CN108864270B - Termite antibacterial peptide and application thereof - Google Patents
Termite antibacterial peptide and application thereof Download PDFInfo
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- CN108864270B CN108864270B CN201810797733.0A CN201810797733A CN108864270B CN 108864270 B CN108864270 B CN 108864270B CN 201810797733 A CN201810797733 A CN 201810797733A CN 108864270 B CN108864270 B CN 108864270B
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- antibacterial peptide
- termite
- klebsiella pneumoniae
- escherichia coli
- antibacterial
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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
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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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
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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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
Abstract
The invention discloses a termite antibacterial peptide and application thereof. The amino acid sequence of the antibacterial peptide is shown in SEQ ID NO.1, and the molecular weight of the antibacterial peptide is 2472 daltons. The antibacterial peptide extracted by the invention has the advantages of small molecular weight, strong bactericidal action and the like, and has the characteristic of small toxicity to normal cells of human.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a termite antibacterial peptide and application thereof.
Background
The antibacterial peptide is a high-efficiency broad-spectrum antibacterial peptide molecule which is rapidly generated by an organism after the organism is invaded by microorganisms and participates in the immune reaction of the organism. Generally consisting of 12 to 100 amino acids, ranging in size from a few kDa to a dozen kDa. Antimicrobial peptides are widely available as defense molecules for the body, and thousands of antimicrobial peptides have been identified in microorganisms, plants, insects, arthropods, amphibians, mammals, and even humans. The antibacterial peptide is different from the traditional antibiotics produced by microorganisms (including bacteria, fungi, streptomyces and the like) in the aspects of synthesis mechanism, amino acid composition, action mechanism and the like. The antibacterial peptide not only has broad-spectrum antibacterial activity, but also shows higher biological activity in the aspects of antivirus, antifungal, antiparasitic, antitumor and the like. The antibacterial mechanism of the antibacterial peptide is complex, but most theories believe that the mechanism involves the action of the cationic property and the hydrophobic property of the antibacterial peptide and the negative charge of microbial cell membranes, and after the antibacterial peptide is contacted with the bacterial cell membranes, the antibacterial peptide causes the membrane permeability to change or transmembrane pores are formed on the bacterial cell membranes, and finally the bacterial contents leak out and die. Therefore, the antibacterial peptide is far higher in the speed of killing bacteria than the traditional antibiotics, and unlike the antibiotics which inhibit the growth of bacteria at low concentration, the effect of the antibacterial peptide on the bacteria is almost lethal.
To date, various antimicrobial peptides have been extracted from termites, but many types of antimicrobial peptides have not been discovered.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the termite antibacterial peptide and the application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a termite antibacterial peptide contains 22 amino acid residues, the amino acid sequence of the termite antibacterial peptide is shown in SEQ ID NO.1, and the molecular weight of the termite antibacterial peptide is 2472 daltons.
The amino acid sequence shown in SEQ ID NO.1 has a difference of less than 10% after one or more amino acids are substituted, deleted and/or added, and expresses the amino acid sequence of the same functional protein.
The amino acid sequence shown in SEQ ID NO.1 has the following sequence after one or more amino acids are substituted, deleted and/or added:
GLGNIPWGKVKEDFLVGGMKAR-NH2；(SEQ ID NO.2)
RLGNIPWGKVKEDFLVGGMKAV-NH2；(SEQ ID NO.3)
RLGNIPWGKVKDFLVGGMKAR-NH2；(SEQ ID NO.4)
RLGNIPWGKVKEDFLVGGMKARV-NH2；(SEQ ID NO.5)
RLGNIPWGKVKEDFLVGGMPKAV-NH2；(SEQ ID NO.6)
the application of the antibacterial peptide in preparing antibacterial drugs.
The application of the antibacterial peptide in preparing antibacterial drugs for inhibiting Escherichia coli and Klebsiella pneumoniae.
Further, the minimum inhibitory concentration of the antibacterial peptide to Escherichia coli is 16.3mg/L, and the minimum inhibitory concentration to Klebsiella pneumoniae is 19.6 mg/L.
The application of the antibacterial peptide in preparing feed additives.
The invention has the beneficial effects that:
the antibacterial peptide extracted by the invention has the advantages of small molecular weight, strong bactericidal action and the like, and has the characteristic of small toxicity to normal cells of human.
Drawings
FIG. 1 is a diagram of the bacteriostatic effect of antibacterial peptide; wherein, FIG. 1a is a bacteriostatic map of Escherichia coli; FIG. 1b is a Klebsiella pneumoniae bacteriostasis pattern;
FIG. 2 is a diagram showing the effect of the antibacterial peptide on the minimum inhibitory concentration; wherein, FIG. 2a is a bacteriostatic map of Escherichia coli; FIG. 2b is the gram showing the inhibition of Klebsiella pneumoniae;
FIG. 3 is the primary mass spectrum test chart of the antibacterial peptide.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1 extraction of antimicrobial peptides
1. Preparation of Termite samples
After drying the termite sample, taking 5-10 g of the termite sample, and crushing the termite sample by using an electric crusher for later use.
2. Degreasing treatment
Wrapping the prepared termite powder with a filter paper cylinder, putting the wrapped termite powder into an extraction cylinder of a Soxhlet extractor, connecting a fat flask dried to constant weight, adding ether or petroleum ether from the upper end of a condenser pipe of the extractor to 2/3 of the volume in the bottle, introducing condensed water, immersing a bottom bottle in hot water at 65 ℃, heating in a water bath, slightly plugging a small group of absorbent cotton into the upper opening of the condenser pipe, extracting for 6-12h, and fully drying a sample in the filter paper cylinder at 50 ℃.
3. Ethanol extraction
Adding 60% ethanol into the product obtained in the step 2 according to the ratio of the material to the liquid of 1:30, leaching for 24h at 4 ℃, filtering, centrifuging the leaching solution for 20min at 4 ℃ at 6000-7000 r/min, and collecting the supernatant.
4. Rotary distillation
And (4) performing rotary evaporation on the supernatant collected in the step (3), and volatilizing ethanol to obtain the termite crude extract.
5. Column chromatography
And (2) filling the Sephadex G-25 serving as a filler into a chromatographic column, carrying out column balance, and after the balance is finished, carrying out column chromatography separation on the crude termite extract by using a phosphate buffer solution with the concentration of 0.05mol/L, pH value of 7.4 as an eluent, wherein the elution volume is 2-3 column volumes, the collection time of each tube of eluent is 15min, the volume is 3-5 mL in the elution process, and numbering is carried out.
6. Screening of antimicrobial peptides
Performing bacteriostasis experiments by using the collected eluent, taking bacteria such as Escherichia coli (ATCC 25922), staphylococcus aureus (ATCC 29213), salmonella enteritidis (ATCC 13076), bacillus subtilis (ATCC11060), Klebsiella pneumoniae (Klebsiella pneumoniae strain WL1309) and the like as test bacteria, performing bacteriostasis experiments by adopting an Oxford cup method, repeating the steps for 3 times, and calculating the average value of the diameter of a bacteriostasis ring; and selecting the eluent with the bacteriostatic action, and concentrating to obtain the purified termite antibacterial peptide.
7. Preservation of antimicrobial peptides
The termite antibacterial peptide substance can be stored for 3-6 months at the temperature of-20 ℃.
8. Sequencing of antimicrobial peptides
Sequencing the separated antibacterial peptide, and determining the sequence structure of the antibacterial peptide by adopting a chromatography, wherein the amino acid sequence of the antibacterial peptide is as follows: RLGNIPWGKVKEDFLVGGMKAR-NH2(SEQ ID NO.1), namely the amino acid sequence of the antibacterial peptide is as follows: arginine (R) -leucine (L) -glycine (G) -aspartic acid (N) -isoleucine (I) -proline (P) -tryptophan (W) -glycine (G) -lysine (K) -valine (V) -lysine (K) -glutamic acid (E) -aspartic acid (D) -phenylalanine (F) -leucine (L) -valine (V) -glycine (G) -methionine (M) -lysine (K) -alanine (a) -arginine (R) -NH2(ii) a The molecular weight is 2472 daltons, and the antibacterial peptide is subjected to primary mass spectrometry detection, and the result is shown in figure 3.
Example 2
Detection of bacteriostatic effects
The method comprises the following steps of detecting the bacteriostasis of the bacteria to be detected by adopting an Oxford cup method, wherein the bacteria to be detected are Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Klebsiella pneumoniae (Klebsiella pneumoniae strain WL1309), Salmonella enteritidis (ATCC 13076) and Bacillus subtilis (ATCC11060) to be used as bacteriostasis experiments, 0.1mL of prepared bacterial suspension is absorbed by an aseptic liquid transfer gun, is respectively added to solid plates corresponding to a culture medium of the bacteria to be detected, is uniformly coated by an aseptic coating rod to prepare a plate with the bacteria (or is uniformly mixed by the bacteria and the culture medium with proper temperature to prepare a pouring plate), a sterilized Oxford cup is placed into the plate by tweezers under the aseptic condition, the antibacterial peptide substance extracted in example 1 is detected whether an antibacterial ring (transparent) appears, and the bacteriostasis effect is shown in figure 1, wherein figure 1a is an Escherichia coli diagram; FIG. 1b is the bacteriostatic map of Klebsiella pneumoniae.
As can be seen from FIG. 1a and FIG. 1b, the antibacterial peptide extracted by the method of the present invention has the effect of inhibiting gram-negative bacteria Escherichia coli and Klebsiella pneumoniae; and no inhibition zone appears in staphylococcus aureus, salmonella enteritidis and bacillus subtilis, which shows that the antibacterial peptide has no inhibition effect on staphylococcus aureus, salmonella enteritidis and bacillus subtilis.
Example 3
Minimum inhibitory concentration detection
Diluting the termite antibacterial peptide purified product extracted in the embodiment 1 in different descending concentrations to respectively obtain antibacterial peptide diluents with the concentrations of 100mg/L, 90mg/L, 80mg/L, 70mg/L, 60mg/L, 50mg/L, 40mg/L, 30mg/L, 20mg/L and 10mg/L, and respectively adopting the antibacterial peptide diluents with the concentrations to detect the minimum inhibitory concentration of Escherichia coli and Klebsiella pneumoniae, wherein the diameter of an inhibition zone is more than 10mm and is the minimum inhibitory mass concentration; the experiment is repeated for 3 times, the average value is calculated, and the detection result is shown in figure 2; wherein, FIG. 2a is a bacteriostatic map of Escherichia coli; FIG. 2b is the bacteriostatic diagram of Klebsiella pneumoniae, and the minimum inhibitory concentration of the antimicrobial peptide to Escherichia coli is 16.3mg/L and the minimum inhibitory concentration to Klebsiella pneumoniae is 19.6mg/L, which are calculated and calculated through the graph of FIG. 2.
Example 4
Security detection
Taking normally growing L-02 cells as a control group, taking an L-02 cell culture solution added with antibacterial peptide as an experimental group, then adding different amounts of antibacterial peptide into the experimental group to enable the concentrations of the antibacterial peptide in the experimental group to be 100mg/L, 50mg/L, 10mg/L, 5mg/L and 1mg/L respectively, after culturing for 12h, detecting the cell activity of each component by an MTT method, wherein the detection result is shown in Table 1, and the notation in Table 1 shows that the relative activities of the hepatocytes of the experimental group and the control group have no significant difference, which indicates that the antibacterial peptide has almost no toxicity to normal human cells.
TABLE 1 Effect of different concentrations of antibacterial peptide stimulation on the level of hepatocyte Activity
Sequence listing
<110> Sichuan institute of technology and technology
<120> termite antibacterial peptide and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213> Termite
<400> 1
Arg Leu Gly Asn Ile Pro Trp Gly Lys Val Lys Glu Asp Phe Leu Val
1 5 10 15
Gly Gly Met Lys Ala Arg-NH2
20
<210> 2
<211> 22
<212> PRT
<213> Termite
<400> 2
Gly Leu Gly Asn Ile Pro Trp Gly Lys Val Lys Glu Asp Phe Leu Val
1 5 10 15
Gly Gly Met Lys Ala Arg-NH2
20
<210> 3
<211> 22
<212> PRT
<213> Termite
<400> 3
Arg Leu Gly Asn Ile Pro Trp Gly Lys Val Lys Glu Asp Phe Leu Val
1 5 10 15
Gly Gly Met Lys Ala Val-NH2
20
<210> 4
<211> 21
<212> PRT
<213> Termite
<400> 4
Arg Leu Gly Asn Ile Pro Trp Gly Lys Val Lys Asp Phe Leu Val Gly
1 5 10 15
Gly Met Lys Ala Arg-NH2
20
<210> 5
<211> 22
<212> PRT
<213> Termite
<400> 5
Arg Leu Gly Asn Ile Pro Trp Gly Lys Val Lys Asp Phe Leu Val Gly
1 5 10 15
Gly Met Lys Ala Arg Val-NH2
20
<210> 6
<211> 22
<212> PRT
<213> Termite
<400> 6
Arg Leu Gly Asn Ile Pro Trp Gly Lys Val Lys Asp Phe Leu Val Gly
1 5 10 15
Gly Met Pro Lys Ala Val-NH2
20
Claims (3)
1. A termite antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO.1, and the molecular weight of the antibacterial peptide is 2472 daltons.
2. The use of the antimicrobial peptide of claim 1 for the preparation of an antimicrobial medicament for inhibiting escherichia coli and klebsiella pneumoniae.
3. The use of the antibacterial peptide according to claim 2 in the preparation of a medicament for inhibiting escherichia coli and klebsiella pneumoniae, wherein the antibacterial peptide has a minimum inhibitory concentration against escherichia coli of 16.3mg/L and a minimum inhibitory concentration against klebsiella pneumoniae of 19.6 mg/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810797733.0A CN108864270B (en) | 2018-07-19 | 2018-07-19 | Termite antibacterial peptide and application thereof |
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| CN108864270B true CN108864270B (en) | 2021-03-30 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102369210A (en) * | 2009-01-29 | 2012-03-07 | Amp治疗学两合有限公司 | Antibiotic peptides |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130332133A1 (en) * | 2006-05-11 | 2013-12-12 | Ramot At Tel Aviv University Ltd. | Classification of Protein Sequences and Uses of Classified Proteins |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102369210A (en) * | 2009-01-29 | 2012-03-07 | Amp治疗学两合有限公司 | Antibiotic peptides |
Non-Patent Citations (3)
| Title |
|---|
| Characterization of Antibacterial Activities of Eastern Subterranean Termite, Reticulitermes flavipes, against Human Pathogens;Yuan Zeng 等;《Plos one》;20160909;第11卷(第9期);e0162249 * |
| 抗菌肽饲料添加剂的研究进展;朱勇 等;《饲料工业》;20060205;第27卷(第3期);第16页左栏第2段 * |
| 野生大黑蚁中抗菌肽类物质的分离纯化及其耐热性;李仁奇 等;《生物资源》;20190102;第40卷(第6期);第536-541页 * |
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