CN116874575B - Apostichopus japonicus antibacterial peptide and application thereof - Google Patents
Apostichopus japonicus antibacterial peptide and application thereof Download PDFInfo
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 49
- 241000965254 Apostichopus japonicus Species 0.000 title abstract description 28
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 5
- 241000607594 Vibrio alginolyticus Species 0.000 claims abstract description 5
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 5
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- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000000022 bacteriostatic agent Substances 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 25
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 20
- UGPMCIBIHRSCBV-XNBOLLIBSA-N Thymosin beta 4 Chemical compound N([C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)C(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(C)=O UGPMCIBIHRSCBV-XNBOLLIBSA-N 0.000 abstract description 19
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Classifications
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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
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- 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
-
- 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
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Abstract
The invention relates to the technical field of bioengineering, in particular to preparation and application of stichopus japonicus antibacterial peptide, and a novel antibacterial peptide family member named StMo _ thymosin-beta4 is obtained from a stichopus japonicus transcriptome library for the first time. And a method of solid-phase chemical synthesis is adopted to prepare the StMo _ thymosin-beta4 alpha polypeptide fragment with the broad-spectrum antibacterial activity of StMo _ thymosin-beta4 antibacterial peptide. The polypeptide fragment has strong inhibitory activity on common pathogenic vibrio of aquatic animals such as vibrio parahaemolyticus and vibrio alginolyticus, and has strong inhibitory activity on common food-borne pathogenic bacteria such as staphylococcus aureus.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a stichopus japonicus antibacterial peptide and application thereof.
Background
Apostichopus japonicus (Stichopus monotuberculatus), also known as Fangshen, yellow-fleshed ginseng, belongs to the genus Apostichopus japonicus, apostichopaceae. The stichopus japonicus is distributed in Guangxi, nansha islands, hainan islands and the like in China, is an important economic category in the south coast in China, has large individual, thick and tender meat, is rich in amino acid, polysaccharide, sea cucumber extract and the like, and is an important food and traditional Chinese medicine raw material. However, in recent years, the offshore habitat of the stichopus japonicus is seriously damaged, meanwhile, because the wild stichopus japonicus is excessively collected with higher economic value, the wild stichopus japonicus population in the coastal of Guangxi province is basically out of track, only the 280642-step island has a small amount of wild resources, and the wild stichopus japonicus population is listed as endangered animals by China species red catalogue. And, up to now, the large-scale artificial breeding technology of stichopus japonicus has not been completely broken through, and the young allergic attachment stage is easy to cause seedling failure due to vibrio outbreak infection. The efficient prevention and control of vibriosis outbreaks in the development of the young stichopus japonicus is an important guarantee of successful artificial breeding.
Echinoderms, like vertebrates, are posterior animals, but they still lack the higher vertebrates' acquired immune system in vivo, and rely primarily on the nonspecific innate immune system to combat bacterial, viral, and fungal disease microorganisms. Antibacterial peptides (antimicrobial peptides, AMPs), also known as host defensive peptides, are a class of small molecule polypeptides that are widely found in nature and have broad-spectrum antibacterial activity, are generally positively charged, have amphiphilicity (lipophilic and hydrophilic), and generally have secondary structures such as alpha helices, beta sheets, beta hairpins, and the like. The antibacterial peptide is used as an important component of an innate immune system, and has obvious inhibiting and killing effects on viruses, bacteria, fungi, parasites and the like. In recent years, with the high-speed development of the aquaculture industry in China, the problems of water environment pollution, outbreak of disease organisms and the like have become main bottlenecks for restricting the healthy development of the aquaculture industry. At present, antibiotics and chemical medicines are mainly adopted in the aquaculture industry to prevent and treat diseases of cultured animals, so that the problems of medicine residues, pathogen resistance and the like are increasingly serious, the production cost is increased, and the prevention and treatment difficulty is increased. The antibacterial peptide is used as a novel biological safety antibiotic, has good heat stability and water solubility, is not easy to generate drug resistance, can replace the traditional antibiotic to directly inhibit and kill disease organisms, can enhance the immunity of cultured animals, improves the production benefit, and has huge application prospect in the aquaculture industry, especially in the field of artificial offspring seed breeding.
Thymus peptide is a biologically active polypeptide extracted from the thymus of the earliest calf, and members of this family are classified into three subtypes, alpha, beta and gamma, from low to high isoelectric points. At present, more than 20 beta-type thymus peptides have been identified and reported, the biological functions of which are evolutionarily conserved, and which are biological peptides of an immune defense in marine echinoderms, capable of binding pathogens and initiating pathogen recognition receptors to resist invasion of pathogenic microorganisms. And the beta-thymus peptide has antibacterial activities of inhibiting and killing bacteria in the research of marine invertebrates such as Pacific oyster, procambarus clarkii, japanese cyst prawn and the like. However, there are no studies on beta-type thymus peptides in Apostichopus japonicus, and the biological functions and application potential thereof are not yet clear.
Disclosure of Invention
In view of the above, the StMo _ thymosin-beta 4-alpha antibacterial peptide provided by the application can be used as an ecological safety antibacterial additive in aquaculture, and has a wide application prospect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a stichopus japonicus StMo-thymosin-beta 4 antibacterial peptide, the amino acid sequence of which is shown as SEQ ID NO. 1; the method comprises the following steps:
MRHAETQEKVALPAKEDIDAEKGQQALRKGIEGFDPANLRKTETQEKNPLPSKEVIEQ EKKA。
Another object of the present invention is to provide a stichopus japonicus StMo _ thymosin-beta4_α antimicrobial peptide, which is a polypeptide fragment selected from StMo _ thymosin-beta4 antimicrobial peptides with antimicrobial activity, wherein the polypeptide fragment can form an amphipathic α -helix structure, has 3 positive charges, and is an α -helix cationic antimicrobial peptide named as: stMo _ thymosin-beta 4. Alpha. The amino acid sequence is SEQ ID NO.2; the method comprises the following steps:
KGQQALRKGIEGFDPANLRK。
The application of the stichopus japonicus StMo _ thymosin-beta 4_alpha antibacterial peptide is also within the protection scope of the invention, and the application of the antibacterial peptide comprises the following steps: is used as an antibacterial additive in the aquaculture industry.
A polypeptide or protein comprising the above-described antimicrobial peptide is also within the scope of the present invention.
The invention has the following beneficial effects:
The invention provides thymosin-beta4 antibacterial peptide family members with broad-spectrum antibacterial activity from stichopus japonicus, the antibacterial peptide active fragments can be prepared in a large amount through solid-phase chemical synthesis, and the antibacterial peptide active fragments have strong inhibition and killing effects on common pathogenic vibrio and food-borne pathogenic vibrio in the aquatic industry, can be used as an ecological safe antibacterial additive in aquaculture, and have wide application prospects.
Drawings
FIG. 1 is a diagram of the tertiary protein structure of the mature peptide of StMo _ thymosin-beta4 antibacterial peptide;
FIG. 2 is a diagram of the amphipathic alpha helix structure of StMo _ thymosin-beta 4. Alpha. Antimicrobial peptides; in the figure, the dark black ground color represents hydrophilic amino acid, the light white ground color represents hydrophobic amino acid, and the direction indicated by the arrow is a hydrophobic surface of an alpha helical structure;
FIG. 3 is a photograph of a culture dish of StMo _ thymosin-beta 4. Alpha. Antimicrobial peptide against Micrococcus solvolcanis.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
Example 1:
obtaining the gene sequence of the stichopus japonicus StMo _ thymosin-beta4 antibacterial peptide:
Collecting tissues such as tentacles, intestines, body walls and respiratory trees of stichopus japonicus, respectively extracting total RNA from the tissues, performing transcriptome library sequencing after quality inspection is qualified, completing the assembly of a reference transcript of a delicious euglena with trinity software, and finally screening the full-length cDNA sequence of the stichopus japonicus StMo _ thymosin-beta4 antibacterial peptide gene for the first time by using blastp software in a homologous comparison mode, wherein the full-length cDNA sequence of the gene is as follows:
ATGCGTCACGCGGAGACCCAAGAGAAGGTCGCTCTTCCGGCCAAAGAAGATATCG
ATGCTGAGAAAGGGCAGCAGGCACTGAGGAAAGGAATCGAGGGGTTCGATCCTGCGAA
TTTGAGGAAGACTGAAACCCAAGAGAAGAATCCTTTGCCAAGCAAGGAAGTCATTGAA
CAGGAGAAGAAGGCATAA。
the full-length cDNA of StMo _ thymosin-beta4 antibacterial peptide gene is translated into an amino acid sequence, and the full-length amino acid sequence of StMo _ thymosin-beta4 antibacterial peptide is SEQ ID NO.1, which is specifically as follows:
MRHAETQEKVALPAKEDIDAEKGQQALRKGIEGFDPANLRKTETQEKNPLPSKEVIEQ EKKA。
Example 2:
Obtaining the tertiary protein structure of the mature peptide and StMo _ thymosin-beta4 alpha polypeptide fragment with antibacterial activity:
Signal peptides and mature peptides of StMo _ thymosin-beta4 antibacterial peptides were predicted using SignalP-6.0 software, and physicochemical properties and tertiary structure of mature peptides were predicted using https:// aps. Un. Edu/and https:// swissmodel. Expasy. Org/website, respectively.
The results show that the mature peptide consists of 62 amino acids and has a molecular formula of C 301H503N87O100S1 and a molecular weight of 6998.9 daltons. The tertiary structure of the protein is shown in FIG. 1 and comprises 2 alpha helices.
Predicting a polypeptide fragment with antibacterial activity of the mature peptide by using an http:// www.camp.bicnirrh.res.in/predict _c/website to obtain a polypeptide fragment with the length of 20 amino acids, which is named StMo _ thymosin-beta4 alpha, wherein the amino acid sequence of the polypeptide fragment is SEQ ID NO.2; the method comprises the following steps:
KGQQALRKGIEGFDPANLRK。
The StMo-thymosin-beta 4-alpha physicochemical properties and structural characteristics were predicted using the https:// helix. Ipmc. Cnrs. Fr/cgi-bin/computParams. Py website.
The result shows that the antibacterial peptide can form an amphipathic alpha-helical structure, as shown in figure 2, has obvious hydrophobic surface and hydrophilic surface, and has 3 positive charges, and is an alpha-helical cationic antibacterial peptide.
Example 3:
preparation of Stichopus japonicus StMo _ thymosin-beta4_α antibacterial peptide:
The StMo-thymosin-beta 4-alpha antibacterial peptide amino acid sequence is submitted to Hubei blaze biotechnology limited company, the company is entrusted to synthesize a StMo-thymosin-beta 4-alpha antibacterial peptide crude product by using a solid phase chemical synthesis method, high performance liquid chromatography is adopted for purification, then a freeze dryer is used for concentration, finally mass spectrometry is carried out by using a mass spectrometer for identification, and the StMo-thymosin-beta 4-alpha antibacterial peptide with high purity is prepared after the identification is qualified.
Example 4:
determination of antibacterial Activity of Stichopus japonicus StMo _ thymosin-beta 4. Alpha. Antibacterial peptide:
In this example, strains such as E.coli (ESCHERICHIA COLI), bacillus subtilis (Bacillus subtilis), staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa), corynebacterium glutamicum (Corynebacterium glutamicum), vibrio alginolyticus (Vibrio alginolyticus), micrococcus luteus (Micrococcus luteus), pseudomonas fluorescens (Psdeuomnodafluoerncnet), micrococcus solvolvatus (Micrococcus lysodeikticus Fleming), shigella flexneri (Shigella flexneri), vibrio parahaemolyticus (Vibrio Parahaemolyticus), vibrio harveyi (Vibrio harveyi), aeromonas hydrophila (Aeromonas hydrophila) and Protous mermairei (Photobacterium damselae) were used as test strains for the antibacterial activity test. Wherein the strains are all purchased from the China academy of sciences of microbiological culture Collection center.
The method for measuring the antibacterial activity is as follows:
(1) Activation of the refrigerated strains: streaking bacterial strain (i.e. the above test strain) stored at-80deg.C on LB plate, culturing at 28deg.C (marine bacteria) or 37deg.C (non-marine bacteria) for 24 hr, picking single colony into 10mL sterile LB liquid medium, shake culturing at 28deg.C or 37deg.C and 180rpm/min for 12 hr;
(2) Preparation of bacterial suspensions: streaking a small amount of activated bacterial liquid on an MHA flat plate, culturing for 12 hours in an inverted mode, picking 1 single colony from the flat plate, streaking on an MHA slant culture medium, culturing for 12 hours at 28 ℃, eluting the slant culture by using NaPB (10 mM), measuring the OD value of the eluent by using an enzyme-labeling instrument, and adding a certain amount of eluent into the NaPB or a working culture medium to ensure that the OD value of the final bacterial suspension is 0.0018;
(3) Preparation of StMo _ thymosin-beta 4_alpha antibacterial peptide working solution: filtering StMo-thymosin-beta 4-alpha antibacterial peptide with a 0.22 mu M filter membrane, measuring protein concentration by using a Bradford method, diluting working solutions with the protein concentration of 1.5, 3.0, 6.0, 12, 24, 48 and 96 mu M by using a multiple ratio, and preserving at 4 ℃ for later use;
(4) Determination of minimum inhibitory concentration (MIC, minimuminhibition concentration): designing an experiment group, a blank control group and a negative control group for experiments, wherein the concentration of each antibacterial peptide is set to be 3 in parallel; the experimental results were observed by culturing in 96-well cell culture plates for 24 hours.
Wherein, each group of the reagents is as follows:
Experimental group: 50. Mu.L of antibacterial peptide working solution+50. Mu.L of bacterial suspension;
Blank control: 50. Mu.L of sterile water+50. Mu.L of bacterial suspension;
negative control: 50. Mu.L of working medium+50. Mu.L of the minimum concentration of the antimicrobial peptide working fluid.
(5) Determination of minimum bactericidal concentration (MBC, minimumbactericidal concentration): mu.L of the mixed culture was aspirated from each well of the cell culture plate after incubation, and the mixture was dropped onto a MHA plate and cultured for 24 hours (culture temperature of non-marine bacteria was 37℃and culture temperature of marine bacteria was 28 ℃), and colony formation was observed.
The specific test results are shown in table 1:
TABLE 1 results of determination of antibacterial Activity of StMo_thymosin-beta 4. Alpha. Antibacterial peptide
The result shows that StMo-thymosin-beta 4-alpha antibacterial peptide has strong inhibition and killing effects on common pathogenic vibrios in aquaculture, wherein the minimum inhibitory concentration on vibrios parahaemolyticus and vibrios alginolyticus is respectively 1.5-3 mu M and 3-6 mu M, and the minimum bactericidal concentration is respectively 3-6 mu M and 12-24 mu M; the bactericidal composition also has stronger inhibiting and killing effect on common food-borne pathogenic bacteria in aquatic products, wherein the minimum inhibitory concentration on staphylococcus aureus is 3-6 mu M, and the minimum bactericidal concentration is 12-24 mu M.
Taking Micrococcus solvolvatus as an example in the plate culture condition of the test strain, a photo of a culture dish of the micrococcus solvolvatus is shown as a figure 3, the plate is divided into 8 fan-shaped areas with the same area size, and the concentration of the antibacterial peptide working solution in each area is as follows: 1.5. Mu.M, 3.0. Mu.M, 6.0. Mu.M, 12. Mu.M, 24. Mu.M, 48. Mu.M, 96. Mu.M, and 0.0. Mu.M (blank medium group), wherein the blank medium group was not inoculated with a wall micrococcus nor with an antimicrobial peptide; as can be seen from FIG. 3, at a concentration of 1.5 to 6.0. Mu.M, the plate can grow the Micrococcus with a wall, while at a concentration of 12. Mu.M, the Micrococcus with a wall cannot grow, thus indicating that the highest concentration of the antimicrobial peptide working solution in which the Micrococcus with a wall can grow is 6. Mu.M, and the lowest concentration of the antimicrobial peptide working solution in which the Micrococcus with a wall can be killed and cannot grow is 12. Mu.M. Thus, the minimum sterilization concentration of StMo _ thymosin-beta4 alpha antibacterial peptide to micrococcus solvolitides is 6-12 mu M.
Example 5:
The stichopus japonicus StMo-thymosin-beta 4-alpha antibacterial peptide is applied to stichopus japonicus seedling production:
The embodiment provides the work of applying the antibacterial peptide as an antibacterial additive in aquaculture, which comprises the following specific steps: the high-purity StMo _ thymosin-beta 4-alpha antibacterial peptide is prepared by using the method described in the embodiment 3, a sterilizing working solution is prepared according to the final concentration of 6 mu M by using the prepared StMo _ thymosin-beta 4-alpha antibacterial peptide, and biological bait spirulina powder of the floating larvae of the stichopus japonicus is soaked for 1h, so that the aim of inhibiting and killing common pathogenic vibrio such as vibrio parahaemolyticus and vibrio alginolyticus in the biological bait produced by the stichopus japonicus seedling is fulfilled, and the survival rate of the larvae is improved.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the present invention.
Claims (2)
1. The antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 2.
2. The use of an antimicrobial peptide according to claim 1 for the preparation of a bacteriostatic agent that inhibits the following pathogenic bacteria: coli, bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa, corynebacterium glutamicum, vibrio alginolyticus, micrococcus luteus, pseudomonas fluorescens, micrococcus solvolens, shigella flexneri, vibrio parahaemolyticus, vibrio harveyi, aeromonas hydrophila and/or mermaid light emitting bacilli.
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