KR20230091424A - Antimicrobial peptide and its uses - Google Patents

Abstract

The present invention relates to a recombinant fusion antibacterial peptide, and the recombinant antibacterial peptide of the present invention artificially has a high positive charge and is recombined to improve antibacterial activity, thereby reducing the problem of antimicrobial activity due to the increase in the number of bacteria of the conventional natural antibacterial peptide and the small amount It solves the problem of insignificant antibacterial effect, is easy to express and purify in Escherichia coli, has excellent antibacterial power against major bacterial disease causative bacteria, and has been confirmed to be safe and effective in pigs, the target animal. It can be usefully used as an antibiotic or feed additive that can be reduced.

Description

Antimicrobial peptide and its uses {Antimicrobial peptide and its uses}

The present invention relates to recombinant fusion antimicrobial peptides.

Bacterial infection is one of the most common and fatal causes of human disease. Unfortunately, the overuse of antibiotics has caused bacterial antibiotic resistance. Currently, compared to countries such as the United States, Japan, and Europe, antibiotic use is high in Korea, and misuse of animal drugs such as antibiotics causes antibiotic retention and the emergence of resistant bacteria, threatening not only animals but also human health. there is. Antibiotic tolerance, which is a phenomenon distinct from resistance to antibiotics, was first discovered in Pneumococcus sp. in the 1970s and provided important clues to the mechanism of action of penicillin. Species resistant to antibiotics stop growing in the presence of normal concentrations of antibiotics, but do not die as a result. Resistance occurs because when antibiotics inhibit cell wall synthesis enzymes, bacterial autolytic enzymes, such as autolysin, do not activate. By doing so, it kills bacteria, and bacteria also inhibit their activity, resulting in survival even when treated with antibiotics. It is of great clinical importance that bacteria have resistance to various antibiotics, because when it is impossible to eradicate resistant bacteria, the effectiveness of antibiotic treatment in clinical infections is reduced. In addition, the development of resistance is considered to be a prerequisite for the development of resistance to antibiotics because it results in strains that survive despite antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics. Therefore, there is a need for the development of novel antibiotics.

On the other hand, antibiotics used in the livestock industry in the past were widely used for the treatment of infectious diseases, prevention of bacterial infections, and growth promoters, but the rate of growth promotion by antibiotics decreased due to resistance due to continued use. In addition, as consumer interest in antibiotic resistance and safe food has recently increased, the use of antibiotics was stopped in Europe in 2006, and in the United States in 2013, it has come to recommend that farmers stop using antibiotics. When such antibiotics are absorbed into the human body, side effects and a vicious cycle of strengthening the bacterial compound's adaptability to resistance occur are repeated. Occurs. Due to these side effects, Korea reduced the number of antibiotics for feed additives from 44 to 28 from May 2005, and banned the use of all antibacterial and antifungal antibiotics for feed additives from July 2011. As the use of antibiotics is banned worldwide, many diseases that have been suppressed due to the use of antibiotics reappear, which is now a threat to the livestock industry, leading to national damage. In the future, due to the ban on the use of antibiotics, the frequency of infectious diseases in the livestock industry is further increasing, and the need to develop antibiotic alternatives to reduce livestock infectious diseases is increasing.

Recently, domestic and foreign researchers are paying attention to antimicrobial peptide (AMP) as a way to solve the existing problems. Antibacterial peptides are the foremost biological defense factors of the innate immune system of living organisms. Unlike acquired immunity that reacts to specific microorganisms or antigens, they act regardless of the type of microorganisms, and the reaction time is very short or within a few hours. fast. Antibacterial peptides found in most animals and plants bind to the cell membranes of various bacteria and create pores in the cell membranes to kill cells. Antimicrobial peptides are called natural antibiotics because they are naturally produced to protect the host from microorganisms in a wide range of biological systems without problems of resistance, immunity or rejection, and are distinguished from conventional antibiotics that always have problems with resistant bacteria.

An object of the present invention is to provide recombinant antimicrobial peptides.

In addition, an object of the present invention is to provide an antibiotic containing the recombinant antimicrobial peptide as an active ingredient.

In addition, an object of the present invention is to provide a feed additive containing the recombinant antibacterial peptide as an active ingredient.

In addition, an object of the present invention is to provide an antibacterial food additive containing the recombinant antimicrobial peptide as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bacterial infection containing the recombinant antimicrobial peptide as an active ingredient.

In addition, an object of the present invention is to provide a method for producing recombinant antimicrobial peptides.

In order to achieve the above object, the present invention is to provide a recombinant antimicrobial peptide.

In addition, the present invention provides an antibiotic containing the recombinant antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide a feed additive containing the recombinant antibacterial peptide as an active ingredient.

In addition, the present invention is to provide an antibacterial food additive containing the recombinant antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide a pharmaceutical composition for preventing or treating bacterial infection containing the recombinant antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide a method for producing a recombinant antimicrobial peptide.

The recombinant antibacterial peptide of the present invention has an artificially high positive charge and is recombined to improve antibacterial activity, thus solving the problem of the decrease in antibacterial activity due to the increase in the number of bacteria and the insignificant antibacterial effect of a small amount of the conventional natural antibacterial peptide, in E. coli It is easy to express and purify, has excellent antimicrobial activity against major bacterial disease causative organisms, and has been confirmed to be safe and effective in pigs, the target animal. Therefore, it is useful as an antibiotic or feed additive that can reduce major bacterial digestive and respiratory diseases. can

1 is a diagram showing the configuration of a vector encoding a recombinant antimicrobial peptide of the present invention.
Figure 2 is a diagram confirming the expression of the recombinant lysozyme-thrombin-HJP34 fusion protein through SDS-PAGE:
M: marker;
Lane 1: before IPTG treatment;
Lane 2: after IPTG treatment;
Lane 3: insoluble protein; and
Lane 4: soluble protein.
Figure 3 is a diagram showing the results of Western blot analysis of the purified recombinant lysozyme-thrombin-HJP34 fusion protein:
M: marker;
Lane 1: insoluble protein; and
Lane 2: soluble protein.
Figure 4 is a diagram showing the survival rate for each group after challenge infection.
Figure 5 is a diagram showing the weight gain on the 7th and 14th days after challenge infection for each group.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

Throughout this specification, '%' used to indicate the concentration of a particular substance is solid/solid (w/w) %, solid/liquid (w/v) %, and Liquid/liquid is (v/v) %.

In one aspect, the present invention relates to a recombinant antimicrobial peptide comprising the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the antimicrobial peptide may have antimicrobial activity against gram-negative bacteria or gram-positive bacteria, and the gram-negative bacteria or gram-positive bacteria may be antibiotic-resistant bacteria.

In the present invention, the term "gram-negative bacteria" refers to bacteria that are stained red when stained by the Gram staining method, and most of the cell walls of Gram-negative bacteria are thin (about 10 nm) and contain a large amount of lipopolysaccharide (LPS) on the outside. am. The Gram-negative bacteria include, for example, Escherichia, Pseudomonas, Salmonella, Leptospira, Rickettsia, etc., but are not limited thereto. In one embodiment of the present invention, Escherichia coli ( E. coli ), which is a gram-negative bacterium among major disease-causing bacteria in pigs, and Actinobacillus pleuropneumoniae (APP) type 2 and 5 strains were used as representative strains of gram-negative bacteria.

In the present invention, the term "Gram-positive bacteria" is a bacterium whose cell wall is stained purple when stained by the Gram staining method, and the cell wall is composed of several layers of peptidoglycan. The Gram-positive bacteria include, for example, Staphylococcus, Listeria, Corynebacterium, Lactobacillus, Bacillus, and Propionibacterium. ), etc., but are not limited thereto. In one embodiment of the present invention, C. perfringens ( Clostridium perfringens ) and S. suis ( Streptococcus suis ), which are Gram-positive bacteria among the major disease-causing bacteria in pigs, were used as representative strains of Gram-positive bacteria.

In one aspect, the present invention relates to a nucleic acid encoding the recombinant antimicrobial peptide of the present invention, a recombinant vector containing the same, and a host cell transformed with the vector.

In one embodiment, the nucleic acid may include the nucleotide sequence of SEQ ID NO: 2.

An expression vector according to the present invention usually includes a protein operably linked to, ie, placed in a functional relationship with, regulatory or control sequences, selectable markers, optional fusion partners, and/or additional elements.

Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose. The vector's promoter may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an α-amylase signal sequence and a subtilisin signal when the host is Bacillus sp. Sequences such as MFα signal sequence, SUC2 signal sequence, etc. can be used when the host is yeast, and insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used when the host is an animal cell. Not limited to this. In addition, the vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.

As used herein, the term "vector" refers to a delivery vehicle into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. A nucleic acid sequence may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

In one embodiment, when constructing the vector, an expression control sequence such as a promoter, terminator, enhancer, etc., for membrane targeting or secretion, according to the type of host cell to produce the antibody. Sequences and the like can be selected appropriately and combined in various ways depending on the purpose.

In the present invention, the term "recombinant vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Recombinant vectors may contain various regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.

In one embodiment, the host cell may be a bacterium or E. coli.

Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used.

In one aspect, the present invention relates to an antibiotic containing an antimicrobial peptide as an active ingredient.

In one embodiment, the antimicrobial peptide may be formulated or used as an antibiotic in combination with one or more antibiotics.

In one embodiment, the antibiotic is methicillin, oxacillin, norfloxacin, vancomycin, amikacin, gentamicin, kanamycin, neo Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin , Rifaximin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cepha Cefazolin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime , Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Cefti Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline Fosamil, Ceftobiprole, Teicoplanin , Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarisro Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam ( Aztreonam), Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B (Polymyxin B), Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Ofloxacin, Trovafloxacin, Grepafloxacin, Spafloxacin, Temafloxacin, Mafenide , Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide ( Sulfanilimide), Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole, TMP-SMX), Sulfonamido chrysoidine, Demeclo Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine ( Cycloserine), Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Arsphenamine , Chloramphenicol, Fosfomycin, Fusidi cacid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, It may be selected from the group consisting of Thiamphenicol, Tigecycline, Tinidazole, and Trimethoprim, and may be Amoxicillin trihydrate.

The antimicrobial peptide of the present invention may include all forms used in various fields requiring antibacterial effect, and may be in the form of, for example, pharmaceuticals, quasi-drugs, food additives, or feed additives. Specifically, for purposes such as antibiotics or antifouling agents in medicine, for antiseptic or antibacterial purposes in food, for antibacterial, sterilization, and disinfection purposes in agriculture, and for suppressing dandruff, preventing athlete's foot, and armpits in cosmetics and household goods. It can be used for products directly related to microorganisms such as collection suppression and anti-acne, or used for preservative, antibacterial or sterilization purposes, such as cleaning detergents or dishwashing detergents, but is not limited to these purposes.

In one aspect, the present invention relates to a feed additive containing the antimicrobial peptide of the present invention as an active ingredient.

In one embodiment, the feed additive may be formulated or used in combination with one or more antibiotics.

In one aspect, the present invention relates to an antibacterial biological pesticide containing the antimicrobial peptide of the present invention as an active ingredient.

In one aspect, the present invention relates to an antibacterial food additive containing the antimicrobial peptide of the present invention as an active ingredient.

When the composition of the present invention is used as a food additive, the antimicrobial peptide may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. In addition to the active ingredient, the composition may include food additives acceptable in food science, and the mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating bacterial infection containing the antimicrobial peptide of the present invention as an active ingredient.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of a bacterial infection by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to the antibacterial peptide of the present invention, or It refers to all activities that ameliorate or beneficially change the symptoms of a bacterial infection by administering a pharmaceutically acceptable salt thereof or a composition containing the same. Those of ordinary skill in the art to which the present invention pertains will be able to determine the degree of improvement, enhancement and treatment by knowing the exact criteria of the disease for which the composition of the present application is effective by referring to the data presented by the Korean Medical Association, etc. will be.

In the present invention, the term "therapeutically effective amount" used in combination with an active ingredient means an effective amount to prevent or treat a target disease, and the therapeutically effective amount of the composition of the present invention depends on several factors, such as the method of administration. , the target site, the condition of the patient, etc. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining an effective amount can be found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's Health condition, cause of bacterial infection, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field It can be determined according to the elements. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological preparations. As used herein, the term "pharmaceutically acceptable" means exhibiting non-toxic properties to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition. For example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injection solutions and sprays, and oral or injection formulations are more preferred.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , Lactose, Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Lead, Synthetic Aluminum Silicate, Stearic Acid, Magnesium Stearate, Aluminum Stearate, Calcium stearate, white sugar, dextrose, sorbitol and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 part by weight to 90 parts by weight based on the composition, but is not limited thereto.

In one aspect, the present invention relates to an antibacterial cosmetic composition containing the antimicrobial peptide of the present invention as an active ingredient.

The cosmetic composition of the present invention may be prepared by including a cosmetically effective amount of the above-described antimicrobial peptide of the present invention and a cosmetically acceptable carrier.

The term "cosmetically effective amount" used in the present invention means an amount sufficient to achieve the above-described anti-inflammatory efficacy of the composition of the present invention.

The appearance of the cosmetic composition contains a cosmetic or dermatologically acceptable medium or base. These are all formulations suitable for topical application, e.g. solutions, gels, solids, anhydrous pasty products, emulsions obtained by dispersing an oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and It may be provided in the form of a non-ionic follicular dispersant, or in the form of a cream, toner, lotion, powder, ointment, spray or conceal stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition according to an embodiment of the present invention is not particularly limited in its dosage form, for example, softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, eye essence, cleansing It can be formulated into cosmetics such as cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil and body essence.

When the formulation of the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

In one aspect, the present invention relates to a method for producing a recombinant antimicrobial peptide, comprising the step of overexpressing the recombinant antimicrobial peptide by transforming a host cell with the recombinant vector of the present invention.

In one aspect, the present invention relates to an antibacterial method in a non-human subject comprising administering to the non-human subject an antibacterial peptide of the present invention in a pharmaceutically effective amount.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.

Example 1. Production of recombinant antimicrobial peptides

In order to improve antibacterial activity while supplementing the disadvantages of natural antimicrobial peptides (AMPs), 34 base sequences were manipulated to obtain a recombinant antimicrobial protein lysozyme- thrombin-HJP34 (SEQ ID NO: 1) was developed, and its gene was optimized for E. coli codons for good expression in E. coli, and chemically synthesized (Biod, Gwangmyeong-si, South Korea) (SEQ ID NO: 2).

Example 2. Construction and confirmation of recombinant antibacterial peptide coding vector

The gene synthesized in Example 1 encoding the recombinant lysozyme-thrombin-HJP34 protein was inserted into pET28a plasmid (Fig. 1), and then transformed into E. coli BL21(DE3)pLysS to express the recombinant protein, and nickel-nitrile lot It was purified by an acetic acid-agarose affinity purification process. As a result of analyzing the purified recombinant protein by electrophoresis on 15% SDS-PAGE, it was confirmed that the recombinant lysozyme-thrombin-HJP34 protein was expressed (FIG. 2). The purified recombinant protein was mixed in 50% glycerol and stored at -70 °C until use.

Example 3. Recombinant Antimicrobial Peptide Expression and Purification

E. coli BL21(DE3)pLysS was transformed to express the pET28a vector containing the recombinant lysozyme-thrombin-HJP34 protein prepared in Example 2, and the recombinant lysozyme-thrombin-HJP34 protein was For overexpression, one colony was inoculated into 100 ml of Luria-Bertani (LB) broth containing 50 μg/ml kanamycin and incubated overnight at 37°C. 10 ml of this culture medium was transferred to 1,000 ml of LB medium containing fresh 50 μg/ml kanamycin and re-cultured until the OD ₆₀₀ reached 0.6. Isopropyl-b-Dthiogalactopyranoside (IPTG) was added to the culture medium to a concentration of 0.5 mM, and incubated at 25 ° C for 20 hours to induce recombinant lysozyme-thrombin-HJP34 protein expression. . The cultured cells were concentrated by centrifugation at 4° C. and 5,000 rpm for 15 minutes, resuspended in 20 ml lysis buffer (10 mM Tris, 1 M NaCl, pH 8.0), and disrupted by a French press method. The soluble protein in the eluate was concentrated by centrifugation at 12,000 rpm at 4° C. for 25 minutes, and purified using a Ni-NTi column connected to an AKTA prime FPLC system (GE healthcare, USA). The reacted column was washed with lysis buffer (buffer A), and soluble protein was removed according to the gradient concentration of buffer B (10 mM Tris-HCl, 1 M NaCl, 300 mM Imidazole, pH 8.0). Separated. Each separated fraction was analyzed by 15% SDS-PAGE, and the purified target protein was dialyzed using buffer C (PBS buffer, GE healthcare, USA), followed by centrcon (cut-off 30 kDa, Amicon, Millipore, Germany) was used to concentrate. Thereafter, the concentration of the concentrated recombinant lysozyme-thrombin-HJP34 protein was measured by a protein quantification method (Bradford protein assay). The purified and concentrated recombinant lysozyme-thrombin-HJP34 protein was electrophoresed on 15% SDS-PAGE, transferred to a PVDF membrane (Millipore, Germany), and then subjected to anti-6-his polyclonal antibody (anti 6,000 dilution). -his polyclonal antibody) (BD, France) was reacted as the primary antibody. After washing, the HRP conjugated goat anti-mouse IgG antibody (Enzo, USA) diluted at 1:12,000 was reacted with the secondary antibody, and the band of the recombinant protein purified with ECL solution (SurModics, USA) was confirmed. As a result, the corresponding fusion protein A band was observed (Fig. 3).

Example 4. Confirmation of antibacterial activity of recombinant antimicrobial peptides

Stx2e ⁺ F18 ⁺ E. coli strains HJL624, 636, 712 strains, APP strains HJL9, 11, 33, 34, 38, 42 strains, and S. suis strains, using slightly modified microfluid medium dilution method recommended by NCCLS The minimum growth inhibitory concentrations (MICs) were measured for strains HJL1221, 1305, and 1307, and strains HJL916, 989, and 987, which are bacterial strains of C. perfringens (CLSI, 2008). Here, the dilution concentration of the recombinant lysozyme-thrombin-HJP34 protein was diluted step by step from 1,200 μg/mL to 18.75 μg/mL by twofold. As a result, the MICs of the recombinant lysozyme-thrombin-HJP34 protein against Stx2e ⁺ F18 ⁺ E. coli , APP, S. suis , and C. perfringens were 75 μg/ml, 300 μg/ml, 75 μg/ml and 35.5 μg/ml, respectively. observed as μg/ml.

Example 5. Confirmation of the effect of recombinant antibacterial peptide as a feed additive

5-1. Manufacture and supply of feed additives

After measuring the lysozyme activity of the recombinant lysozyme-thrombin-HJP34 protein expressed and purified in Example 3, it was diluted to 50,000 units/g and freeze-dried to prepare a feed additive. In addition, 25 domestic breeding piglets were divided into 5 groups in Table 1 below, isolated and reared, and in the case of the recombinant lysozyme-thrombin-HJP34 protein feed additive administration group, fed with feed every day for 4 weeks from 3 weeks of age And, the total content of the feed additive was 0.1% (50 unit / g) was mixed with feed and fed. Specifically, in the case of groups A, B and C, recombinant lysozyme-thrombin-HJP34 protein non-feeding groups were fed feed for pigs currently marketed as pig feed, and groups D and E were fed recombinant lysozyme-thrombin-HJP34 protein As a group, the feed additive of the present invention was mixed to 0.1% (50 unit / g) in the same feed and fed daily (Table 1).

group Whether recombinant feed additives are provided challenge infection Antibiotic treatment after spot infection A commercial feed - - B commercial feed E. coli, A. pleuropneumoniae, S. suis - C commercial feed E. coli, A. pleuropneumoniae, S. suis Broad-spectrum antibiotics (muscularly and orally administered) D Commercial Feed + Recombinant Protein E. coli, A. pleuropneumoniae, S. suis Broad-spectrum antibiotics (muscularly and orally administered) E Commercial Feed + Recombinant Protein E. coli, A. pleuropneumoniae, S. suis -

5-2. stability evaluation

In order to evaluate the safety of feeding the recombinant lysozyme-thrombin-HJP34 protein feed additive of the present invention, Side effects such as feed intake, diarrhea, fever, behavioral abnormalities, and mortality were observed twice a day for 4 weeks during which the feed additive was mixed with the feed and fed. As a result, the feed intake of the feed additive administration group and the control group was statistically similar, and side effects such as diarrhea, fever, abnormal behavior, and death were not observed.

5-3. Disease reduction effect evaluation

As in 5-1 above, gram-negative bacteria Stx2e ⁺ F18 ⁺ Escherichia coli ( E. coli ), Actinobacillus pleuropneumoniae (APP) among the major disease-causing bacteria in all weaned piglets on the 4th week after feeding for 4 weeks under the conditions of Table 1 above A challenge inoculation test was performed by challenge infection with type 2 and type 5 strains, Gram-positive bacteria Clostridium perfringens ( C. perfringens ) and Streptococcus suis ( S. suis ). Specifically, E. coli HJL624 strain was diluted to a concentration of 1 × 10 ^8.0 cfu / 1 ml and 2 ml was orally inoculated, A. pleuropneumoniae HJL67 and HJL263 strains, and S. suis HJL1305 strain were diluted to a concentration of 1 × 10 ^8.0 cfu / 1 ml , and inoculated intranasally by 1ml into each nasal cavity (Table 2). The control group was inoculated with 1 ml of sterile PBS in the same way. 24 hours after challenge infection, all surviving piglets in groups C and D were intramuscularly inoculated with a broad-spectrum antibiotic [Vetrimoxin _® LA (amoxicillin trihydrate), Seba Korea] at 1 ml per 10 kg, and 48 hours later, 1 ml per 10 kg was additionally inoculated. In addition, after 96 hours of challenge infection, 1 g per 10 kg of body weight of Yuhan Kimoxin Power (Yuhan Corporation), an oral broad-spectrum antibiotic, was mixed with feed and fed to Groups C and D for one day. All piglets were observed daily and any abnormal reactions or behaviors were observed for 14 days. All pigs showing abnormal gross findings were euthanized, necropsied, and grossly examined. In order to isolate the attacking inoculum, ileum and rectal swab samples were smeared on EMB agar, and any abnormal findings were observed. Lung cross-sectional swabs were smeared on Chocolate agar and Columbia agar containing 6% sheep blood, and 100 μl of heparinized blood was smeared on Chocolate agar and Columbia agar containing 6% sheep blood. After that, it was confirmed whether the strains isolated from each plate were challenged with a primer for identifying each strain (Table 3).

Strain/plasmid Description strains E. coli BL21(DE3)pLysS F ^- , omp T, hsd S _B (r _B ^- ,m _B ^- ), dcm, gal , λ(DE3), pLysS, Cm ^r BL21-lysozyme-HJP34 BL21(DE3)pLysS with pET28a-lysozyme-HJP34 HJL624 LT ⁺ Stx2e ⁺ F18 ⁺ E. coli isolate from piglets with edema disease HJL636 LT ⁺ Sta ⁺ Stx2e ⁺ F18 ⁺ E. coli isolate from piglets with edema disease HJL712 LT ⁺ Sta ⁺ Stx2e ⁺ F18 ⁺ E. coli isolate from piglets with edema disease A. pleuropneumoniae HJL9 Actinobacillus pleuropneumoniae serotype 5 isolate from piglet with pleuropneumonia HJL11 Actinobacillus pleuropneumoniae serotype 1 isolate from piglet with pleuropneumonia HJL33 Actinobacillus pleuropneumoniae serotype 7 isolate from piglet with pleuropneumonia HJL34 Actinobacillus pleuropneumoniae serotype 2 isolate from piglet with pleuropneumonia HJL67 Actinobacillus pleuropneumoniae serotype 2 isolate from piglet with pleuropneumonia HJL263 Actinobacillus pleuropneumoniae serotype 5 isolate from piglet with pleuropneumonia S. suis HJL1221 Streptococcus suis serotype 12 isolate from piglets HJL1305 Streptococcus suis serotype 2 1/2 isolate from piglets HJL1307 Streptococcus suis serotype 8 solate from piglets C. perfringens HJL916 Clostridium perfringens A type isolate from piglet with pleuropneumonia HJL986 Clostridium perfringens A type isolate from piglet with pleuropneumonia HJL987 Clostridium perfringens A type isolate from piglet with pleuropneumonia Plasmids pET28a IPTG-inducible expression vector; Km ^r pET28a-lysozyme-HJP34 pET28a containing the gene for lysozyme-thrombin-HJP34

Target gene Primer name Sequence Size F18 F18-L TTG CAC TGT AGG AGA TAC CAT TCA GC 334bp F18-R GGT TTG ACC ACC TTT CAG TTG AGC AG S. suis JP4 GCA GCG TAT TCT GTC AAA CG 688bp JP5 CCA TGG ACA GAT AAA GAT GG apxIV apxIVA1 TTA TCC GAA CTT TGG TTT AGC C 418bp apxIVA3 CAT ATT TGA TAA AAC CAT CCG TC APP 2 AP2F GAG TGT GAT GAT GAT GCT CTG GTT C 247bp AP2R TAC CAA TAA CTG TTG CAA CTA ACG C APP 5 AP5F AGC CAC AAG ACC CGA ATG GTA TAA TG 825bp AP5R CCA TCA AAT GCA GCT TCA AGG AGC

As a result, 5 piglets belonging to Group B died from the day after the challenge infection, and all piglets died on the 3rd day. Two of them died on the 4th day after challenge infection, and all survived until the 14th day of challenge infection when the experiment ended. In the group in which antibiotics were inoculated and fed from 24 hours after challenge infection after feeding only commercial feed without feed additives, and in the group inoculated with antibiotics and fed after mixing feed additives with feed, 1 within 24 hours after challenge infection Only two died (FIG. 4).

5-4. Check growth rate

As a result of confirming the growth rate of the surviving piglets in the challenge inoculation experiment of Example 5-3, on the 7th day after the challenge infection, recombinant lysozyme-thrombin-HJP34 protein (feed additive) was mixed with feed and inoculated with antibiotics after feeding The growth rate was the highest in the group fed, and the group fed antibiotics after feeding by mixing recombinant lysozyme-thrombin-HJP34 protein with feed also showed high growth rate. In addition, at the end of the experiment, the recombinant lysozyme-thrombin-HJP34 protein was mixed with the feed, and the piglets of the group inoculated with antibiotics after feeding showed the greatest increase in body weight compared to the pigs of other groups (FIG. 5)

Through the above examples, the recombinant lysozyme-thrombin-HJP34 protein has an antibacterial effect on various bacteria, and when fed by mixing it with feed, there is an effect of reducing diseases caused by pathogenic bacteria that cause diseases in the digestive and respiratory organs in pigs. In addition, the group in which the recombinant lysozyme-thrombin-HJP34 protein was mixed with the feed and antibiotics were used after feeding showed the highest increase in weight gain compared to the other groups, and the recombinant lysozyme-thrombin-HJP34 protein of the present invention was used as a feed additive If it is used and used in combination with antibiotics, it is judged that it will be of economic help to pig farmers.

<110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> Antimicrobial peptide and its uses <130> DHP21-550 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 528 <212> DNA <213> artificial sequence <220> <223> lysozyme-thrombin-HJP34 <400> 1 gaattcatga aggtgttcga gcgttgcgaa ctggcgcgta ccctgaaacg tctgggtatg 60 gacggctacc gtggtatcag cctggcgaac tggatgtgcc tggcgaagtg ggagagcggc 120 tacaacaccc gtgcgaccaa ctataacgcg ggcgaccgta gcaccgatta cggtatcttt 180 cagattaaca gccgttattg gtgcaacgat ggtaagacgc cgggtgcggt taacgcgtgc 240 cacctgagct gtagcgcgct gctgcaagac aacatcgcgg atgcggtggc gtgcgcgaaa 300 cgtgtggttc gtgacccgca gggcattcgt gcgtgggttg cgtggcgtaa ccgttgccaa 360 aaccgtgatg tgcgtcagta tgttcaaggt tgcggtgtgc tggttccgcg tggtagcggc 420 cgtttccgtc gtctgcgtaa gaaaacccgt aaacgtctga agaaaatcgg caaggtgctg 480 aaatggattc cgccgattgt tggcagcatt ccgctgggtt aaaagctt 528 <210> 2 <211> 171 <212> PRT <213> artificial sequence <220> <223> lysozyme-thrombin-HJP34 <400> 2 Met Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu 1 5 10 15 Gly Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu 20 25 30 Ala Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala 35 40 45 Gly Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr 50 55 60 Trp Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu 65 70 75 80 Ser Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys 85 90 95 Ala Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala 100 105 110 Trp Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly 115 120 125 Cys Gly Val Leu Val Pro Ala Gly Ser Gly Arg Phe Arg Arg Leu Arg 130 135 140 Lys Lys Thr Arg Lys Arg Leu Lys Lys Ile Gly Lys Val Leu Lys Trp 145 150 155 160 Ile Pro Pro Ile Val Gly Ser Ile Pro Leu Gly 165 170

Claims (12)

A recombinant antimicrobial peptide comprising the amino acid sequence of SEQ ID NO: 1. The recombinant antimicrobial peptide according to claim 1, wherein the antimicrobial peptide has antibacterial activity against gram-negative bacteria or gram-positive bacteria. A nucleic acid encoding the recombinant antimicrobial peptide of claim 1. A recombinant vector comprising the nucleic acid of claim 3. A host cell transformed with the recombinant vector of claim 4. An antibiotic containing the recombinant antimicrobial peptide of claim 1 as an active ingredient. The antibiotic according to claim 6, which is formulated or used in combination with one or more antibiotics. A feed additive containing the recombinant antimicrobial peptide of claim 1 as an active ingredient. The feed additive according to claim 8, which is formulated or used in combination with one or more antibiotics. An antibacterial food additive containing the recombinant antimicrobial peptide of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating a bacterial infection comprising the recombinant antimicrobial peptide of claim 1 as an active ingredient. A method for producing a recombinant antimicrobial peptide comprising the step of transforming a host cell with the recombinant vector of claim 4 to overexpress the recombinant antimicrobial peptide.

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