CN111471094B - Antimicrobial peptide MSPiscidin-2, and coding gene and application thereof - Google Patents
Antimicrobial peptide MSPiscidin-2, and coding gene and application thereof Download PDFInfo
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- CN111471094B CN111471094B CN202010364173.7A CN202010364173A CN111471094B CN 111471094 B CN111471094 B CN 111471094B CN 202010364173 A CN202010364173 A CN 202010364173A CN 111471094 B CN111471094 B CN 111471094B
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- mspiscidin
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/13—Prevention or treatment of fish diseases
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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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
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
-
- 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 an antimicrobial peptide MSPiscidin-2, a coding gene and application thereof, wherein the MSPiscidin-2 is a straight-chain polypeptide, contains 27 amino acid residues, has a theoretical isoelectric point of 12.69, a molecular weight of 3.34kDa and a net charge of + 8. The gene encoding the antimicrobial peptide MSPiscidin-2 precursor Piscidin consists of 465 nucleotides. MSPiscidin-2 has strong antibacterial activity to gram-negative bacteria and gram-positive bacteria, including various aquaculture pathogenic bacteria; it does not contain disulfide bond and cyclic structure, and is convenient for chemical synthesis and genetic engineering preparation; can be used for preparing clinical medicines for resisting pathogenic microorganism infection and diminishing inflammation; or as a substitute for antibiotics in animal feed; or as an additive in cosmetics, health products and foods to replace the traditional preservative, and has good application prospect.
Description
Technical Field
The invention discloses an antimicrobial peptide MSPiscidin-2, a coding gene and application thereof, and particularly provides a Piscidin family broad-spectrum antimicrobial peptide MSPiscidin-2 from Micropterus salmoides and a gene thereof, a preparation method and application thereof in pathogenic microorganism infection resistance, medical treatment, bio-pharmaceuticals and aquaculture disease control, belonging to the technical field of biomedicine.
Background
The Piscidin family antibacterial peptide is a kind of antibacterial peptide from fish, is an important component of a non-specific immune system in fish bodies, generally consists of 20-50 amino acid residues, has an alpha helical structure, is rich in cations, has hydrophobicity and amphipathy, and has broad-spectrum bactericidal, anti-tumor and antiviral activities. After Piscidin was first discovered from mast cells of hybrid zebra weever, Piscidin was successively found in many fishes such as flounder, gilhead sea bream, and large yellow croaker. Fish belongs to primitive lower vertebrates, the living environment of the fish is relatively complex, the self immune organs are relatively incomplete in development and evolution, the self acquired immune system is very fragile relative to other higher vertebrates, and adaptive immune immunoglobulin and immune memory of the fish are relatively deficient, so that when the fish faces the complex living environment and the harm of pathogenic bacteria, the self innate immune system is mainly relied on to resist the invasion of exogenous factors for self protection. Research shows that the antibacterial peptide is an important component in the innate immune system of fish, and when the fish is invaded by exogenous factors, the antibacterial peptide can induce the fish to generate a large amount of antibacterial peptide so as to perform defense protection. With the research on more and more antibacterial peptides separated from fish, the antibacterial peptides are predicted to be the most important defense line in the innate immune system of fish. At present, a large number of antibacterial peptides have been found from the saliva tissues, mucus tissues and other parts and immune organs of fishes which are easily attacked by foreign factors, including Piscidin and Hepcidin antibacterial peptides found from hybrid zebra weever, Pleurocidin antibacterial peptides found from flatfish mucus tissues, and cathelicidin antibacterial peptides found from Atlantic hagus anguillarum by researchers.
At present, the application of the antibacterial peptide is mainly focused on the development of the medical field, and a plurality of satisfactory results are obtained, and a plurality of novel medicines are gradually brought into the medical market. For example, Pexiganan from Magainin, a xenopus antibacterial peptide, developed by Genaera corporation, is currently used in the form of an external cream for treating pustulosis and plantar ulcers in diabetic patients, and has entered the clinical phase III trial, which is also the first commercially developed antibacterial peptide; daptomycin is an anionic antimicrobial peptide, developed by Cubit Pharmaceuticals, approved by the U.S. Food and Drug Administration (FDA) for marketing in 9 months 2003; iseganan from porcine leukocytes, developed by Intrabiotics, is administered as an oral liquid or spray for the treatment of lung infections in patients with stomatitis and cystic fibrosis induced by antineoplastic therapy, and is currently also in phase III clinical trials; MBI-peptide from cattle, in the form of a topical cream for the treatment of urinary tract-related bloodstream infections, by Micrologix Biotech, in phase III clinics; histatin derived peptides from human saliva in the form of a mouth wash for treating dental inflammation and oral infections (phase II-III clinical), oral Candida infections (phase II clinical) and chronic Pseudomonas aeruginosa infections (phase I clinical). Therefore, the antibacterial peptide has good application prospect in the field of medicine, is not limited to the field of medicine, and has huge application potential in the fields of agriculture, animal husbandry and daily chemical products.
China is a fishery big country, and the total yield of aquatic products is the first place in the world. However, in recent years, with the development of high-density and intensive culture mode, the pollution degree of the water environment is increased, various bacterial and viral diseases are frequent, and a large amount of antibiotics are used for preventing and treating the diseases to destroy the micro-ecological balance of the water environment, so that certain pathogens generate drug resistance to drugs and harm human health. The 'malachite green event', 'turbot event', 'chloramphenicol event' and the like caused by excessive use of fishery drugs or addition of forbidden drugs in the process of aquaculture or aquatic product processing which occur successively can cover the aquatic industry with a layer of thick shadow, thus threatening the personal safety of consumers, and the food safety problem is concerned by people. In recent years, China has studied that antibacterial peptides are used for replacing traditional antibiotics to have better antibacterial effects on common pathogenic bacteria in the aquiculture process, such as Aeromonas hydrophila (Aeromonas hydrophylla), Vibrio metschnikovi (Vibrio metschnikovi), Edwardsiella tarda (edwards siella tarda) and Aeromonas sobria (Aeromonas sobria). The antibacterial peptide is taken in the feed, so that the effect of feeding the parent shrimps of the penaeus vannamei on egg laying, hatching rate and young shrimps and adult shrimps is obviously superior to that of aureomycin. The antibacterial peptide product can control microorganisms in water in the feed or water body of the prawns, so that pathogenic microorganisms such as vibrio and the like can be killed, and the feed can improve the immunity and promote the growth of the prawns after being taken into the bodies of the prawns. The research on the piscidins shows that the oral administration or injection of the medicine containing the piscidins to the groupers and the zebra fishes can improve the vibrio infection resistance of the groupers and the zebra fishes, and the research reports that the piscidins can effectively inhibit the growth of escherichia coli, Staphylococcus aureus and streptococcus, and the research also indicates that the mice are infected with a drug-resistant strain Staphylococcus aureus (MRSA), and the bacterial number in the mice can be effectively inhibited by administering the piscidins to the mice, so that the mice can survive. The antibacterial peptide also has a remarkable effect on the aspect of resisting tumors, and the Epinecidin-1 can effectively inhibit the growth of tumor cells. Research reports that when the antibacterial peptide is added into feed for feeding the carp, the carp serum phosphatase activity can be obviously improved, the contents of immune factors, immune globulin and interleukin in serum are also obviously improved, and the immunity of the carp is effectively improved.
Lateolabrax japonicus (Micropterus salmoides), also known as Micropterus salmoides, belongs to the family Sungloredae, genus Pericaceae, genus Plectrus, genus Panthenocissus, native North America Missippi river basin. Guangdong province is introduced in 1983, and the fish is mainly cultured in Guangdong, Jiangsu, Zhejiang, Jiangxi, Sichuan and Fujian provinces, and is an important freshwater culture variety in China. The antimicrobial peptide MSPiscidin-2 of the weever californica of the invention is searched and compared by a NCBI protein database in a complete sequence amino acid structure, and no identical polypeptide is found. The inventor searches and compares the encoding gene of Micropterus salmoides MSPiscidin-2 of the invention with NCBI gene database, and does not find any identical gene.
Disclosure of Invention
The invention provides an antimicrobial peptide, MSPiscidin-2, a gene thereof, a chemical synthesis method and application, wherein the antimicrobial peptide has strong antimicrobial activity under submicromolar dose and is derived from Micropterus salmoides. The invention aims to provide the micropterus salmoides MSPiscidin-2 which has strong antibacterial activity and particularly aims at various common pathogenic bacteria in aquaculture and application thereof based on the theoretical research and the prior art, and emphasizes the aquaculture field.
In order to realize the purpose of the invention, the invention provides the following technical scheme:
in a first aspect, the present invention provides an antimicrobial peptide MSPiscidin-2 having the amino acid sequence phenylalanine1-leucine2-lysine3-histidine4-isoleucine5-lysine6-serine7-phenylalanine8-tryptophan9-arginine10-Glycine11-alanine12-lysine13-alanine14-isoleucine15-phenylalanine16-arginine17-Glycine18-alanine19-arginine20-glutamine21-Glycine22-tryptophan23-arginine24-glutamic acid25-histidine26-arginine27(FLKHIKSFWRGAKAIFRGARQGWREHR) as shown in SEQ ID NO: 1.
Further, the antimicrobial peptide MSPiscidin-2 is derived from spleen and head kidney tissues of Micropterus salmoides, and the spleen and head kidney tissues of Micropterus salmoides are selected, washed and homogenized to obtain a crude tissue protein extract. Subsequently, Sephadex G-50 gel filtration chromatography was performed, 3 mL/tube was collected with an automatic fraction collector, and the antibacterial activity was measured by detecting at 220nm and collecting each peak, and lyophilized for use. Then gradient elution is carried out by reversed phase high pressure liquid chromatography (RP-HPLC), each polypeptide sample peak is collected, and is dissolved by sterilized deionized water, and the antibacterial activity is detected. And finally, analyzing the primary structure of the obtained polypeptide sample, wherein the analysis comprises the step of determining the molecular weight by adopting an electrospray quadrupole time-of-flight tandem mass spectrometry. By isoelectric focusingThe isoelectric point is determined by focusing electrophoresis, and the polypeptide amino acid sequence composition is determined by Edman degradation. The primary structure of the polypeptide complete sequence is as follows: phenylalanine1-leucine2-lysine3-histidine4-isoleucine5-lysine6-serine7-phenylalanine8-tryptophan9-arginine10-Glycine11-alanine12-lysine13-alanine14-isoleucine15-phenylalanine16-arginine17-Glycine18-alanine19-arginine20-glutamine21-Glycine22-tryptophan23-arginine24-glutamic acid25-histidine26-arginine27。
Further, MSPiscidin-2 is a linear polypeptide encoded by the micropidin gene of weever, california, contains 27 amino acid residues, has a theoretical isoelectric point (pI) of 12.69, a theoretical molecular weight of 3.34kDa, contains 10 basic amino acid residues (5 arginine, 3 lysine and 2 histidine), contains two acidic amino acid residues (1 glutamic acid, 1 glutamine), and has a net charge of +8, indicating that it is a stronger basic polypeptide. MSPiscidin-2 does not contain cysteine, so that intramolecular and intermolecular disulfide bonds are avoided, and the structure is simple. The MSPiscidin-2 complete sequence is as follows: phenylalanine1-leucine2-lysine3-histidine4-isoleucine5-lysine6-serine7-phenylalanine8-tryptophan9-arginine10-Glycine11-alanine12-lysine13-alanine14-isoleucine15-phenylalanine16-arginine17-Glycine18-alanine19-arginine20-glutamine21-Glycine22-tryptophan23-arginine24-glutamic acid25-histidine26-arginine27。
In a second aspect, the present invention provides a nucleic acid molecule encoding the antimicrobial peptide MSPiscidin-2 described above.
Specifically, the cloning of the Micropterus salmoides MSPiscidin-2 gene comprises the following steps: extracting total RNA of the head kidney and the spleen of the Micropterus salmoides, purifying mRNA, carrying out reverse transcription on the mRNA, constructing a cDNA library, designing a primer, and screening the Micropterus salmoides antibacterial peptide MSPiscidin-2 gene by using a PCR method. Obtaining positive single clone to proceed gene nucleotide sequence determination. The gene sequencing result shows that the gene for coding the antibacterial peptide MSPiscidin-2 precursor Piscidin consists of 465 nucleotides, and the sequence from the 5 'end to the 3' end is shown as SEQ ID NO. 7:
encoding the mature peptide MSPiscidin-2 of weever Californis Piscidin is 67 th to 147 th nucleotides.
In a third aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the antimicrobial peptide MSPiscidin-2.
In a fourth aspect, the present invention provides a host cell transformed with the above recombinant vector, said host cell being selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells.
The antimicrobial peptide MSPiscidin-2 of the invention can be prepared by a genetic engineering method or by a chemical method.
The gene engineering preparation method of the MSPiscidin-2 comprises the following steps:
the method comprises culturing the host cell in a medium suitable for the growth of the host cell to form and accumulate the antimicrobial peptide MSPiscidin-2, and recovering the accumulated antimicrobial peptide MSPiscidin-2 from the culture.
The chemical preparation method of the MSPiscidin-2 comprises the following steps:
the synthesis of the antimicrobial peptide MSPiscidin-2 comprises the following steps:
(1) synthesizing the full sequence of the MSPiscidin-2 amino acid sequence by using an automatic polypeptide synthesizer according to the gene code;
(2) desalting and purifying by HPLC reversed phase column chromatography, and determining the purity to be more than 95%;
(3) analyzing ionization time-of-flight mass spectrometry by matrix-assisted laser to determine the molecular weight; isoelectric point is determined by isoelectric focusing electrophoresis, and amino acid sequence structure is determined by automatic amino acid sequencer. The synthesized MSPiscidin-2 antibacterial peptide can be dissolved in sterilized ultrapure water and is used for pharmacological activity detection.
In a fifth aspect, the invention provides the use of the antimicrobial peptide MSPiscidin-2, the nucleic acid molecule provided by the second aspect, and the recombinant vector provided by the third aspect in the preparation of a medicament for resisting microbial infection.
Further, the microorganism is a gram-positive and/or gram-negative bacterium. The gram-positive bacteria are selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio Brazilian, Vibrio cholerae, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio splendens, Vibrio vulnificus, Aeromonas hydrophila, Aeromonas sobria and Aeromonas veronii. The gram-positive bacteria are selected from Staphylococcus aureus and Nocardia asteroides.
Further, the medicine is used for preventing and treating aquaculture diseases.
In a sixth aspect, the invention provides the antimicrobial peptide MSPiscidin-2, the nucleic acid molecule provided by the second aspect and the recombinant vector provided by the third aspect, wherein the recombinant vector is used as an additive in cosmetics, health products and foods to replace antibiotics or preservatives.
In a seventh aspect, the present invention provides a pharmaceutical composition, which comprises an effective amount of the above antimicrobial peptide MSPiscidin-2, the nucleic acid molecule provided by the above second aspect or the recombinant vector provided by the above third aspect, and a pharmaceutically acceptable carrier.
The invention has the beneficial effects that:
the gene of the coding Micropterus salmoides Piscidin antimicrobial peptide is obtained by gene cloning, and the mature peptide MSPiscidin-2 is obtained by a chemical synthesis method. The antimicrobial peptide is rich in basic amino acid, has strong bactericidal effect on common pathogenic bacteria of aquaculture, has a simple structure, does not contain disulfide bonds and cyclic structures, is convenient for chemical synthesis and genetic engineering preparation, and is not easy to cause drug resistance.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the technical solutions, but the present invention is not limited thereto. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The term "nucleic acid molecule" as used herein refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
The term "vector" refers to a construct capable of delivering one or more genes or sequences of interest and preferably expressing it in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells. In one embodiment of the present disclosure, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along with the host genome (e.g., non-episomal mammalian vectors).
The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include microbial (e.g., bacterial), plant, or animal cells. Bacteria susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.
As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.
As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a particular portion of nucleic acid, RNA, and/or DNA are amplified as described, for example, in U.S. patent No. 4,683,195. In general, it is desirable to obtain sequence information from the ends of or beyond the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotide of the 2 primers may coincide with the end of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al (1987) Cold Spring Harbor Symp. Ouant. biol.51: 263; erlich editors, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.
"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.
An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. In one embodiment of the present disclosure, an "effective amount" is the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial results that eliminate or reduce the risk, lessen the severity, or delay the onset of a disorder (including, but not limited to, the disorder, its complications, and biochemical, histological, and/or behavioral symptoms of intermediate pathological phenotypes present during the development of the disorder); in another embodiment, an "effective amount" is the amount of a drug, compound, or pharmaceutical composition necessary to achieve a beneficial or desired clinical result (including, but not limited to, such as reducing the incidence of or ameliorating one or more symptoms of various thrombin-related disorders, reducing the dosage of other agents required to treat a disorder, enhancing the therapeutic efficacy of another agent, and/or delaying the progression of a thrombin-related disorder in a patient).
EXAMPLE 1 isolation and purification of antimicrobial peptide MSPiscidin-2
(1) Fresh and alive Lateolabrax japonicus was purchased from a fresh market, and fresh and intact spleen and head kidney tissues were dissected and collected, and the surfaces of the spleen and head kidney tissues were slightly washed with physiological saline. After homogenization, the mixture is dissolved by a small amount of normal saline, and the ratio of the homogenate tissue dissolving solution to n-butanol 1: stirring the homogenate with n-butanol at 50(V/V) ratio at room temperature for 60min, 13000r/min, centrifuging twice for 20min, and freeze-drying the precipitate. Subsequently, a first step of Sephadex G-50 gel filtration chromatography: 0.9G of the lyophilized powder was dissolved in 10mL of 0.1M phosphate (Na2HPO4-NaH2PO4, pH 6.0) buffer, centrifuged at 12000rpm for 10min, the supernatant was applied to a well-equilibrated Sephadex G-50 gel exclusion chromatography column (1.6 cm. times.90 cm, Amersham Bioscience), eluted with the same buffer at a flow rate of 3mL/10min, collected in 3 mL/tube using an automatic fraction collector, detected at 220nm and each peak for detection of antibacterial activity, and lyophilized for use.
(2) Reverse phase high pressure liquid chromatography (RP-HPLC): dissolving the peak of the active component obtained by Sephadex G-50 gel exclusion chromatography in pure water again, centrifuging at 12000rpm for 15min at 4 ℃, taking the supernatant, filtering with a 0.45 μm filter membrane, collecting the filtrate, loading the filtrate on a C18 reverse phase column (Hypersil BDS C18,30cm x 0.46cm) fully balanced by ultrapure water containing 1 ‰ trifluoroacetic acid, performing gradient elution by an elution system composed of acetonitrile (containing 1 ‰ trifluoroacetic acid), and detecting the polypeptide concentration at 215 nm. The resulting peaks were collected, lyophilized, concentrated, redissolved with sterile deionized water and tested for antimicrobial activity.
(3) Primary structure analysis of active polypeptide. Purified MSPiscidin-2 was measured for molecular weight using electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS, Biosystems/MDS Sciex, Toronto, Canada). The isoelectric point is determined by isoelectric focusing electrophoresis, and the amino acid sequence component is determined to be MSPiscidin-2 by Edman degradation method: FLKHIKSFWRGAKAIFRGARQGWREHR (SEQ ID NO: 1).
Example 2 cloning and Gene sequencing of the MSPiscidin-2 precursor Gene
First, total RNA extraction of a mixture of the head kidney and spleen of the Micropterus salmoides (instruments and reagents used in the following experiments are treated and no RNase is used):
A. cutting about 1g of small pieces from fresh tissues of a head kidney and a spleen of a live dissection of a micropterus salmoides, putting the small pieces into a cell cryopreservation tube precooled by liquid nitrogen, and then quickly putting the tube into the liquid nitrogen for preservation;
B. taking out the tissue material preserved in the liquid nitrogen, putting the tissue material into a precooled mortar, quickly and fully grinding the tissue material, and continuously adding a little liquid nitrogen into the mortar in the period; approximately 30mg of the tissue powder was transferred to a pre-cooled 1.5mL centrifuge tube, to which 1mL of total RNA extraction buffer (Trizol, product of Life, USA) was added, mixed well, and then centrifuged at 12000rpm for 10min at 4 ℃;
C. the centrifuged supernatant was transferred to a new 1.5mL centrifuge tube, 200. mu.L of chloroform solution was added thereto, mixed vigorously, left at room temperature for 10 minutes, centrifuged at 12000rpm at 4 ℃ for 10 minutes, and the precipitate was discarded.
D. Adding equal volume of isopropanol into the supernatant, standing at room temperature for 10min, centrifuging at 4 deg.C and 12000rpm for 10min, collecting precipitate, washing with 75% (V/V) ethanol, and air drying to obtain the final product, which is total RNA of mixture of head, kidney and spleen of Japanese sea Perch.
The second step, cDNA library construction, uses the In-Fusion SMARTer of CLONTECH companyTMSynthesis of the directive cDNA Library Construction Kit.
First strand cDNA Synthesis (reverse transcription of mRNA):
A. in a new 0.2mL PCR tube (without DNase and RNase) the following mix was prepared:
mixing well, and centrifuging briefly (2000rpm, 30 s); after centrifugation, the PCR tube is incubated for 3min at 72 ℃ in a PCR instrument, and then the PCR tube is incubated for 2min at 42 ℃; after brief centrifugation, the following reverse transcription reaction solutions were prepared in the tubes:
after mixing well, centrifuge briefly (2000rpm, 30 s); the following procedure was performed in a PCR instrument:
at 42 ℃ for 90 min; at 68 ℃ for 10 min; the first strand synthesis was then terminated by placing the centrifuge tube on ice. mu.L of the first strand of the synthesized cDNA was taken from the centrifuge tube and used.
Second chain synthesis: amplifying the second strand by long-terminal polymerase chain reaction (LD-PCR) (all reagents are In-Fusion SMARTer from CLONTECH)TMPrepared in the Library Construction Kit of the directive cDNA Library Construction Kit)
The above PCR tubes were mixed in a pre-heated PCR tube at 95 ℃.
Then, the following procedure was followed for amplification in a PCR instrument:
at 95 ℃ for 1 min; 18 cycles: 95 ℃, 15s, 65 ℃,30 s, 68 ℃ and 6 min. After the circulation was completed, the cDNA double strand synthesized in the centrifuge tube was stored at-80 ℃.
Thirdly, cloning the encoding gene of Micropterus salmoides antimicrobial peptide MSPiscidin-2
According to the conserved sequence of the signal peptide region of the antibiotic peptide of the Piscidin family, a forward primer P1 is artificially designed and synthesized: 5 ' -GTATTRTGATCTTTCTTGTGYTGTC-3 ' (SEQ ID NO:2), and the reverse primer was the 3 ' -PCR primer from the In-Fusion SMARTer TM directed cDNA Library Construction Kit from CLONTECH having the sequence 5'-CGGGGTACGATGAGACACCAT-3' (SEQ ID NO: 3). The primers were centrifuged at 12000rpm for 5min before use, and then the corresponding volume of ddH was added in the indicated molar amounts2O was dissolved to a concentration of 20. mu.M. First PCR amplification was performed using the synthesized cDNA as a template and P1 and In-fusion SMARTERCDS as primers. The following reagents (20 μ L in total volume) were added to a 0.2ml PCR tube:
after mixing, centrifugation was carried out briefly. The PCR conditions were: denaturation at 95 deg.C for 4 min; 30 cycles: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 1 min; extending for 10min at 72 ℃; storing at 4 ℃. After the reaction, 5. mu.L of the product was electrophoresed on 1% agarose gel to detect the band of interest.
Taking 1 mu L of PCR product obtained in the previous step, adding 99 mu L of ddH2O diluted 100-fold as template, P2: 5'-GGTCGTCCTCATGGCTGAACC-3' (SEQ ID NO:4) and CDS III: 5'-CGGGGTACGATGAGACACCAT-3' (SEQ ID NO:3) as primers, a second PCR amplification was performed. The following reagents (20 μ L in total volume) were added to a 0.2ml PCR tube:
the PCR conditions were: denaturation at 95 deg.C for 4 min; 25 cycles: denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1 min; extending for 10min at 72 ℃; storing at 4 ℃. After the reaction, 5. mu.l of the sample was subjected to electrophoresis on 1% agarose gel to detect the desired band.
After the amplification, the target fragment was recovered with a gel recovery kit (Tiangen). The recovered DNA fragment of interest was ligated with sequencing vector pMD19-T Vecter (Takara, Dalian) to transform DH 5. alpha. competent cells. 100 mu L of the transformant was applied evenly to LB agar plates containing 100. mu.g/mL ampicillin (Amp), and the plates were placed in a 37 ℃ incubator and cultured upside down for 12-16 hours. Single colonies were picked and tested for insert size by PCR using M13 primer (forward M13-F: 5'-GTAAAACGACGGCCAGTG-3' (SEQ ID NO: 5); reverse M13-R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO: 6)). Positive colonies were picked, plasmids were extracted by shake culture, and nucleotide sequencing was performed using an Applied Biosystems DNA sequencer, model ABI PRISM 377.
Fourthly, determining the gene sequence of the bass Piscidin and obtaining the result:
the gene sequencing result shows that the gene coding the MSPiscidin-2 precursor Piscidin consists of 465 nucleotides, and the sequence from the 5 'end to the 3' end is (SEQ ID NO:7):
the cDNA nucleotide sequence of the Micropterus salmoides Piscidin coding region is as follows: the sequence length is 465 bases, and the sequence types are as follows: nucleic acid, strand number: single strand, topology: linear, sequence type: cDNA, source: the kidneys and spleen of the micropterus salmoides.
The encoding Micropterus salmoides Piscidin mature peptide MSPiscidin-2 is 67-147 nucleotides, and the amino acid sequence is as follows: FLKHIKSFWRGAKAIFRGARQGWREHR (SEQ ID NO:1), namely: phenylalanine1-leucine2-lysine3-histidine4-isoleucine5-lysine6-serine7-phenylalanine8-tryptophan9-arginine10-Glycine11-alanine12-lysine13-alanine14-isoleucine15-phenylalanine16-arginine17-Glycine18-alanine19-arginine20-glutamine21-Glycine22-tryptophan23-arginine24-glutamic acid25-histidine26-arginine27。
Example 3 chemical preparation of MSPiscidin-2:
(1) the full sequence was synthesized using an automated polypeptide synthesizer (433A, Applied Biosystems) based on the deduced amino acid sequence of the mature peptide MSPiscidin-2 encoding the Piscidin gene from Micropterus salmoides.
(2) By HPLC reverse phase C18Desalting and purifying the synthesized polypeptide by column chromatography: the column used in the process is Gemini-NX 5 mu C18100A, 4.6 multiplied by 250 mm; solvent A is 0.1% Trifluoroacetic in 100% acetonitrile, solvent B is 0.1% Trifluoroacetic in 100% water; the gradient is set as: 0.01min (A10%, B90%), 25min (A70%, B30%), 25.1min (A100%, B0%), 30min (stop); the flow rate is 1.0mL/min, the wavelength is 220nm, the volume is 20 muL,the purity of the synthesized polypeptide sequence is more than 95 percent.
(3) And (3) determining the molecular weight by adopting matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF): firstly, dispersing an MSPiscidin-2 sample in matrix molecules to form crystals, then irradiating the crystals with laser, absorbing energy from the laser by the matrix, desorbing the sample, ionizing sample molecules by charge transfer between the matrix and the sample, flying the ionized sample through a vacuum flight tube under the action of an electric field, and detecting according to different flight times of a detector, namely analyzing ions by the fact that the mass-to-charge ratio (M/Z) of the ions is in direct proportion to the flight time of the ions, and measuring the molecular weight of the sample molecules to obtain the molecular weight of the MSPiscidin-2 sample of 3339.86.
(4) Isoelectric focusing electrophoresis determination of isoelectric point: preparing glue: the glue solution is composed of a carrier ampholyte, gel liquid storage, distilled water, a mixed sample, a dyeing substance and the like, the glue is filled, an electrode solution is added, electrophoresis is carried out, and the sample is collected for detection to obtain the MSPiscidin-2 isoelectric point.
(5) The amino acid sequence structure was determined using an automated amino acid sequencer. The synthesized MSPiscidin-2 antibacterial peptide can be dissolved in sterilized ultrapure water or PBS for pharmacological activity detection.
The Micropterus salmoides antimicrobial peptide MSPiscidin-2 is a small molecular polypeptide encoded by a gene, consists of 27 amino acid residues, has the molecular weight of 3.34kDa and the isoelectric point of 12.69. The complete sequence of the Micropterus salmoides antimicrobial peptide MSPiscidin-2 is as follows: FLKHIKSFWRGAKAIFRGARQGWREHR (SEQ ID NO:1), namely: phenylalanine1-leucine2-lysine3-histidine4-isoleucine5-lysine6-serine7-phenylalanine8-tryptophan9-arginine10-Glycine11-alanine12-lysine13-alanine14-isoleucine15-phenylalanine16-arginine17-Glycine18-alanine19-arginine20-glutamine21-Glycine22-tryptophan23-arginine24-glutamic acid25-histidine26-arginineAcid(s)27。
Test example antimicrobial peptide of weever, california MSPiscidin-2 antimicrobial activity assay:
(1) and (3) detecting the antibacterial activity of the MSPiscidin-2:
dissolving chemically synthesized MSPiscidin-2 in sterile ultrapure water at a concentration of 2 mg/mL; picking the newly activated microorganism with an inoculating loop, and then spreading evenly on a new MH (Mueller-Hinton agar plate) agar plate; placing a circular sterile filter paper sheet with the diameter of 0.5cm on the agar plate, and then slowly dropwise adding 10 mu of LMSpiscidin-2 sample solution onto the paper sheet; culturing in a 37 deg.C constant temperature incubator for 12-24 hr; observing whether a zone of inhibition is formed, and recording strains sensitive to the MSPiscidin-2.
(2) MSPiscidin-2 was assayed for sensitive strain Minimum Inhibitory Concentration (MIC):
the experiment uses sterile liquid MH culture medium as a negative control, and the minimum inhibitory concentration is defined as the lowest polypeptide concentration which can completely inhibit the growth of microorganisms observed by naked eyes or the lowest concentration of which the light absorption value is not higher than 5% of that of the negative control. Selecting newly activated microorganisms, inoculating to sterile liquid MH culture medium, and culturing in a 37 deg.C constant temperature oscillator at 200rpm for 10-16h to logarithmic phase; measuring the light absorption value of the bacteria solution at 600nm light wave position by using an ultraviolet spectrophotometer, wherein the concentration is about 10 when the light absorption value is 19CFU/mL, diluting the bacterial liquid to 10 with sterile liquid MH medium6CFU/mL, on ice for standby; preparing MSPiscidin-2 sample solutions (i.e., 59.8828. mu.M, 29.9414. mu.M, 14.9707. mu.M, 7.4853. mu.M, 3.7427. mu.M, 1.8713. mu.M, 0.9357. mu.M, 0.4678. mu.M, 0.2339. mu.M) with concentration gradients of 200. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, 1.5625. mu.g/mL on sterile 96-microwell plates through a 0.22 μ M-pore sterile filter, at 50. mu.L per well; adding 50 μ L of the diluted bacteria solution into each well, mixing well to obtain final concentration of MSPiscidin-2 sample of 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL, 1.5625 μ g/mL, 0.78125 μ g/mL (i.e. 29.9414 μ M, 14.9707 μ M, 7.4853 μ M, 3.7427 μ M, 1.8713 μ M, 0.9357 μ M, 0.4678 μ M, 0.2339 μ M), culturing in 37 deg.C incubatorCulturing for 10-16 h; measuring the light absorption value at 600nm by using an enzyme-linked immunosorbent assay (ELISA) reader or observing by naked eyes; the above experiment was repeated 3 times and the average was taken.
(3) Kinetics of sterilization of MSPiscidin-2 against Aeromonas hydrophila and Vibrio parahaemolyticus:
inoculating Aeromonas hydrophila to MH solid culture medium plate, and performing inverted culture in an incubator at 37 ℃ until bacterial colony grows out. Single colonies were picked with an inoculating loop and inoculated into MH liquid medium, and cultured in a shaking incubator at 37 ℃ to logarithmic phase. The culture broth was diluted to 1X 10 with fresh MH broth6CFU/mL, the MSPiscidin-2 sample was added to the diluted bacterial suspension to a final concentration of 5 × MIC (negative control added to corresponding volume of sterilized ultrapure water). Adding the bacterial liquid into the sample, quickly placing into a shaking incubator at 37 ℃, shaking and culturing at 150rpm, respectively taking 10 mu L of bacterial liquid at 0min, 15min, 30min, 60min, 120min and 180min, diluting with sterilized normal saline by 500 times, and taking 50 mu L of MH solid plate coated. Neomycin sulfate was used as a positive control. The plates were placed in a 37 ℃ incubator for inverted culture for 16h, after which colony counting was performed.
The kinetics of the sterilization of Vibrio parahaemolyticus by MSPiscidin-2 was also described above. :
see tables 1-3 below for results:
TABLE 1 Minimum Inhibitory Concentration (MIC) of MSPiscidin-2
MIC: minimum inhibition Concentration, the Concentration value is the average value of 3 repeated experiments; the above results are the average of three independent replicates.
As can be seen from Table 1, MSPiscidin-2 has strong antibacterial activity on tested gram-positive bacteria and gram-negative bacteria, most of tested strains are aquaculture pathogenic bacteria, and the MIC value of the MSPiscidin-2 is in the range of 2.8085-11.2280 mu M.
TABLE 2 kinetics of the Sterilization of MSPiscidin-2 against Staphylococcus aureus CMCC26003
TABLE 3 kinetics of the Sterilization of MSPiscidin-2 against Vibrio vulnificus
As can be seen from the table 2, the MSPiscidin-2 can kill all staphylococcus aureus CMCC26003 within 15 minutes, the positive control neomycin sulfate can kill all staphylococcus aureus CMCC26003 within 30 minutes, and the table 3 shows that the MSPiscidin-2 kills all vibrio vulnificus within 120 minutes, and is equivalent to the positive control neomycin sulfate, and the result proves that the MSPiscidin-2 not only has strong bactericidal activity, but also has the bactericidal speed which is faster than or equivalent to that of the antibiotic drug neomycin sulfate; moreover, the effect of the MSPiscidin-2 on bacteria is lethal, the staphylococcus aureus CMCC26003 and the vibrio vulnificus die after the effect of the MSPiscidin-2, no bacteria recover to grow after 3 hours, and the good and durable bactericidal effect of the MSPiscidin-2 is shown.
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Claims (8)
1. An antimicrobial peptide MSPiscidin-2 is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the antimicrobial peptide MSPiscidin-2 according to claim 1.
3. A recombinant vector comprising the nucleic acid molecule of claim 2.
4. A host cell transformed with the recombinant vector of claim 3, said host cell selected from the group consisting of prokaryotic cells and eukaryotic cells.
5. The host cell of claim 4, which is selected from mammalian cells.
6. Use of the antimicrobial peptide MSPiscidin-2 according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3 for the preparation of a medicament for combating microbial infections; the microorganism is gram-positive bacteria and/or gram-negative bacteria; the gram-positive bacteria are selected from Vibrio alginolyticus, Vibrio anguillarum, Vibrio Brazilian, Vibrio cholerae, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio splendens, Vibrio vulnificus, Aeromonas hydrophila, Aeromonas sobria and Aeromonas veronii; the gram-positive bacteria are selected from staphylococcus aureus and nocardia asteroides.
7. The use according to claim 6, wherein the medicament is for aquaculture disease control.
8. A pharmaceutical composition comprising an effective amount of the antimicrobial peptide MSPiscidin-2 of claim 1, the nucleic acid molecule of claim 2 or the recombinant vector of claim 3, and a pharmaceutically acceptable carrier.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013078217A2 (en) * | 2011-11-21 | 2013-05-30 | Board Of Regents Of The University Of Nebraska | Anti-microbial peptides and methods of use thereof |
| CN103804482A (en) * | 2014-02-26 | 2014-05-21 | 中国科学院海洋研究所 | Antibacterial peptide and application thereof |
| CN105727258A (en) * | 2016-02-02 | 2016-07-06 | 深圳大学 | Application of marine-organism antimicrobial peptide and chemical composition with marine-organism antimicrobial peptide |
| CN105801680A (en) * | 2016-05-17 | 2016-07-27 | 中国科学院南海海洋研究所 | Grouper piscidin 4 polypeptide and application thereof |
| EP3096616A2 (en) * | 2014-01-24 | 2016-11-30 | The Regents of the University of Colorado, a Body Corporate | Dermaseptin-type and piscidin-type antimicrobial peptides |
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| CN105566483A (en) * | 2014-10-15 | 2016-05-11 | 深圳大学 | Epinephelus malabaricus Piscidin transformation body antimicrobial peptide and application thereof |
| CN106367429A (en) * | 2016-08-30 | 2017-02-01 | 福建福鼎海鸥水产食品有限公司 | Construction and secretory expression of tandem piscidin antimicrobial peptide PSP |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013078217A2 (en) * | 2011-11-21 | 2013-05-30 | Board Of Regents Of The University Of Nebraska | Anti-microbial peptides and methods of use thereof |
| EP3096616A2 (en) * | 2014-01-24 | 2016-11-30 | The Regents of the University of Colorado, a Body Corporate | Dermaseptin-type and piscidin-type antimicrobial peptides |
| CN103804482A (en) * | 2014-02-26 | 2014-05-21 | 中国科学院海洋研究所 | Antibacterial peptide and application thereof |
| CN105727258A (en) * | 2016-02-02 | 2016-07-06 | 深圳大学 | Application of marine-organism antimicrobial peptide and chemical composition with marine-organism antimicrobial peptide |
| CN105801680A (en) * | 2016-05-17 | 2016-07-27 | 中国科学院南海海洋研究所 | Grouper piscidin 4 polypeptide and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Piscidin: Antimicrobial peptide of rock bream, Oplegnathus fasciatus;Jin-Sol Bae等;《Fish Shellfish Immunol》;20160430;第51卷;136-142 * |
| piscidin-like protein[Micropterus salmoides];Genbank Database;《Genbank Database》;20210914;Accession No:UAD82650.1 * |
| 基于转录组的大口黑鲈（Micropterus salmoides）piscidins基因鉴定及感染胁迫响应研究;杨怀欣;《中国优秀硕士学位论文全文数据库农业科技辑》;20210612;1-92 * |
| 大弹涂鱼(Boleophthalmus pectinirostris)piscidin 1的分子特征及功能分析;管峰等;《海洋与湖沼》;20180315(第02期);194-203 * |
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