zone carries out MIC measurement experiment in step experiment in selection.Test strain is inoculated into the training of NB liquid
It supports in base (OXOID company, Britain), 37 DEG C of shaken cultivations will then be cultivated extremely with fresh NB fluid nutrient medium to logarithmic growth phase
The culture solution of logarithmic growth phase is diluted to 2 × 105Cfu/ml is stand-by.
According to the dilution process in table 1,100 μ l NB fluid nutrient mediums are previously added in sterile each hole of 96 orifice plate, then
Be added in the first hole 100 μ l be diluted to NB fluid nutrient medium it is certain density through 0.22 μm of hole membrane filtration
Alligatorin4 sample solution takes 100 μ l that the 2nd hole is added, successively doubling dilution (referring to table 1) after mixing, be sucked out from the 9th hole
100 μ l are discarded, the 10th hole system control tube.
1 dilution process of table
96 orifice plates are placed 37 DEG C slow oscillation culture 18 hours, light absorption is measured at 600nm wavelength.It is minimum antibacterial dense
Degree is the minimum sample concentration of invisible bacterial growth, and the results are shown in Table 2.
Table 2.Alligatorin4 is to aquatic pathogenic bacteria antibacterial activity
As can be seen from Table 2, Alligatorin4 shows extremely strong antibacterial activity to the aquatic pathogenic bacteria in table 2, wherein
Bacterial strain including a variety of pairs of ampicillin-resistants.MIC value is in the range of 4.69-37.5 μ g/ml.
The measurement of 3 Alligatorin4 sterilization speed of embodiment
Vibrio parahaemolytious is cultivated 12 hours with NB fluid nutrient medium (OXOID company, Britain) at 37 DEG C, then with fresh
NB fluid nutrient medium is diluted to 106The bacteria suspension of CFU/ml.The Alligatorin4 sample that will be dissolved in the deionized water of sterilizing
Product are added in bacteria suspension, make final concentration of 5 × MIC (23.45 μ g/ml).The bacterium solution that Alligatorin4 sample is added is put
It is placed in shake culture in 37 DEG C of incubators, 50 μ l bacterium solutions was taken to dilute at 0,10,20,30,45,60,90,120 and 180 minute respectively
1000 times, the 50 diluted bacterium solutions of μ l are then taken to be applied in NB solid medium tablets, bacterium colony after 37 DEG C of incubator overnight incubations
It counts.The experiment uses ampicillin as positive control, and the deionized water of sterilizing is as negative control.
The results are shown in Table 3, and Alligatorin4 is rapid to the sterilization speed of vibrio parahaemolytious, can kill in 90 minutes
Dead all bacteriums, and positive control ampicillin sterilization speed is slower, needs could kill all bacteriums within 180 minutes.
Sterilization speed of 3 Alligatorin4 of table to vibrio parahaemolytious
4 Alligatorin4 of embodiment is to steamed crab immune system determination of activity
Steamed crab used is purchased from Suzhou Yangcheng Lake steamed crab farm in following embodiment, after laboratory is temporarily supported 1 week, chooses
Individual (1-2g/ is only) random grouping of neat specification, health is selected to raise in finishing box, every group 15.Experiment sets 4 groups altogether:
Blank control group, 10,25,50mg/kg Alligatorin4 group.
Alligatorin4 polypeptide sample is dissolved with PBS, is injected from the Ji Guanjiemochu of the 3rd of steamed crab the or step 4 foot
Enter in vivo (50 μ l/ are only), blank control group injects the PBS buffer solution of same volume.After injection for 24 hours, it is taken at random from each group
Steamed crab 3, serum is made in acquisition hemolymph, and with acid phosphatase and alkaline phosphatase assay kit, (biology is built up in Nanjing
Research institute's production) detect acid phosphatase and alkaline phosphatase activities in steamed crab serum.Specific experiment method is referring to kit
Specification carries out.
As a result as shown in Figure 1, compared with blank control group, 10,25,50mg/kg Alligatorin4 group steamed crab serum
Alkaline phosphatase and activity of acid phosphatase significantly increase (P < 0.01 * P < 0.05, * *), illustrate that Alligatorin4 can increase
The immune function of strong steamed crab.
5 Alligatorin4 of embodiment is to steamed crab Alligatorin4 to steamed crab premunition (immune protective rate)
Measurement
Vibrio parahaemolytious is cultivated 12 hours with NB fluid nutrient medium (OXOID company, Britain) at 37 DEG C, is washed with PBS buffer solution
It washs twice and is resuspended at 1 × 106The bacteria suspension of CFU/ml is spare.
Steamed crab be purchased from Suzhou Yangcheng Lake steamed crab farm, laboratory temporarily support 1 week after, select neat specification, health
Individual (3-5g/ is only) random grouping raising is in finishing box, and every group 25.Experiment sets 2 groups: blank control group and 50mg/ altogether
Kg Alligatorin4 group (in experiment used Alligatorin4 concentration be 50mg/kg).Alligatorin4 polypeptide sample is used
PBS dissolution, is injected into vivo from the Ji Guanjiemochu of the 3rd of steamed crab the or step 4 foot, and blank control group injects same volume
PBS buffer solution.12h after sample injection injects the good bacterial solution (1 × 10 of above-mentioned dilution with same method5CFU/g) infection is big
Lock crab.Steamed crab continues temporarily to support 5 days, observes steamed crab vigor, calculates final survival rate.
As a result as shown in Fig. 2, Alligatorin4 can provide effective immanoprotection action for steamed crab.Preparatory 12h
The Alligatorin4 of injection 50mg/kg can significantly improve the survival rate of steamed crab after bacterium infection.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>University Of Suzhou
<120>application of natural host defense peptide Alligatorin4
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 38
<212> PRT
<213>amino acid sequence
<400> 1
Arg Arg Gly Leu Phe Lys Lys Leu Arg Arg Lys Ile Lys Lys Gly
5 10 15
Phe Lys Lys Ile Phe Lys Arg Leu Pro Pro Val Gly Val Gly Val
20 25 30
Ser Ile Pro Leu Ala Gly Arg Arg
35
Claims (1)
1. natural host defense peptide Alligatorin4 is preparing the application in aquiculture animal immunoactivator；It is described natural
The amino acid sequence of host defense peptide Alligatorin4 is as shown in SEQ ID No.1.
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| CN201710383361.2A CN107252475B (en) | 2017-05-26 | 2017-05-26 | The application of natural host defense peptide Alligatorin4 |
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| CN201710383361.2A CN107252475B (en) | 2017-05-26 | 2017-05-26 | The application of natural host defense peptide Alligatorin4 |
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| CN108467426B (en) * | 2018-05-28 | 2020-08-04 | 苏州大学 | Taihu lake whitefish host defense peptide and application thereof |
| CN110305890A (en) * | 2019-07-02 | 2019-10-08 | 河南城建学院 | The novel 5 kinds of antibacterial peptides expressing in series recombination engineering construction method of one kind and its application |
| CN111374168B (en) * | 2019-12-18 | 2022-08-30 | 苏州大学 | Application of natural antibacterial peptide As-CATH4 in food preservation and fresh-keeping |
| CN111374169B (en) * | 2019-12-18 | 2022-08-30 | 苏州大学 | Application of natural antibacterial peptide As-CATH5 in food preservation and fresh-keeping |
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| CN1238378C (en) * | 2003-11-07 | 2006-01-25 | 中山大学 | An antibiotic peptide and its coded sequence and use |
| CN104177485B (en) * | 2014-07-03 | 2017-06-06 | 苏州大学 | A kind of Chinese alligator cecropin A lligatorin6 and its application |
| CN104163861B (en) * | 2014-07-03 | 2017-04-26 | 苏州大学 | Reptile antibacterial peptide Alligatorin5 and applications thereof |
| CN104151415B (en) * | 2014-07-03 | 2017-06-06 | 苏州大学 | A kind of natural antibacterial peptide Alligatorin4 and its application |
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Non-Patent Citations (2)
| Title |
|---|
| antimicrobial peptides in reptiles;monique l. van hoek;《pharmaceuticals》;20140610;第7卷(第6期);723-753 * |
| characterization of bacillus islated from the gut of rohu, labeo rohita, fingerling and its signification in digestion;ghosh k等;《journal of applied aquaculture》;20021231;第12卷(第3期);33-42 * |
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Effective date of registration: 20201215 Address after: 401-03, building 10, Xibei District, Suzhou nano City, No. 99, Jinjihu Avenue, Suzhou Industrial Park, Jiangsu Province Patentee after: SiGe (Suzhou) Biotechnology Co.,Ltd. Address before: 215000 8 Ji Xue Road, Xiangcheng District, Suzhou, Jiangsu. Patentee before: Suzhou University |
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