阅读说明：本技术 一种抗微生物肽MSPiscidin-3、其编码基因及用途 (Antimicrobial peptide MSPiscidin-3, and coding gene and application thereof ) 是由 于海宁 王义鹏 杨怀欣 于 2020-04-30 设计创作，主要内容包括：本发明公开了一种抗微生物肽MSPiscidin-2、其编码基因及用途,MSPiscidin-3是一种直链多肽,含有25个氨基酸残基,理论等电点为12.81,分子量是2.97kDa,净电荷为+9。编码抗微生物肽MSPiscidin-3前体Piscidin的基因由351个核苷酸组成。MSPiscidin-3对革兰氏阴性菌、革兰氏阳性菌均有很强的抗菌活性,包括多种水产养殖致病菌；其不含有二硫键及环状结构,方便化学合成及基因工程制备；可用于制备抗病原微生物感染以及消炎用的临床药物；或作为动物饲料中抗生素的替代品；或作为化妆品、保健品、食品中的添加剂以替代传统防腐剂,有很好的应用前景。(The invention discloses an antimicrobial peptide MSPiscidin-2, a coding gene and application thereof, wherein the MSPiscidin-3 is a straight-chain polypeptide, contains 25 amino acid residues, has a theoretical isoelectric point of 12.81, a molecular weight of 2.97kDa and a net charge of + 9. The gene encoding the antimicrobial peptide MSPiscidin-3 precursor Piscidin consists of 351 nucleotides. MSPiscidin-3 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.)

1. An antimicrobial peptide MSPiscidin-3 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-3 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; preferably eukaryotic cells; more preferably mammalian cells.

5. Use of the antimicrobial peptide MSPiscidin-3 according to claim 1, the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 3 for the preparation of a medicament for combating microbial infections.

6. Use according to claim 5, wherein 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 lautus, Vibrio vulnificus, Aeromonas hydrophila, Aeromonas sobria and Aeromonas veronii;

the gram-positive bacteria are selected from staphylococcus aureus, staphylococcus aureus and nocardia asteroides.

7. The use according to claim 5 or 6, wherein the medicament is for aquaculture disease control.

8. Use of the antimicrobial peptide MSPiscidin-3 according to claim 1, the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 3 as an additive in cosmetics, nutraceuticals or foodstuffs.

9. A pharmaceutical composition comprising an effective amount of the antimicrobial peptide MSPiscidin-3 of claim 1, the nucleic acid molecule of claim 2 or the recombinant vector of claim 3, and a pharmaceutically acceptable carrier.

Technical Field

The invention discloses an antimicrobial peptide MSPiscidin-3, a coding gene and application thereof, and particularly provides a Piscidin family broad-spectrum antimicrobial peptide MSPiscidin-3 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 mice are infected by a drug-resistant strain staphylococcus aureus (MRSA), and the quantity of bacteria 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-3 of the weever californica of the invention is searched and compared by a NCBI protein database in a full sequence amino acid structure, and no identical polypeptide is found. The inventor searches and compares the encoding gene of Micropterus salmoides MSPiscidin-3 of the invention with NCBI gene database, and does not find any identical gene.

Disclosure of Invention

The invention provides an antimicrobial peptide, MSPiscidin-3, 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-3 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:

an antimicrobial peptide MSPiscidin-3 with amino acid sequence of phenylalanine¹-isoleucine²-phenylalanine³-histidine⁴-valine⁵-isoleucine⁶-lysine⁷-Glycine⁸-leucine⁹-phenylalanine¹⁰-histidine¹¹-alanine¹²-Glycine¹³-lysine¹⁴-methionine¹⁵-isoleucine¹⁶-histidine¹⁷-Glycine¹⁸-leucine¹⁹-valine²⁰-threonine²¹-arginine²²-arginine²³-arginine²⁴-histidine²⁵(FIFHVIKGLFHAGKMIHGLVTRRRH) is shown in SEQ ID NO: 1.

Further, the antimicrobial peptide MSPiscidin-3 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 measuring the molecular weight by adopting an 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 polypeptide amino acid sequence composition is determined by Edman degradation. The primary structure of the polypeptide complete sequence is as follows: phenylalanine¹-isoleucine²-phenylalanine³Group ofAmino acid⁴-valine⁵-isoleucine⁶-lysine⁷-Glycine⁸-leucine⁹-phenylalanine¹⁰-histidine¹¹-alanine¹²-Glycine¹³-lysine¹⁴-methionine¹⁵-isoleucine¹⁶-histidine¹⁷-Glycine¹⁸-leucine¹⁹-valine²⁰-threonine²¹-arginine²²-arginine²³-arginine²⁴-histidine²⁵。

Further, MSPiscidin-3 is a linear polypeptide encoded by the California weever Piscidin gene, contains 25 amino acid residues, has a theoretical isoelectric point (pI) of 12.81 and a theoretical molecular weight of 2.97kDa, contains 9 basic amino acid residues (3 arginine, 2 lysine and 4 histidine), does not contain acidic amino acid residues, and has a net charge of +9, indicating that it is a stronger basic polypeptide. MSPiscidin-3 does not contain cysteine, so that intramolecular and intermolecular disulfide bonds are avoided, and the structure is simple. The MSPiscidin-3 complete sequence is as follows: phenylalanine¹-isoleucine²-phenylalanine³-histidine⁴-valine⁵-isoleucine⁶-lysine⁷-Glycine⁸-leucine⁹-phenylalanine¹⁰-histidine¹¹-alanine¹²-Glycine¹³-lysine¹⁴-methionine¹⁵-isoleucine¹⁶-histidine¹⁷-Glycine¹⁸-leucine¹⁹-valine²⁰-threonine²¹-arginine²²-arginine²³-arginine²⁴-histidine²⁵。

In a second aspect, the present invention provides a nucleic acid molecule encoding the antimicrobial peptide MSPiscidin-3 described above.

Specifically, the cloning of the Micropterus salmoides MSPiscidin-3 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-3 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-3 precursor Piscidin consists of 351 nucleotides, and is shown as SEQ ID NO. 7, and the sequence from the 5 'end to the 3' end is as follows:

1 ATGAGGTGCATCACCCTCTTTCTTGTGTTGTCGCTGGTGGTCCTCATGGC

51 TGAACCCGGGGAGGGTTTTATCTTCCACGTCATCAAAGGACTCTTTCACG

101 CTGGCAAGATGATCCATGGACTTGTCACCAGGAGACGACATGGCATGGAA

151 GAGCTGCAAGACCTGGACCAACGTGCCTTTGAACGAGAGTAGACTAAAGG

201 GGCCATTGGAATGAAGGATGCTTTGAATGAAAAGAAATGCTTCTTGTTCA

251 CCACTTAATATATATATTGCACTGCAGCTAACTGATTGAAATTGTTTGGG

301 AATAAATTTGCATTGGAGTGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAA

351 A

encoding mature peptide MSPiscidin-3 of weever California Piscidin is 67-141 nucleotides.

In a third aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the antimicrobial peptide MSPiscidin-3.

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-3 of the invention can be prepared by a genetic engineering method or by a chemical method.

The gene engineering preparation method of the MSPiscidin-3 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-3, and recovering the accumulated antimicrobial peptide MSPiscidin-3 from the culture.

The chemical preparation method of the MSPiscidin-3 comprises the following steps:

the synthesis of the antimicrobial peptide MSPiscidin-3 comprises the following steps:

(1) synthesizing the full sequence of the MSPiscidin-3 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-3 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-3, 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 lautus, Vibrio vulnificus, Aeromonas hydrophila, Aeromonas sobria and Aeromonas veronii. The gram-positive bacteria are selected from the group consisting of Staphylococcus aureus, and Nocardia asteroides.

Further, the medicine is used for preventing and treating aquaculture diseases.

In a sixth aspect, the invention provides the use of the antimicrobial peptide MSPiscidin-3, the nucleic acid molecule provided by the second aspect and the recombinant vector provided by the third aspect as additives of 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-3, 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 then the mature peptide MSPiscidin-3 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.