Bacteria-targeted capsid particles, therapeutic composition, bactericide, food, method for eliminating bacteria, sterilization method, corrosion prevention method, animal treatment method, gene transfer method, method for adding bacterial function, method for producing bacteria-targeted capsid particles, and method for producing nucleic acid for bacteria-targeted capsid particles

Abstract

Provided are bacterium-targeted capsid particles which are non-proliferative and have a high bactericidal effect and the like. The capsid protein of the bacteriophage is prepared by a capsid nucleic acid element that is segmented from the bacteriophage genome, does not include a packaging region, mainly includes a virion region, and synthesizes the capsid. An element for bacterial targeting capsid particles (B-CAP) is separated from a region of a non-capsid nucleic acid element in a bacteriophage genome, and includes a nucleic acid injection region, a replication region required for nucleic acid replication, and a packaging region. The assembled bacterium-targeted capsid particles (B-CAP) are capable of carrying long-chain DNA that imparts a novel biological function including a bactericidal effect, despite not having proliferative properties.

Description

Technical Field

[0002] The present invention particularly relates to a bacteria-targeted capsid particle, a therapeutic composition, a bactericide, a food, a method for eliminating bacteria, a bactericidal method, a corrosion prevention method, an animal treatment method, a gene transfer method, a bacterial function-adding method, a method for producing bacteria-targeted capsid particles, and a method for producing nucleic acids for bacteria-targeted capsid particles. Background Art

[0003] Many antibacterial drugs have been developed to treat various bacterial infections. However, soon after the use of antibacterial drugs, reports of drug-resistant bacteria (bacteria that are ineffective against antibacterial drugs and antibiotics) began to emerge. Now, drug-resistant bacteria are everywhere on the earth, and there are drug-resistant bacteria to almost all antibacterial drugs used clinically. Drug-resistant bacteria are emerging at a speed far exceeding the development of antibacterial drugs, and bacterial infections are once again a global problem threatening human health. In other words, given the history of antibacterial drug development and the evolution of drug-resistant bacteria, the emergence of drug-resistant bacteria seems inevitable, and there is also a worrying problem that even if new drug-resistant bacteria emerge, there are no evolutionary adaptive pharmaceuticals that can cope with them. In such a crisis situation, countries around the world have formulated action plans for drug-resistant countermeasures to promote the development of new prevention, diagnosis, and treatment methods for drug-resistant bacteria.

[0004] Phage therapy is an antibacterial treatment method that uses bacteriophage (hereinafter referred to as "phage"). Bacteriophage is a virus that infects bacteria and proliferates through the following process. (1) Attaching to the host bacteria, (2) Injecting its own nucleic acid (DNA or RNA), (3) Replicating its own nucleic acid in the bacteria. Then, (4) Synthesizing the constituent proteins (capsid) that become the outer shell, (5) Assembling progeny phage, and (6) Release the progeny phage outside the bacteria by lysis or the like. Phage therapy is a treatment method that uses the bacteriophage, which is a virus that infects bacteria, to kill bacteria. It was attempted with the discovery of bacteriophage in 1915, and its clinical application has been accelerating in recent years.

[0005] However, since phages proliferate and spread their genes, there are concerns that they may induce unexpected bacterial evolution, spread toxin genes, and cause adverse effects on the ecosystem.

[0006] Therefore, a bacteriophage sterilization method equipped with non-proliferating CRISPR-Cas13 is described in Patent Document 1. In addition, the development of phage therapy using gene-deficient phages and phagemids is also being attempted.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2014 / 204726

[0010] Non-patent document 1: Kiga K. et al., "Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria", Nature Communications, 2020, vol. 11(1): 2934

[0011] Non-patent document 2: De Graaf, FK and Oudega, B., "Production and release of cloacin DF13 and related colicins", Curr. Top. Microbiol. Immunol, 1986, vol. 125, p. 183-205 Summary of the invention

[0012] Problem that the invention aims to solve

[0013] However, as described in Patent Document 1, conventional non-proliferative phages are limited and can only sterilize infected bacteria, and more effective phages are needed for the treatment of drug-resistant bacteria.

[0014] The present invention has been made in view of such circumstances, and an object of the present invention is to solve the above-mentioned problems.

[0015] Solutions to the problem

[0016] The bacteria-targeting capsid particle of the present invention is characterized in that it is non-proliferative and has: a capsid protein of a bacteriophage; an element for bacteria-targeting capsid particles, which includes a nucleic acid injection region of the genome of the bacteriophage; a replication region required for replicating nucleic acid; and a packaging region.

[0017] The bacteria-targeting capsid particle of the present invention is characterized in that the element for bacteria-targeting capsid particle contains a foreign gene.

[0018] The bacteria-targeting capsid particle of the present invention is characterized in that the exogenous gene comprises any one or any combination of a bactericidal gene, a biofilm degradation gene, an antigen presentation gene and a transfection gene.

[0019] The bacteria-targeting capsid particle of the present invention is characterized in that the bactericidal gene is used to produce a secretory bactericidal product that kills surrounding bacteria.

[0020] The bacteria-targeted capsid particle of the present invention is characterized in that the foreign gene contains a resistance factor, which is used to inhibit the sterilization of the targeted bacteria.

[0021] The therapeutic composition of the present invention is characterized by containing the bacteria-targeting capsid particles.

[0022] The fungicide of the present invention is characterized by comprising the bacteria-targeting capsid particles.

[0023] The food of the present invention is characterized by containing the bacteria-targeting capsid particles.

[0024] The method for eliminating bacteria of the present invention is characterized in that the targeted bacteria are eliminated using the bacteria-targeting capsid particles.

[0025] The method for eliminating bacteria of the present invention is characterized in that the target bacteria are present in a bacterial population (microbiota) in humans, animals and / or the environment.

[0026] The method for eliminating bacteria of the present invention is characterized in that the targeted bacteria are present in food.

[0027] The sterilization method of the present invention is characterized in that the targeted bacteria are sterilized using the bacteria-targeting capsid particles.

[0028] The corrosion prevention method of the present invention is characterized in that the targeted bacteria are sterilized by using the bacteria-targeted capsid particles to prevent corrosion of articles.

[0029] The animal treatment method of the present invention is characterized in that the animal is treated using the bacteria-targeting capsid particles.

[0030] The gene transfer method of the present invention is characterized in that the foreign gene contained in the bacteria-targeting capsid particle is transferred to the targeted bacteria.

[0031] The method for adding function to bacteria of the present invention is characterized in that the foreign gene contained in the bacteria-targeting capsid particle is transferred to the target bacteria to add function to the target bacteria.

[0032] The method for producing bacteria-targeting capsid particles of the present invention is characterized in that the capsid protein of the bacteriophage is prepared by using a capsid nucleic acid element that is separated from the genome of the bacteriophage and does not contain a packaging region and a synthetic capsid, and an element for bacteria-targeting capsid particles that is separated from a site other than the capsid nucleic acid element of the genome of the bacteriophage and contains a nucleic acid injection region, a replication region required for replicating nucleic acid, and the packaging region is packaged into the capsid protein to generate non-proliferative bacteria-targeting capsid particles.

[0033] The method for producing bacteria-targeted capsid particles of the present invention is characterized in that the capsid nucleic acid element and the element for bacteria-targeted capsid particles are transplanted into production bacteria for production, and the bacteria-targeted capsid particles are produced in the production bacteria.

[0034] The method for producing bacteria-targeted capsid particles of the present invention is characterized in that the capsid nucleic acid element is transferred via the chromosome or artificial chromosome for preparing bacteria.

[0035] The method for producing bacteria-targeting capsid particles of the present invention is characterized in that the element for bacteria-targeting capsid particles is transferred via the chromosome or plasmid.

[0036] The method for producing a nucleic acid for bacteria-targeting capsid particles of the present invention is characterized in that a capsid nucleic acid element that does not include a packaging region and a synthetic capsid is split from a phage genome and prepared, and an element for bacteria-targeting capsid particles that includes a nucleic acid injection region, a replication region required for nucleic acid replication, and the packaging region is split from a portion of the phage genome other than the capsid nucleic acid element to construct a non-proliferative nucleic acid for bacteria-targeting capsid particles.

[0037] Effects of the Invention

[0038] According to the present invention, by having a bacteriophage capsid protein and an element for bacteria-targeting capsid particles including a nucleic acid injection region, a replication region required for nucleic acid replication, and a packaging region, a non-proliferative bacteria-targeting capsid particle that is effective for the treatment of drug-resistant bacteria can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a diagram showing the structure of a bacteria-targeting capsid particle according to an embodiment of the present invention.

[0040] Figure 2 This is a conceptual diagram showing a comparison between a conventional phage therapy and an antibacterial therapy method using bacteria-targeting capsid particles according to an embodiment of the present invention.

[0041] Figure 3 This is a conceptual diagram of the sequence structure of T7 phage and Helper B-CAP involved in the examples of the present invention.

[0042] Figure 4 This is a conceptual diagram of the sequence structure of T7 bacteriophage and B-CAP involved in the examples of the present invention.

[0043] Figure 5 It is a schematic diagram of confirming the construction of B-CAP by PCR method and a photograph of the electrophoresis result involved in the embodiment of the present invention.

[0044] Figure 6 This is an electron microscope photograph of B-CAP according to an example of the present invention.

[0045] Figure 7 These are photographs showing the bactericidal activity of B-CAP according to Examples of the present invention.

[0046] Figure 8 The diagram shows the sequence structure of B-CAP derived from Tan2 phage of Staphylococcus aureus and a conceptual diagram and photograph of B-CAP synthesis according to an embodiment of the present invention.

[0047] Fig. 9 This is a conceptual diagram of an experiment in which a long-chain DNA is carried on B-CAP according to an embodiment of the present invention.

[0048] Fig.10 It is shown in Fig. 9 Graph of plaque numbers obtained in the experiments shown.

[0049] Fig.11 is Fig. 9 Photographs of plaques obtained in the indicated experiments.

[0050] Fig.12 The PCR method was used to confirm Fig. 9 Schematic diagram of the long-chain DNA on the plaque obtained in the experiment and a photograph of the electrophoresis results.

[0051] Fig.13 It is shown that the sequencing confirmed Fig. 9 This is a diagram showing the results of the experiment in which long DNA was carried on plaques.

[0052] Fig.14 This is a conceptual diagram of the construction of the bactericidal B-CAP involved in the embodiment of the present invention.

[0053] Fig.15 It is a schematic diagram of the sequence structure of the bactericidal B-CAP involved in the embodiment of the present invention.

[0054] Fig.16 These are photographs showing the bactericidal effect of the bactericidal B-CAP sheet according to the example of the present invention.

[0055] Fig.17 This is a graph showing the bactericidal effect of the bactericidal B-CAP according to the example of the present invention in a liquid culture medium.

[0056] Fig.18 These are photographs showing the bactericidal effect of the bactericidal B-CAP according to the examples of the present invention on drug-resistant Escherichia coli.

[0057] Fig.19 This is a conceptual diagram of an antibacterial treatment experiment on bacterially infected mice using bactericidal B-CAP according to an embodiment of the present invention.

[0058] Fig. 20 : is a graph showing the antibacterial therapeutic effect of the bactericidal B-CAP according to the example of the present invention on mice infected with bacteria. DETAILED DESCRIPTION

[0059] <Implementation Method>

[0060] There are concerns that proliferative phages could induce unexpected bacterial evolution, spread toxic genes, and cause adverse effects on the ecosystem.

[0061] Therefore, the present inventors considered producing a non-proliferative phage that is effective in treating drug-resistant bacteria. Furthermore, the present inventors repeatedly conducted intensive experiments and envisioned constructing a "non-proliferative phage capsid particle capable of injecting DNA into bacteria" that has phage genome elements required for DNA injection and DNA replication. Thus, the present inventors generated a DNA structure (factor, element) with a specific sequence, excluding the constituent genes of the virus particle (Virion) including the region for synthesizing the capsid related to proliferation; and successfully packaged the element into a lysogenic phage capsid, thereby completing the present invention. The element packaged in the phage capsid is injected into the cells of the target host bacteria, just like the bacterial infection of wild phages. Therefore, the packaged element is named a non-proliferative bacteria-targeting capsid particle (hereinafter referred to as "B-CAP").

[0062] Hereinafter, the B-CAP, therapeutic composition, bactericide, food, method for eliminating bacteria, bactericidal method, corrosion prevention method, animal treatment method, gene transfer method, bacterial function-enhancing method, method for producing B-CAP, and method for producing nucleic acid for B-CAP involved in this embodiment will be specifically and detailedly described.

[0063] (Construction of B-CAP)

[0064] First, the construction method (manufacturing method) of B-CAP is described. First, as the phage genome segmentation method involved in this embodiment, the chromosome (genome) of the phage is appropriately segmented to construct a B-CAP element, which is specifically used for transduction, DNA replication and packaging by a lytic phage. Specifically, the genome of the phage is segmented to generate a B-CAP element, which only contains the gene group required for DNA injection and DNA replication. The B-CAP element excludes the viral particle constituting genes including the region for synthesizing the capsid related to proliferation, including the nucleic acid injection region, the replication region required for nucleic acid replication, and the packaging region.

[0065] The elements for B-CAP can be constructed via chromosome or plasmid. The number of copies of the plasmid can be greater than that of the artificial chromosome.

[0066] On the other hand, from the genome of the phage, a capsid nucleic acid element (hereinafter referred to as "capsid nucleic acid element (capsimid)") is also constructed, which is a DNA element for constructing the capsid that is used to synthesize the phage capsid, except for the element for B-CAP. The capsid nucleic acid element contains the virion but does not contain the packaging region, and becomes an element dedicated to synthesizing the capsid.

[0067] In this embodiment, since the capsid nucleic acid element is stably maintained in the prepared bacteria, it can be constructed using chromosomes for the prepared bacteria or bacterial artificial chromosomes (BAC).

[0068] By constructing the B-CAP element and the capsid nucleic acid element in this way, a non-proliferative B-CAP nucleic acid can be produced.

[0069] These B-CAP elements and capsid nucleic acid elements can be transferred into the production bacteria for production to produce B-CAP in the production bacteria. Specifically, the B-CAP elements and capsid nucleic acid elements can be transferred into the production bacteria by electroporation, liposomes, injection, or other gene transfer methods.

[0070] The prepared bacteria can be, for example, general bacteria such as Escherichia coli and Bacillus subtilis. Alternatively, bacteria of the same species or closely related species as the target bacteria to be infected with B-CAP and injected with DNA can be used as prepared bacteria.

[0071] On this basis, in the method for producing B-CAP involved in this embodiment, the B-CAP element is proliferated in the preparation bacteria. In addition, in the same preparation bacteria, the capsid protein is also produced by the capsid nucleic acid element, and the proliferated B-CAP element is packaged in the capsid protein. Thus, non-proliferative B-CAP particles are produced. The B-CAP particles can be obtained in large quantities by methods commonly used by those skilled in the art, such as releasing them into the lysate of the preparation bacteria and / or outside the bacteria and purifying them by ultracentrifugation.

[0072] according to Figure 1 An example of constructing the B-CAP involved in the present embodiment using T7 phage that mainly infects Escherichia coli is described.

[0073] In this embodiment, as shown in the examples described later, the genome of the T7 phage, which is a model of a proliferating phage, is divided and assembled into regions of the B-CAP element and the Helper B-CAP, which is a capsid nucleic acid element. The B-CAP element is a plasmid-like circular DNA. On the other hand, the Helper B-CAP is inserted into the artificial chromosome (BAC) of Escherichia coli and named pKLC172. On this basis, by electroporating the DNA of the B-CAP element and pKLC172 into the Escherichia coli HST08 strain, MC1061 strain, and MC1061R strain, respectively, it is possible to produce B-CAP packaged as a T7 phage capsid and transfer the DNA to the target bacteria.

[0074] The B-CAP produced in this way is non-reproductive and can only grow in the production bacteria of the gene complemented host with the other divided capsid nucleic acid element, and the production bacteria become a "factory" for synthesizing B-CAP.

[0075] On the other hand, although the prepared B-CAP is non-proliferative, it has the same infection mode as the wild-type phage and can deliver long-chain DNA to the target bacteria. That is, B-CAP can load exogenous DNA and inject B-CAP element DNA into the target bacteria. In addition, the injected B-CAP element includes amplification in the target bacteria and can exert biological activity derived from its own DNA.

[0076] Therefore, B-CAP can be used more effectively than ever before as an antibacterial therapeutic drug for bacterial infections, a disinfectant, probiotics, an oral component, an antiseptic, and the like.

[0077] Here, the B-CAP element of the B-CAP according to the present embodiment may contain a foreign gene, and the foreign gene may include any one or any combination of biofilm degradation genes, antigen presentation genes, transfer genes, and bactericidal genes.

[0078] Specifically, as shown in the examples described below, a foreign long DNA of at least 18.0 kb can be inserted into the produced B-CAP. The foreign gene can be contained in the long DNA.

[0079] Among them, the biofilm degradation gene involved in this embodiment can also be, for example, dextranase, protease, etc., which cuts or decomposes the molecular structure of the biofilm composed of bacterial polysaccharides, peptidoglycans, proteins, etc. The gene product of the biofilm degradation gene can decompose the bacterial biofilm, making it impossible for the bacteria themselves to survive and / or easy for antibacterial agents to penetrate.

[0080] In addition, the antigen presentation gene involved in this embodiment can also be a gene that makes it easy for the immune system to recognize or present an antigen that becomes a target for attack. The antigen is not necessarily an antigen for the bacteria infected by B-CAP, but can also be an antigen for other pathogenic organisms, viruses, animal tumors, etc. present in the lesion. That is, since the B-CAP involved in this embodiment can freely transfer DNA sequences in the capsid, it can also be used in the field of phage vaccines that carry pathogenic bacteria, bacteria of intestinal flora, and other bacteria, or present antigens on the surface of phage capsids. In addition, it is also suitable for the treatment of cancer and genetic diseases.

[0081] In addition, the gene for transformation involved in this embodiment can also be a gene that is transformed into bacteria. The gene for transformation can use various genes for adding functions to the bacteria infected by B-CAP. For example, as the function addition, genes related to the synthesis and secretion of proteins and various substances can be added. In addition, as mentioned above, since exogenous long-chain DNA can be carried in B-CAP, multiple genes can also be transformed. The gene for transformation can also be a "gene" that shows, for example, a drug gene, a drug resistance gene, a gene that encodes a protein related to attenuation or potentiation, a toxin gene, a specific metabolite gene, an enzyme gene for producing a specific metabolite, a gene for determining bacteria, a reporter gene for transformation, a sequence containing a restriction enzyme or bonding end for gene recombination, a repetitive sequence, and other broad genotypes.

[0082] In addition, the exogenous gene involved in this embodiment can also lose drug resistance or develop drug resistance by acquiring nucleic acid and / or nucleic acid mutation.

[0083] Specifically, since drug-resistant bacteria are sometimes generated by the acquisition of exogenous DNA or RNA (nucleic acid) and / or nucleic acid mutation, the antibacterial effect can be improved by using exogenous genes that can inhibit the acquisition of nucleic acid and / or nucleic acid mutation.

[0084] In addition, the bactericidal gene involved in this embodiment can also generate a secretory bactericidal product. Specifically, the bactericidal gene involved in this embodiment can generate a secretory bactericidal product that kills surrounding bacteria. That is, by carrying a bactericidal gene that generates a secretory bactericidal product in the B-CAP involved in this embodiment, it is possible to sterilize the target bacteria and the bacterial group around the target bacteria (collective sterilization).

[0085] For example, in B-CAP, as an exogenous gene, a secretory bactericidal DNA machine can be carried. As the bactericidal DNA machine, for example, a bacteriocin gene cluster (colicin E1genecluster) for producing and secreting bacteriocin can also be carried. Bacteriocin is an antibacterial protein or peptide produced by bacteria, secreted outside the bacteria, and shows antibacterial activity against Bacteroides. Therefore, by carrying a bacteriocin gene cluster in B-CAP, it is possible to sterilize the targeted bacteria and the bacterial groups around the targeted bacteria, and obtain a significant antibacterial effect.

[0086] The present inventors named B-CAP used as a bactericide as nonproliferative mass-killing antibacterial capsid (hereinafter referred to as "NM-AB capsid").

[0087] Here, the NM-AB capsid according to the present embodiment exhibits a very strong collective bactericidal effect and becomes a non-proliferative and effectively therapeutic B-CAP.

[0088] Specifically, as shown in the examples described below, B-CAP (B-CAP_ColE1), an example of NM-AB capsid, which carries a bacteriocin gene group, is non-proliferative but has a strong bactericidal activity against carbapenem-resistant Enterobacteriaceae. In addition, it also showed a significant survival effect on mice infected with the bacteria.

[0089] according to Figure 2 , the concept of the antibacterial treatment method of B-CAP containing NM-AB capsid involved in this embodiment is explained.

[0090] Figure 2 The differences in antimicrobial treatment of T7 phage, B-CAP and B-CAP_ColE1 are shown.

[0091] T7 phage is proliferative, multiplying in the body of the target bacterium and lysing the host during the process of proliferation. Due to its proliferation, there are concerns about adverse effects on the environment or the spread of genes to other bacteria.

[0092] On the other hand, B-CAP can kill targeted bacteria by injecting DNA derived from bacteriophages, but since it has no proliferation, its bactericidal power itself is very weak. On the other hand, B-CAP can carry long-chain genes and can express the genes in large quantities in targeted bacteria at one time. Therefore, as a non-proliferative capsid, it can be used in various ways. Furthermore, it can also be effectively used for sterilization by carrying the above-mentioned biofilm elimination gene, etc.

[0093] B-CAP_ColE1 is an example of NM-AB capsid, which is a secretory antimicrobial protein / peptide (AMPs) carried in B-CAP. B-CAP_ColE1 carries bacteriocins as AMPs. NM-AB capsids amplify after entering the target bacteria and synthesize a large amount of AMPs. AMPs are secreted outside the bacteria and can also kill surrounding bacteria.

[0094] Here, in B-CAP_ColE1 according to the present embodiment, B-CAP_ColE1 was prepared by loading ColicinE1 (ColE1), immunity protein (immunity gene), and colicin release lysis protein (secretion gene) into B-CAP as a bacteriocin gene group.

[0095] Among them, immunity protein (immunity gene) is an example of a resistance factor that inhibits the bactericidal effect of targeted bacteria. Thus, by including the gene of the resistance factor as the exogenous gene of B-CAP, the bactericidal effect of the targeted bacteria can be inhibited until the secretory bactericidal product is fully produced after the NM-AB capsid infects the targeted bacteria.

[0096] In addition, the B-CAP involved in the present embodiment may be configured to carry multiple exogenous genes. That is, the B-CAP involved in the present embodiment may contain multiple exogenous genes in one bacteriophage. In this case, multiple exogenous genes may be contained within the range that can be contained in the capsid. The multiple exogenous genes may be configured to be contained in multiple by being arranged continuously in the sequence or as multiple parts.

[0097] For example, by carrying multiple bactericidal genes to be applicable to a variety of bacteria, one B-CAP can be applied to a variety of drug-resistant bacteria. In addition, in the case of multi-drug resistant bacteria with multiple drug resistance genes, multiple genes corresponding to all of them can also be carried to improve the antibacterial efficiency. In addition, by carrying any combination of the above-mentioned bactericidal genes, biofilm degradation genes, antigen presentation genes, and transfer genes in B-CAP, the antibacterial effect can be further improved.

[0098] (Therapeutic composition)

[0099] The B-CAP involved in the embodiment of the present invention can also be used in the application of a therapeutic composition comprising the B-CAP.

[0100] That is, by applying the B-CAP involved in this embodiment for treatment, it can be used in an antibacterial treatment method for killing bacteria. Specifically, by using B-CAP, an antibacterial treatment method for killing bacteria can be achieved. As a result, antibacterial treatment can be performed on infections containing drug-resistant bacteria and infections that are difficult to treat using antibiotics.

[0101] In the preparation of the therapeutic composition, various compounds similar to those used in the conventional antibacterial therapy using bacteriophages can be used.

[0102] In addition, the therapeutic composition involved in the embodiment of the present invention can be administered together with any acceptable carrier in the formulation (for example, physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc.; appropriate dissolution auxiliary agents, such as alcohol, specifically, ethanol, polyols, such as propylene glycol, polyethylene glycol; non-ionic surfactants, such as polysorbate 80 (TM), HCO-50, etc., but not limited thereto). In addition, it can also contain appropriate excipients, etc.

[0103] In addition, the therapeutic composition of the present embodiment is an acceptable carrier for preparing a preparation, and may also contain an appropriate pharmaceutically acceptable carrier. As the carrier, it may contain biocompatible materials such as silicone, collagen, gelatin, etc. Alternatively, it may be various emulsions. In addition, it may also contain one or more preparation additives selected from, for example, diluents, spices, preservatives, excipients, disintegrants, lubricants, adhesives, emulsifiers, plasticizers, etc.

[0104] The therapeutic composition according to the embodiment of the present invention can be formulated in a dosage form suitable for oral administration using a pharmaceutically acceptable carrier known in the art.

[0105] The administration route of the pharmaceutical composition of the present invention is not particularly limited, and can be administered orally or parenterally. For parenterally, for example, it can be administered intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, or intraperitoneally, or it can be directly administered, dripped, or applied to the bacterial colony or the infected site. This administration method can also be carried out in the same manner as conventional phage therapy.

[0106] Furthermore, in order to use the therapeutic composition according to the embodiment of the present invention for the above-mentioned treatment, the administration interval and the administration amount can be appropriately selected and changed according to various conditions such as the state of the disease and the state of the subject.

[0107] The single dose and the frequency of administration of the therapeutic composition according to the embodiment of the present invention can be appropriately selected and changed according to the purpose of administration, and various conditions such as the age and weight of the patient, symptoms, and severity of the disease.

[0108] The frequency and period of administration may be once, or may be once to several times a day for several weeks, and the condition of the disease may be monitored and administered again or repeatedly according to the condition.

[0109] The therapeutic composition according to the embodiment of the present invention may be used in combination with other compositions, etc. In addition, the composition of the present invention may be administered simultaneously with other compositions or may be administered at intervals, but the order of administration is not particularly limited.

[0110] In the embodiments of the present invention, the period during which the disease is improved or alleviated is not particularly limited, and the improvement or alleviation may be temporary or last for a certain period of time.

[0111] In addition, the therapeutic composition according to the embodiment of the present invention can be used as a target for treatment of an organism, a part of an organism, or a part extracted or excreted from an organism.

[0112] The organism is not particularly limited, and may be, for example, an animal, a plant, a fungus, etc. The animal includes, for example, humans, domestic animals, wild animals, and the like.

[0113] Therefore, the therapeutic composition according to the embodiment of the present invention can also be used for animal therapy for treating animals. That is, the B-CAP according to the present embodiment can also be used for animal therapy methods for various animals other than humans.

[0114] Specifically, the animal treatment method of the present embodiment can treat infections caused by bacteria in animals other than humans using a therapeutic composition containing B-CAP.

[0115] The animal is not particularly limited and widely includes vertebrates and invertebrates. Vertebrates include fish, amphibians, reptiles, birds and mammals. Specifically, for example, the mammal can be a rodent, such as a mouse, rat, ferret, hamster, guinea pig or rabbit, dog, cat, sheep, pig, cow, horse or non-human primate. In addition, as wild animals, in addition to mammals, fish, birds including poultry, reptiles, etc. are also included. In addition, crustaceans including shrimps and insects, other invertebrates such as squid, etc. are also widely included.

[0116] That is, the therapeutic composition according to the embodiment of the present invention can be used for methods such as the treatment of animals and the promotion of growth of livestock, in addition to the treatment of humans.

[0117] In addition, the sterilization method according to the embodiment of the present invention is characterized in that the target bacteria are eliminated (sterilized, sterilized) by B-CAP.

[0118] In this way, the composition containing the B-CAP involved in this embodiment can be used as a bactericide (proliferation inhibitor). As mentioned above, the bactericide can also be contained in a suitable carrier, solution, etc. in advance. That is, the composition containing the B-CAP involved in this embodiment can be provided as a disinfectant by being contained in various antibacterial products such as lotions, hand soaps, gargles, masks or napkins, and filters for air purifiers.

[0119] In addition, the composition containing the B-CAP according to the present embodiment can also be used for eliminating bacterial peptidoglycan, biofilm, etc. In this case, as described above, it is preferable to carry a biofilm degradation gene as the exogenous gene of B-CAP.

[0120] By applying, spreading, etc., the disinfectant according to this embodiment can eliminate bacteria from lesions, bacterial colonies, and the environment and inhibit bacterial proliferation. That is, it is possible to reduce bacteria in bacterial colonies existing in humans, animals, and / or the environment.

[0121] In addition, in the method for eliminating bacteria involved in the embodiment of the present invention, bacteria present in or outside of food can also be eliminated. Specifically, food characterized by containing B-CAP can also be provided. That is, the B-CAP involved in this embodiment can eliminate bacteria in food. This is because the B-CAP involved in this embodiment will not infect humans even if it is obtained. In addition, food in which bacteria are eliminated in this way can also be provided. Thus, for example, safe food that reliably eliminates toxin-producing food poisoning bacteria can be provided. Here, the B-CAP involved in this embodiment can be included in any food that is cultivated, raised, collected, etc., including various vegetables, edible meats, fish and shellfish, processed foods, dairy products, etc., and used.

[0122] Furthermore, the B-CAP according to the present embodiment can also be used in a corrosion prevention method for sterilizing target bacteria and preventing corrosion of food and articles other than food.

[0123] By configuring as described above, the following effects can be obtained.

[0124] Antimicrobial drugs have always played the most important role in the treatment of bacterial infections. However, due to the emergence and rapid spread of resistant bacteria that are ineffective against antibiotics, existing antibiotics are becoming ineffective. As resistant bacteria spread around the world, new antibiotics need to be developed. However, the problem of resistant bacteria cannot be solved because resistant bacteria are emerging at a rate far exceeding the development of antibiotics and the development of new antibiotics is at a stalemate. As a result, bacterial infections have once again become a global problem that threatens human health.

[0125] In addition, the bactericidal effect of phages on drug-resistant bacteria has been confirmed, and there is a trend to develop phage therapy. Since the phages used in phage therapy proliferate in the bacteria present in the infection focus, they exert a strong bactericidal power. However, from the perspective of the biological property of the phage itself, which is the proliferation, clinical use is worrying, and there is no progress in practical application. That is, when the proliferation of phages is used in antibacterial treatment, although the bactericidal power is ensured, there is a concern that it will cause adverse effects and unexpected side effects to the ecosystem. In addition, the spread of drug-resistant genes and toxin genes through phages is also worrying.

[0126] Therefore, in order to enable the use of phage therapy in society, improving treatment accuracy, controlling targeting, and improving safety have become issues.

[0127] Therefore, attempts to solve these problems have been made by modifying phages. Specifically, it is also conceivable to partially delete the genome of the phage to form a non-proliferative phage.

[0128] For example, as for the synthesis of non-proliferative phages, it is known that the proliferation ability is lost due to the functional defect of essential genes in the transformation of Amber mutations. In addition, it is known that gene defects can restore the proliferation ability through gene complementation of plasmids. However, it is considered technically difficult to delete a large number of phage genomes and to defect multiple genes, so the work has not been carried out at all.

[0129] In fact, the present inventors also tried to use plasmids to synthesize long-chain gene-deficient phages, but failed. Specifically, the terS gene of the phage was deleted and complemented with a plasmid to produce a non-proliferative phage capsid. At this time, the method using phagemids and the method using SaPI synthesized non-proliferative phages, but there were disadvantages in that an inducer such as Mitomycin C was required during the synthesis or the amount of synthesis was small.

[0130] In addition, it is known that non-proliferative phages do not proliferate, so their bactericidal power is generally weak. In fact, when using non-proliferative phages, since they do not proliferate, a very large dose of drug is required for treatment. In the examples described below, even if only non-proliferative B-CAP is allowed to act on Escherichia coli, almost no bactericidal effect can be obtained at MOI = 1.

[0131] That is, non-proliferative phages lose the advantage of phages that can proliferate at the infection site, and are not realistic for use in actual antibacterial treatment, since a large amount of phages is required.

[0132] For these reasons, it is difficult to use ordinary phages or non-proliferative phages as antimicrobial therapeutic agents. Ideal phage preparations require "effectiveness against drug-resistant bacteria", "high safety" with little impact on the ecosystem like non-proliferative phages, "mass production", and "strong antimicrobial power" with high antimicrobial therapeutic effects.

[0133] In contrast, the B-CAP according to the embodiment of the present invention can carry foreign genes in long-chain DNA while maintaining non-proliferative properties through the phage genome segmentation method, so it can be effectively used. That is, by appropriately selecting the foreign genes carried to make B-CAP, bacteria can be reliably sterilized. For example, a single bactericidal gene sometimes develops drug resistance due to bacterial evolution, but by carrying multiple genes on B-CAP, it can be suppressed, thereby maintaining the effect for a long time.

[0134] In addition, B-CAP can kill any bacteria according to the foreign genes it carries, so its "efficacy against drug-resistant bacteria" becomes higher. That is, B-CAP according to the embodiment of the present invention can deal with the emergence of new drug-resistant bacteria by simply changing the genes it carries, so it can become an evolutionary adaptive pharmaceutical. In addition, genetic engineering technology can also be used to change B-CAP itself to deal with bacteria.

[0135] In addition, the B-CAP involved in the embodiment of the present invention is "highly safe" and does not produce self-reproducing phages (progeny pharge), so it is considered to be superior to general phages in terms of safety. In addition, in traditional non-proliferative phages, if a single gene is missing, there is a high possibility that it can be proliferated after supplementation, while the B-CAP involved in this embodiment lacks a long-chain phage genome, and it is almost impossible to supplement long-chain DNA from nature. That is, B-CAP has a high safety because the probability of obtaining proliferation is extremely low. In addition, B-CAP can be prepared only in a preparation bacterium that has a complementary gene for the capsid nucleic acid element (capsimid) that synthesizes the segmented capsid. That is, B-CAP is not generated in nature, and can only be prepared in a dedicated preparation bacterium that serves as a "factory" for B-CAP synthesis.

[0136] For these reasons, B-CAP has a “high safety profile” and is able to address the biosafety issues of antimicrobial phage preparations.

[0137] Moreover, since the B-CAP involved in this embodiment is a synthetic capsid that does not self-replicate, it is possible to suppress the effects caused by the mutation of B-CAP itself. For example, since B-CAP does not proliferate, it does not introduce new drug resistance genes or toxin genes, nor does it spread. In addition, since B-CAP does not self-replicate, the possibility of the applied organisms and bacteria in the environment remaining prophages is extremely low, and the impact on the ecosystem is small. Moreover, in principle, it is difficult to produce drug-resistant bacteria. As a result, safety can be improved. Moreover, since B-CAP does not self-replicate, the application amount can be accurately estimated.

[0138] In addition, since the B-CAP according to the embodiment of the present invention is derived from natural substances, it is safe and has a low environmental impact, so it is also possible to provide a safe disinfectant or food with a low environmental impact.

[0139] In addition, regarding "mass productivity", conventionally, the genome assembly of non-proliferative phages has been performed using a cumbersome method using yeast, but the B-CAP involved in this embodiment can perform the genome assembly in a test tube during synthesis, and thus can ensure mass productivity. That is, in this embodiment, a B-CAP including a replication region and a packaging region required for nucleic acid replication of a phage is constructed by using the new concept of the genome segmentation method for segmenting the genome of a lytic phage, and it is possible to carry any long-chain DNA on the phage capsid and synthesize it in large quantities.

[0140] By assembling the B-CAP genome directly in a test tube, B-CAP can be synthesized efficiently, greatly reducing the cost and time of its synthesis. In addition, regarding formulation, since large quantities (about 1010 PFU / ml or more), thus reducing costs and having great advantages in manufacturing.

[0141] Although the B-CAP involved in this embodiment is non-proliferative, it has the same infection mode as the wild-type bacteriophage and can deliver long-chain DNA to the target bacteria. Therefore, according to the B-CAP involved in this embodiment, various biological functions including bactericidal activity can be imparted by the properties of the DNA carried, so that new modalities can be created. That is, B-CAP can construct a phage preparation with high bactericidal power.

[0142] The B-CAP synthesized in this embodiment can carry exogenous DNA and inject the DNA into the target bacteria. In addition, the injected DNA can exert biological activities derived from the self-DNA, including amplification in the target bacteria.

[0143] In addition, by utilizing the phage genome segmentation method of capsimid, the length of the DNA sequence of B-CAP that can be maintained in the bacterium can be reduced to about half of that of the conventional phage. Therefore, B-CAP also functions as a cloning vector for long-chain DNA, and can maintain highly toxic long-chain genes and synthesize them in large quantities in a short period of time. That is, exogenous long-chain DNA can be carried on B-CAP. In fact, in the examples described below, 18kb long-chain DNA from Staphylococcus aureus was successfully carried on B-CAP.

[0144] That is, B-CAP can be used to impart new biological functions in addition to bactericidal effects and the like.

[0145] Here, although the B-CAP involved in this embodiment is non-proliferative, it carries a bactericidal gene and produces a bactericidal substance with strong bactericidal activity. The product is secreted to kill the surrounding bacterial population, thereby compensating for the disadvantage of non-proliferative phages that "the antibacterial activity is weak due to non-proliferation".

[0146] For example, by loading a bactericidal gene that produces a secretory bactericidal product that kills surrounding bacteria as an exogenous gene into B-CAP, an antibacterial capsid (NM-ABCapsid) that is non-proliferative but can kill bacteria surrounding the host bacterium can be constructed.

[0147] In fact, in the examples described below, B-CAP_ColE1, which is a NM-ABCapsid, synthesizes a secretory bactericidal protein, i.e., bacteriocin (colicinE1), in the target bacteria, and shows a very strong collective bactericidal effect. That is, B-CAP_ColE1 also sterilizes the bacterial group around the target bacteria, and exerts a sufficient bactericidal effect even when the same amount as the number of bacteria is applied. Specifically, according to the examples, in the test tube experiment, the bactericidal effect of B-CAP_ColE1 is 1000 times that of the monomer B-CAP.

[0148] In addition, in the mouse infection experiment, B-CAP_ColE1 showed a significant antibacterial therapeutic effect. Specifically, B-CAP_ColE1 can treat the infection of carbapenem-resistant Escherichia coli, which is a very important drug-resistant bacteria in the clinic, as shown in the mouse infection model, and can make lethally infected mice survive. That is, in the examples described below, mice intraperitoneally administered carbapenem-resistant Escherichia coli also showed a significant survival effect on mice infected with the bacteria.

[0149] That is, the B-CAP according to the present embodiment can construct a phage preparation having a high bactericidal activity although it is non-proliferative.

[0150] In addition, the strong antibacterial activity of bacteriocins is expected to be applied to the treatment of bacterial infections, but in order to use them for treatment, bacteriocins need to be synthesized and purified in large quantities. In addition, since bacteriocins show concentration-dependent bactericidal activity, it is necessary to maintain the concentration at the infection site. Therefore, in the creation of antibacterial drugs using bacteriocins, it is important to establish a method for synthesizing a large amount of bacteriocins and to construct an effective delivery system for bacteriocins to the infection site.

[0151] In contrast, the B-CAP_ColE1 of this embodiment carries bacteriocins on safe, non-proliferative phages, and can secrete bacteriocins at a concentration that can be maintained at the infection site by inhibiting the immune gene, a resistance factor of targeted bacteria, thereby significantly improving the antibacterial therapeutic effect of the capsid.

[0152] In addition, in the above-mentioned embodiment, an example is described in which the Helper B-CAP region of the capsid nucleic acid element (capsimid) is carried on the BAC when preparing B-CAP.

[0153] However, the capsid nucleic acid element can also be carried on the genome or plasmid of the bacteria. In this case, the general plasmid has a large number of copies, and it is difficult to maintain a large number of phage genes or long-chain genes with genes that are toxic to the host. Therefore, a plasmid with a small copy number can be used to make the toxic phage gene inductive, and stability can be maintained by dividing the long-chain DNA and inserting it into multiple plasmids.

[0154] In addition, it is also possible to delete non-essential genes from B-CAP or transfer some essential genes to the HelperB-CAP side, thereby allowing longer DNA chains to be carried.

[0155] In addition, when preparing a B-CAP of a phage that is lysogenized on the genome, the region required for lysogeny may be deleted. In this case, it is also possible to construct a B-CAP using only the region required for B-CAP (replication region and packaging region required for nucleic acid replication) while leaving the region (virion, etc.) that is not required to be placed on the chromosome as B-CAP.

[0156] In addition, in B-CAP, as the replication region required for nucleic acid replication, the minimum required region other than the RNA polymerase, DNA replication region, and packaging region can be maintained.

[0157] In the above embodiment, although the phage genome is directly fragmented when forming B-CAP, other sequences may be used.

[0158] For example, when constructing B-CAP, a region sequence of about 20 kb in general, called PICI (Phage-inducible chromosomal island) contained in the bacterial genome, can be used. This PICI is also called SaPI (Staphylococcus aureus pathogenicity islands) in the case of Staphylococcus aureus.

[0159] PICI or SaPI is similar to a lysogenic phage that can enter the bacterial genome, but it does not basically have the viral particle constituting gene of the phage. Therefore, it becomes about half the genome length of the lysogenic phage. Although PICI has a compact structure, it can also be induced and replicated from the genome during phage infection, and the genome is packaged in the capsid of the phage. This packaging requires a packaging sequence derived from PICI. If this region is carried on a plasmid, the plasmid can be inserted into the lysogenic phage. In addition, it is known that the PICI that enters the capsid of the phage is transfected into other bacteria like the phage, and PICI is spread through the phage, so it can be used for B-CAP.

[0160] Alternatively, a structure in which phage DNA and the PICI or SaPI DNA are used simultaneously may be employed.

[0161] In the above embodiment, B-CAP was prepared based on T7 phage, but it can also be constructed using phages other than T7 or other bacteria.

[0162] For example, T4 phage and other giant phages, which are model phages of around ∼170 kb, can carry longer DNA.

[0163] In addition, the present inventors prepared B-CAPs for Escherichia coli phage T7 as an example of Gram-negative bacteria and Staphylococcus aureus Tan2 phage as an example of Gram-positive bacteria. That is, phage preparations using B-CAPs can be applied to a wide range of bacterial species.

[0164] In the above embodiment, an example of using a general bacteriophage as B-CAP is described. However, viruses (Virus), RNA viruses, phagemids, etc. that infect bacteria, which are different from general bacteriophages, can also be used as B-CAP. In addition, B-CAP can also be provided in a state of integration with the genome or plasmid of bacteria in the state of prophage. In addition, phagemids can be transferred to cells by any method using electrotransformation and nanoparticles, for example, in the bacteria discrimination method of this embodiment.

[0165] In the above-mentioned embodiment, examples in which a bactericidal gene, a biofilm degradation gene, a gene for antigen presentation, and a gene for colonization are used as foreign genes are described.

[0166] However, the exogenous gene can be any gene as the object. In addition, the exogenous gene can include genes that are incidentally expressed, regulated in expression, or associated with drug resistance genes or toxin genes. In addition, the exogenous gene can include pathogenic genes related to other pathogenicity and genes that inhibit them.

[0167] Furthermore, the exogenous gene of the present embodiment may also be a broadly defined "gene" such as a sequence of gene mutation, single base substitution, or repetition that is targeted for cancer treatment or genetic diseases. In this case, the bacteria themselves may also be used as a carrier (vector) of the drug for treatment. That is, it may also be applicable to the use of including a drug corresponding to the cell of the exogenous gene in the cytoplasm of the bacteria of the object, and distributing it to the tissue or the periphery of the targeted cells by lysis. In this case, the bacteria themselves containing the prophage may also be provided. Prophages activated by specific immune responses may also be included.

[0168] In addition, in the above embodiment, an example of using B-CAP as a therapeutic composition for treating infectious diseases is described.

[0169] However, this treatment can be used not only for the treatment of infectious diseases itself, but also for the modification of bacterial flora, pretreatment of fecal transplantation, etc. Specifically, the antigen presentation gene can also be used as an exogenous gene to induce immunity to bacteria required for treatment in the bacterial flora.

[0170] Moreover, just as the FDA (U.S. Food and Drug Administration) allows bacteriophages to be added to food, B-CAP can also be used to sterilize and prevent spoilage in food.

[0171] In addition, the B-CAP involved in the embodiment of the present invention can also be used in combination with other compositions, etc. In addition, it can also be provided as a "mixture" containing multiple B-CAPs. In addition, the composition of the present invention can also be applied, spread, applied, etc. simultaneously with other compositions.

[0172] [Example]

[0173] Hereinafter, the construction (production), sterilization (elimination of bacteria), and animal treatment methods of B-CAP according to the embodiment of the present invention will be described in more detail as examples based on specific experiments. However, this example is only an example and is not limited thereto.

[0174] [Technique]

[0175] (Strains and culture conditions)

[0176] The strains used in this example are shown in Table 1 below. The strains were grown in LB medium (manufactured by BDDifco) at 37°C. Unless otherwise specified, appropriate antibiotics were added to the growth medium at the following final concentrations: ampicillin (Amp) at 100 μg / ml, kanamycin (Km) at 30 μg / ml, and chloramphenicol (Cm) at 34 μg / ml.

[0177]

Table 1

[0178] Table S1. List of strains used in this study

[0179]

[0180] Among them, MC1061 and USA300 were manufactured by Lucigen, and HST08 was manufactured by Takara-Bio. ESBL (Extended Spectrum β-Lactamase) Escherichia coli Ec320 and Ec353 were obtained from Dr. Zenzo Nagasawa.

[0181] (DNA used)

[0182] DNA used in this example, such as plasmid vectors and BAC (Bacterial Artificial Chromosome) DNA, is shown in Table 2 below.

[0183]

Table 2

[0184] Table S2. List of DNA used in this study

[0185]

[0186] (Primers used)

[0187] The primers used in this example are shown in the following Table 3 and Sequence Listing.

[0188]

Table 3

[0189] Table S3 List of primers used in this study

[0190]

[0191] (Helper B-CAP construct)

[0192] From the DNA of T7 phage, the region from gp6.5 to gp12 (8.7 kb) and the region from gp13 to gp17 (9.1 kb) were amplified in two steps using KOD Fx Neo (manufactured by TOYOBO). In addition, the region from rrnBT1terminator to TTe terminator (7.5 kb) of pSMART BACv2.0 (manufactured by Lucigen) was also amplified. The amplified PCR fragments were gel-extracted using FastGene Gel / PCR Extraction kit (manufactured by NIPPON Genetics) and the concentration was measured. Each fragment was made into about 100 ng and 10 μl of NEBuilder (manufactured by New England Biolabs), for a total of 20 μl, and assembled at 50°C for 1 hour. Next, prepare the electroporation competent cells of MC1061 Escherichia coli. First, the OD 600= 0.5 MC1061 Escherichia coli 100ml was placed on ice for 20 minutes, and then washed 3 times with ice-cold 10% glycerol. Finally, 200-400μl of 10% glycerol was used for suspension. 5μl of DNA was added to 50μl of electrotransformation cells, transferred to a 1mm ice-cold test tube, and electrotransformed by ELEPO21. The conditions for electrotransformation are: Poring Pulse voltage: 1500V, pulse width: 2.5m seconds, pulse interval: 50m seconds, number of times: 1 time, polarity: +, Transfer Pulse voltage: 150V, pulse width: 50m seconds, pulse interval: 50m seconds, number of times: 5 times, polarity: + / -. Then, the electrotransformed Escherichia coli was placed in 1ml of SOC medium, cultured at 37°C for 30 minutes, and 50μl was inoculated onto an LB plate containing chloramphenicol. Eight colonies were obtained, and the strain with high B-CAP synthesis efficiency was called Escherichia coli with Helper B-CAP (hereinafter referred to as the "MC1061 (pKLC172)" strain). In addition, for the MC1061R strain with ColE1 resistance, electrotransformation was also performed in pKLC172 to form the "MC1061R (pKLC172)" strain. The pKLC172 contained in these strains was confirmed by PCR method and deep sequencing. In addition, the MC1061 (pKLC172) strain or MC1061R (pKLC172) strain is an example of the preparation bacteria used in each test described later. That is, the MC1061 (pKLC172) strain or MC1061R (pKLC172) strain is used as a "Propagation Strain" for the proliferation and preparation of B-CAP or B-CAP_ColE1.

[0193] Helper B-CAP for Staphylococcus aureus was prepared in two plasmids. The plasmid pLC-tailTan2 was constructed to make the tail part of the lysogenic phage of Staphylococcus aureus inducible to tetracycline, and the plasmid pNL-headTan2 was constructed to make the head part inducible to cadmium. Chloramphenicol and erythromycin were used for each drug resistance.

[0194] The primer sequences used for PCR are described in Table 3 and the Sequence Listing above.

[0195] (Generation of B-CAP)

[0196] For B-CAP for Escherichia coli, the region from gp17.5 to gp1 (6.8 kb), the region from gp1 to gp2.8 (6.9 kb), and the region from gp3 to gp6.3 (8.2 kb) were amplified from the DNA of T7 phage using KOD Fx Neo (manufactured by TOYOBO) in a two-step method. The amplified PCR fragments were colloid-extracted using FastGene Gel / PCR Extraction kit (manufactured by NIPPON Genetics) and the concentrations were measured. Each fragment was made into about 100 ng, and a total of 20 μl was assembled using 10 μl of NEBuilder (manufactured by New England Biolabs) and assembled at 50°C for 4 hours. The MC1061 (pKLC172) strain containing Helper B-CAP was cultured in LB medium containing chloramphenicol and the OD was 0.1444. 600 =0.5, cool the E. coli and wash three times with ice-cold 10% glycerol. Resuspend with ice-cold 10% glycerol to concentrate to about 500 times when shaken to prepare electrotransformation competent cells. Add 5μl of the prepared B-CAP reaction solution to 50μl of the electrotransformation competent cells, and transfer the mixed solution to an ice-cold 1mm test tube. Perform electrotransformation under the same conditions as the preparation of B-CAP, place in 1ml of SOC medium, and culture at 37°C for 30 minutes. Then, mix all the culture products with 3ml of LTA (LB0.5% agarose containing 1mM CaCl2, 56°C) and overlap on the LB plate. After drying the LB plate for about 10 minutes, after about 8 hours in a 37°C incubator, B-CAP plaques appear. The prepared B-CAP was confirmed by PCR method and deep sequencing.

[0197] The B-CAP of Staphylococcus aureus was constructed by electroporating the B-CAP DNA on RNA 4220 strains carrying Helper B-CAP DNA. 150 μl of overnight culture was added to 4 ml TSA top agar medium supplemented with erythromycin (15 μg / ml), chloramphenicol (15 μg / ml), CaCl2 (2.5 μM), and ATc (1 μg / ml), mixed, and poured onto a TSA plate. The plate was incubated overnight in a 37°C incubator.

[0198] The primer sequences used for PCR are described in Table 3 and the Sequence Listing above.

[0199] (Generation of B-CAP carrying foreign genes)

[0200] First, DNA of Helper B-CAP and B-CAP were prepared. In order to insert the foreign gene into B-CAP, a gene in which B-CAP was fragmented into three parts by PCR and a gene in which the foreign gene was amplified by PCR were prepared. PCR of Staphylococcus aureus and ColE1 was performed as the foreign gene. The template for each PCR was the genomic DNA of Staphylococcus aureus, pKLC187. Homologous sequences were added to the ends of each fragment so that they could be assembled with NEBuilder. The four gene fragments were assembled by NEBuilder through their homologous sequences. The assembled DNA and Helper B-CAP were electrotransformed into competent cells HST08 (manufactured by TaKaRa) of Escherichia coli. The conditions for electrotransformation were the same as above. The electrotransformed product was transferred to a total of 1 ml of SOC medium and cultured with shaking at 37°C for 30 minutes. 1 ml of Escherichia coli MC1061 (pKLC172) containing Helper B-CAP after overnight culture was added, and 8 ml of LB medium containing 1 mM CaCl2 was added. Cultivate at 37°C for 30 minutes, take out 1 ml from about 10 ml of culture solution, mix with 3 ml of LTA (LB 0.5% agarose containing 1 mM CaCl2, 56°C), and overlay on LB plate containing chloramphenicol. After drying the plate for 10 minutes, culture in an incubator at 37°C.

[0201] The primer sequences used for PCR are described in Table 3 and the Sequence Listing above.

[0202] (Preparation of Staphylococcus aureus and ColE1 sequences)

[0203] Prepare the DNA sequences of Staphylococcus aureus (MRSA) and ColE1 for loading onto B-CAP. Shake overnight in 20 mL at 37°C, centrifuge and suspend the pellet with 1 mL LTE Buffer. Add 10 μl of Achromopeptidase (50 U / μl), 5 μl of Lysostaphin (10 mg / ml) and 1 μlof RNase, incubate at 37°C for 2 hours, add 165 μl of 10% SDS and 12.5 μl of proteinase K. Incubate at 57°C for 2 hours, add 1.2 ml of TE-saturated phenol and mix well. After centrifugation at 12,000 × g for 10 minutes, transfer the supernatant to a new test tube. Add an equal amount of chloroform isoamyl alcohol (25:1) to the sample to mix, and centrifuge again at 12,000 × g for 10 minutes. 2.5 times the amount of ethanol and 1 / 10 of 3M sodium acetate were added to the supernatant, cooled at -20°C, centrifuged, and finally washed with 70% ethanol to serve as a DNA sample of Staphylococcus aureus. The ColE1 gene sequence was amplified from ColE1 DNA (manufactured by NIPPON GENE) by PCR, and the gene cassette of ColE1 (including immune gene and release gene) was cloned into pKLC23 described in Non-Patent Document 2 as an arabinose-inducible expression vector, and named pKLC146. pKLC146 was designed so that ColE1 was immediately downstream of the arabinose-inducible promoter, and then the immune gene and release gene were sequenced.

[0204] The pKLC146 plasmid was transformed into the E. coli MC1061 strain, and after culturing for 12 hours in LB medium containing 0.2% L-arabinose, the supernatant was collected. After centrifugation at 5000×g for 10 minutes, the supernatant was filtered with a 0.22μM filter. The filtrate was added to MC1061, which strongly inhibited the proliferation of bacteria. In other words, it was confirmed that ColE1 was synthesized by pKLC146. Afterwards, in order to facilitate the loading of ColE1 into B-CAP, a plasmid with a portion of the T7 phage sequence inserted into pKLC146 was made and named pKLC187.

[0205] The primer sequences used for PCR are described in the above Table 3 and the sequence listing.

[0206] (Preparation of T7 phage-resistant Escherichia coli)

[0207] 200 μl of E. coli MC1061 cultured overnight was mixed with 3 ml of LTA (containing 1 mM CaC l2 LB 0.5% agarose, 56°C) was mixed and overlapped on the LB plate. After drying for about 10 minutes and the agarose solidified, 5 μl of T7 phage solution (10 8 PFU (Plaque Forming Unit) / ml). After culturing at 37°C for 20 hours, the colonies that emerged were obtained and spread on a new LB plate. The colonies that emerged were spread on a new LB plate and cultured in LB medium. The colonies were confirmed to be resistant to T7 phage by experiments using LTA agar and were preserved.

[0208] (TEM imaging of bacteriophage and B-CAP)

[0209] First, add 1 drop of concentrated phage suspension onto a 400-mesh copper mesh (Nissin EM Co., Ltd., Japan) coated with collodion, and allow it to adsorb the phage for 10 minutes. Then, remove the residual liquid with filter paper, and drop 1% uranyl acetate (negative staining solution) onto the copper mesh. Wet the mesh completely for about 5 seconds, and then dry it using blotting. After staining, observe using an 80kV transmission electron microscope (TEM), i.e., HT7700 (manufactured by Hitachi). Take transmission electron microscope photos of phages using an 8M pixel master digital camera.

[0210] (DNA extraction from bacteriophage and B-CAP)

[0211] Phage and B-CAP were added to logarithmically growing host cells at an MOI of 0.1 to 0.01, and cultured at 37°C until the turbidity of the solution became thinner. Then, the supernatant was centrifuged at 5000g for 10 minutes, and the supernatant was recovered in a new test tube. DNase (manufactured by Sigma-Aldrich) and 10μg / ml RNase (manufactured by Sigma-Aldrich) were added at a final concentration of 10μg / ml, and stirred at 37°C for 1 hour, and an equal amount of PEG solution (10% (w / v) PEG6000, 5mM Tris-HCl (pH7.5), 1M NaCl, 5mM MgSO4·7H2O) was gradually added. After culturing overnight at 4°C, centrifugation was performed at 15000g, 30 minutes, and 4°C to recover the precipitated phage. The phage particles were resuspended in TE buffer (10mM Tris-HCl (pH8), 1mM EDTA), and 1 / 100 of 1M EDTA and 1 / 100 of 10% SDS were added to the phage suspension, and incubated at 68°C for 15 minutes. After incubation, an equal amount of phenol:chloroform:isoamyl alcohol (25:24:1, v / v / v) was added, and the solution was thoroughly mixed. After centrifugation, the aqueous phase was transferred to a new test tube, and an equal amount of chloroform:isoamyl alcohol was added at a ratio of 24:1 (v / v), and the mixture was thoroughly mixed again. After centrifugation, the aqueous phase containing the phage DNA was transferred to a new test tube, and 2.5 times the amount of ethanol was added. After incubation at -20°C for 1 hour, centrifugation was performed at 4°C and 12000g for 10 minutes. The pelleted DNA was washed twice with 1ml of 70% ethanol, and the obtained DNA was slightly dried and resuspended in an appropriate amount of TE buffer.

[0212] (Test for inhibition of bacterial proliferation by plate)

[0213] 100 μl of bacteria cultured overnight were mixed with 3 ml of LB soft agarose (LB, 0.5% agarose, 1 mM CaCl, 1 mM CaCl2, 56°C) and quickly poured into a round LB plate. In the case of a square plate, the volume of the inflow was doubled. After standing at room temperature for 5 minutes, 2 μl of B-CAP or phage was directly spotted on the upper agar containing bacterial cells, and the plate was incubated at 37°C. After incubation for about 6 hours, the formation of plaques was observed and photographed.

[0214] (Test on inhibition of bacterial growth by culture medium)

[0215] The MC1061 strain or the MC1061R strain with ColE1 tolerance was inoculated on an LB plate and incubated at 37°C for 12 hours. The culture was transferred to the LB medium containing the colonies on the plate and cultured at 37°C for 8 hours. After confirming that the bacteria had fully proliferated, each culture solution was diluted 100 times with LB medium and cultured at 37°C for 4 hours while shaking vigorously (400 rpm). When the OD (650nm) reached about 0.5, the culture was started again in the LB medium to reach an OD 600 = 0.25. After culturing for 2 hours, B-CAP / B-CAP(ColE1) was added to the growing E. coli solution to make MOI=0.001, 0.01, 0.1, 1 (10 6 , 10 7 , 10 8 , 10 9 PFU / ml), and continued shaking culture for 24 hours. During this period, the turbidity of bacteria was measured every 1 hour.

[0216] (Measurement of phage titer)

[0217] T7 phage and B-CAP were incubated for 10 -4 Up to 10 -7 On the other hand, the overnight culture of E. coli MC1061 strain and MC1061(pKLC172) strain diluted 1:100 with LB culture medium was cultured at 37°C with stirring until OD 600 Then, 10 μl of each dilution of T7 phage / B-CAP was added to 100 μl of bacterial suspension and cultured at 37°C for 20 minutes. Then, the total amount of culture solution was mixed with 0.5% LTA containing 1 mM CaCl2 and poured onto the surface of LB plate. Then, the plate was incubated at 37°C overnight. The number of plaques on the plate was counted and PFU / mL was calculated as the titer of phage.

[0218] (Whole genome sequencing of extracted DNA)

[0219] The extracted DNA sequence was read by the next generation sequencer (sequencing). First, each genomic DNA was randomly sheared into short fragments, the ends of the obtained fragments were repaired, A-tail was added, and then connected with Illumina adapters. The fragments with adapters were amplified by PCR, the size was selected and purified. The library was quantified by Qubit and real-time PCR, and the size distribution was detected by bioanalyzer. A pair-end sequence library was constructed from the plasmid using the Nextera XT Library Prep Kit (manufactured by Illumina). Sequencing was performed in the Illumina MiSeq platform (2×301bp) using the MiSeq reagent kit version3 (manufactured by Illumina). The sequence numbers were 7059496 and 8277786 in B-CAP, 8000044 and 8691738 in B-CAP (ColE1), and 88147334 and 8173908 in pKLC172. The sequence array was trimmed and assembled do novo in contigs using CLC Genomics Workbench (Qiagen). Sequence errors and DNA circularity were evaluated using CLC Genomics Workbench, and the resulting genome was annotated using RAST ver. 2.038.

[0220] (Mouse survival test)

[0221] In order to evaluate the effect of B-CAP / B-CAP_ColE1 on the treatment of E. coli infection, 7-week-old Balb / c (Clea, Japan) were used for survival test. Before starting the test, the mice were raised for one week to adapt to the laboratory environment. First, the overnight culture of carbapenem-resistant E. coli was diluted to 1:1000 with fresh LB medium and then cultured at 37°C with stirring until OD 600 Then the cells were washed twice with PBS and adjusted to about 1×10 10 Density of CFU / mL. B-CAP, B-CAP(ColE1), and T7 phage were dissolved in SM buffer after PEG precipitation and dialyzed against PBS for 1 day. Each titer was measured using MC1061 or MC1061(pKLC172).

[0222] After the mice were anesthetized, 6 mice were selected from each group and 200 μl of bacterial suspension (2×10 9CFU / mouse) was directly injected into the peritoneal cavity. Anesthesia was performed again, and 1.5 hours later, 200μl of B-CAP, B-CAP (ColE1), PBS containing T7 phage, or only PBS was injected into the same part injected with bacterial solution. 200μl of a mixed anesthetic of medetomidine hydrochloride, midazolam, and butorphanol tartrate was injected subcutaneously on the back of the mouse, and then atipamezole was used to wake up from anesthesia, and the longest survival was observed to be 5 days. The cumulative mortality rate and body weight of each treatment group were recorded at 48 hours and 72 hours after infection. Then, Kaplan-Meier survival curves were prepared based on the data of 3 independent experiments, and analyzed by log-rank test using software Prism8.

[0223] 〔result〕

[0224] (Construction of B-CAP by phage genome segmentation)

[0225] according to Figure 3 and Figure 4 , the construction (synthesis, production) of B-CAP is described.

[0226] Figure 3 (a) shows the results of extracting DNA of T7 phage, a model of lytic phage, reading the sequence with a next-generation sequencer, and annotating it using RASTver2.0. The sequence length of this T7 phage is 39956 bp.

[0227] In this example, for the purpose of B-CAP synthesis, the genome of T7 phage was divided into B-CAP DNA as a B-CAP element and Helper B-CAP DNA as a capsid nucleic acid element (capsimid), and the divided DNAs were assembled separately.

[0228] Among them, B-CAP DNA is designed to include regions responsible for Early genes (Host-virus interaction and RNA polymerase), DNA replication, lysis and packaging.

[0229] Helper B-CAP DNA used to construct B-CAP was designed to include a viral particle component region. This Helper B-CAP DNA was cloned into BAC (Bacterial artificial chromosome) to prepare pKLC172 to maintain stability in the bacteria.

[0230] Figure 3(b) shows an example of cloning the viral particle structural gene region into BAC as the Helper B-CAP of this embodiment to prepare pKLC 172. This pKLC172 is a combination of the replication system of BAC and the viral particle constituent genes of T7 phage. The sequence of pKLC172 was read by the next generation sequencer and confirmed to be 25289 bp. In addition, it was annotated using RAST ver2.0.

[0231] Next, according to Figure 4 , the sequence structure of the produced B-CAP is explained.

[0232] B-CAP DNA was electroporated into the E. coli HST08 strain carrying the Helper B-CAP DNA to produce B-CAP plaques. After isolating the plaques, we investigated whether B-CAP was constructed. Specifically, we extracted the genomic DNA of T7 phage and B-CAP, read the sequences with a next-generation sequencer, and annotated them with RAST ver2.0.

[0233] According to the results of the whole genome sequence survey, B-CAP is 22132bp, retaining the Early gene, DNA replication, Lysis, and Packaging regions, but lacking the viral particle gene.

[0234] according to Figure 5 The results of confirming the construction of B-CAP by PCR using purified T7 phage and B-CAP DNA as mold are described below.

[0235] Figure 5 (a) shows the structure of primers for checking T7 phage and B-CAP. Specifically, PCR was performed using the primer sets of "B-CAP det1274bp-f" to "T7check det5-mini3(det6)7009bp mini5221bp-r, det6 as23,027bp" in Table 3 above.

[0236] Figure 5 (b) shows the results of electrophoresis using 0.5% agarose gel. It was confirmed that the size of the genomic DNA of B-CAP was reduced to about 22 kb. It was also confirmed that the length of the fragment of the PCR (PCR3) of the Virion, lysis, and packaging regions of T7 phage was 23.0 kb, and the length of the fragment of the corresponding region in B-CAP was 5.2 kb.

[0237] Next, T7 phage and B-CAP were purified, negatively stained, and photographed using an electron microscope.

[0238] Figure 6 An electron microscope photograph is shown. Figure 6 (a) is a photo of T7 bacteriophage. Figure 6 (b) in the figure is a photo of B-CAP.

[0239] The results confirmed that the shape of B-CAP was basically the same as that of T7 phage.

[0240] Next, the bactericidal activity of the constructed B-CAP was measured. Specifically, a dilution series of T7 phage and B-CAP was prepared and dropped into E. coli MC1061 and MC1061 (pKLC172) cultured on soft agar plates. After culturing at 37°C for 6 hours, the plate was photographed. All tests were performed three times each.

[0241] Figure 7 The photographs are shown. Escherichia coli MC1061 strain was infected with B-CAP, and only 2×10 5 When the above spots were placed on the bacterial lawn of MC1061, bactericidal spots without phage spots were observed. It is believed that since B-CAP contains endolysin and toxic genes derived from T7 phage, bactericidal spots different from those of phage were formed.

[0242] On the other hand, in the E. coli MC1061 (pKLC172) strain supplemented with the genome region of the phage that does not have B-CAP with Helper B-CAP (pKLC172), bactericidal plaques were produced even at a lower B-CAP concentration due to the restoration of the proliferative properties of B-CAP. However, the plaque size of B-CAP was smaller than that of the complete T7 phage.

[0243] In this example, the synthesis of B-CAP derived from Staphylococcus aureus was further performed.

[0244] Here, the genome of the Tan2 phage of Staphylococcus aureus was divided into B-CAP DNA and Helper B-CAP DNA. Specifically, the structural gene was deleted in the lysogenic Tan2 phage of Staphylococcus aureus. In addition, a Helper B-CAP plasmid expressing the structural gene was constructed and transformed into Staphylococcus aureus RN4220 in which the structural gene-deleted Tan2 phage was lysogenic. Mitomycin C was added to the B-CAP synthesizing bacteria to assemble B-CAP by inducing Tan2 phage.

[0245] Figure 8 An example of constructing B-CAP from Tan2 phage of Staphylococcus aureus is shown.

[0246] Figure 8 (a) shows the gene structure of Tan2 phage and the concept of cloning into Helper B-CAP region. The Tan2 phage was cloned from the structural gene minor capsid to the 20140 bp of the tail fiber and inserted into the pLC1 plasmid, thereby constructing a Helper B-CAP plasmid for Tan2 phage.

[0247] Figure 8 (b) in the figure shows an example of using the Helper B-CAP plasmid to construct B-CAP. Here, first, a Staphylococcus aureus RN4220 strain in which the Tan2 phage is lysogeny is prepared. Then, a "Tan2without structure genes" bacterium in which the structural genes of the phage are deleted is prepared from the bacterium. On this basis, the Helper B-CAP plasmid is transformed into RN4220 having a Tan2 phage in which the structural genes are deleted, and a Staphylococcus aureus strain (named "S.aureus RN4220::Tan2 (pHelper B-CAP)") that synthesizes a Tan2-based B-CAP is constructed. By adding mitomycin C to the B-CAP synthesizing bacteria, the Tan2 phage is induced, thereby assembling B-CAP.

[0248] Figure 8 In (c), serial 10-fold dilutions of the prepared wild-type Tan2 phage and B-CAP were spotted on agar medium containing RN4220 or RN4220 with Helper B-CAP plasmid (hereinafter referred to as "RN4220 (Helper B-CAP)"), and lytic plaques were observed after overnight culture. Lytic plaques were observed only in RN4220 (Helper B-CAP), indicating that B-CAP was successfully constructed.

[0249] (Carrying long-chain DNA on B-CAP)

[0250] B-CAP can use the infection system of bacteriophage to inject DNA into target bacteria. Therefore, the inventors decided to investigate how long DNA can be carried in B-CAP. Specifically, the B-CAP genome was made into three fragments by PCR, and a DNA sequence derived from Staphylococcus aureus was inserted.

[0251] according to Fig. 9, an experiment of carrying long-chain DNA on B-CAP is described. Here, a long-chain DNA fragment derived from the chromosome of Staphylococcus aureus is inserted into B-CAP to obtain B-CAP (S. aureus DNA). Specifically, DNA fragments derived from Staphylococcus aureus are prepared at about 17, 18, 19, and 20 kb, respectively, and assembled into B-CAP DNA to be inserted into the downstream region of gp19, which is the late gene of T7 phage. In this embodiment, B-CAP DNA is amplified into three fragments by PCR, and circularized by combining with DNA fragments derived from Staphylococcus aureus of 17kb, 18kb, 19kb, and 20kb. The circular DNA of B-CAP containing the sequences derived from these assembled Staphylococcus aureus is electrotransformed into the Escherichia coli HST08 strain together with pKLC172 as a Helper B-CAP plasmid. Then, in order to detect the synthesized B-CAP (S. aureus DNA) as a plaque, the electro-transformed sample, E. coli MC1061 (pKLC172) and soft agar were mixed and injected onto an LB plate containing chloramphenicol (Cm). The reason for adding chloramphenicol is to prevent the wild-type strain of E. coli HST08 from proliferating, and only the B-CAP helper E. coli MC1061 (pKLC172) can proliferate. Plaques were obtained after culture.

[0252] Fig.10 The number of plaques obtained by transforming long-chain DNA of various lengths is shown. Each bar indicates the standard deviation and the mean value (n=3).

[0253] Fig.11 Representative photographs of plaques of B-CAP (S. aureus DNA) are shown.

[0254] Plaques formed on the colony of B-CAP helper Escherichia coli MC1061 (pKLC172) were counted, and as a result, about 150 and 40 plaques were confirmed from B-CAP inserted with 17 kb and 18 kb DNA, respectively.

[0255] On the other hand, several plaques were confirmed from B-CAPs into which 19 kb and 20 kb were inserted, but all of these plaques were wild-type B-CAPs into which no DNA was inserted.

[0256] Here, it was confirmed by PCR and sequencing using the Sanger method that the 17 kb and 18 kb DNA sequences derived from Staphylococcus aureus were inserted into B-CAP.

[0257] Fig.12 The results are shown in which the obtained plaques were isolated and amplified, and the insertion of long-chain DNA was confirmed by the PCR method. Fig.12(a) is a schematic diagram of each fragment. Fig.12 (b) shows the result of electrophoresis using 0.5% agarose gel.

[0258] Fig.13 The results of similarly obtained plaques being subjected to block separation, amplification, and sequencing using a next-generation sequencer are shown. Fig.13 (a) shows a schematic diagram in which the positions of the sequences subjected to B-CAP (S. aureus DNA) are represented as sequence 1 and sequence 2. Fig.13 (b) in FIG. 1 shows a portion of the actual sequence.

[0259] From the above results, it can be seen that at least 18kb of exogenous DNA can be inserted into B-CAP.

[0260] (Equipped with bactericidal genes to construct a powerful bactericidal B-CAP (NM-AB capsid))

[0261] Next, attempts were made to develop antimicrobial agents using B-CAP. Figure 7 As shown, B-CAP contains lysozyme and genes that alter the host's metabolism, so although it cannot proliferate, it is able to kill infectious bacteria.

[0262] Here, since B-CAP does not increase progeny unlike ordinary bacteriophages, there is no concern about biological risks such as accidental infection and gene transmission. However, since it cannot be amplified at the infection site like bacteriophages, it is expected that the antibacterial activity is inferior to that of ordinary bacteriophages. Therefore, the present inventors considered giving B-CAP antibacterial activity.

[0263] In this example, as a factor secreted outside the bacteria, the bacteriocin of Escherichia coli, i.e., colicin E1 (hereinafter referred to as "ColE1"), which is encoded in the plasmid of Escherichia coli and described in Non-Patent Document 2, was used. ColE1 is encoded with an immunity protein so that the bacteria carrying ColE1 are not bactericidal themselves, and has a release protein to allow the bacteria to secrete the bacteriocin outside the bacteria.

[0264] according to Fig.14 , the construction of the bactericidal B-CAP (NM-AB capsid) of this example is described.

[0265] In this embodiment, the gene region of ColE1 composed of Escherichia coli, immunity protein and release protein was loaded into B-CAP. The NM-AB capsid structure obtained by loading the gene box composed of the ColE1 gene, immunity gene and release lysis protein gene on B-CAP was named B-CAP_ColE1. B-CAP_ColE1 infects the target bacteria, injects DNA into the cells, produces bactericidal ColE1 and secretes it to the surrounding.

[0266] Fig.15 The structure of the B-CAP (ColE1) sequence of B-CAP_ColE1 is shown. Here, all DNA of T7 phage and B-CAP were extracted, and the sequence was read by next generation sequencer and annotated using RASTver2.0. The genome length of B-CAP_ColE1 is 24322bp. Thus, it was confirmed that ColE1, immunity protein, and release protein were all inserted.

[0267] In the bacterial host injected with B-CAP_ColE1, the DNA of B-CAP_ColE1 is transcribed. Then, ColE1, immunity protein, and release protein are produced, and the host bacteria secrete ColE1 outside the bacteria through the release protein under the protection of the immunity protein. It is expected that the secreted ColE1 can kill bacteria around the host bacteria in a wide range.

[0268] On the other hand, bacteria injected with the DNA of B-CAP_ColE1 are killed by toxic genes such as endolysin derived from B-CAP.

[0269] As described above, it was considered that an antibacterial agent capable of killing a wide range of bacteria despite being non-proliferative could be prepared, and the bactericidal activity of B-CAP_ColE1 was investigated.

[0270] Fig.16The results of investigating the bactericidal power are shown. The photo shows the result of planting E. coli MC1061 and MC1061R (pKLC172) on soft agar plates, and then placing solutions containing B-CAP and B-CAP_ColE1 on the plates for culturing. The "Propagation strain" is a bacteria used to obtain B-CAP_COLE1 in MC1061R (pKLC172) that was transduced with pKLC172 in the MC1061R strain with ColE1 resistance, and is a comparative example. Host: MC106 is the bacteria used to actually investigate the bactericidal power. These tests were repeated 3 times.

[0271] As a result, since B-CAP is non-proliferative, its bactericidal effect was not much confirmed, but B-CAP_ColE1 showed a very strong collective bactericidal effect.

[0272] Specifically, the results of adding B-CAP to E. coli MC1061 showed that at 2×10 5 Bactericidal plaques were observed below PFU, but almost no bactericidal plaques were observed below PFU. 1 Bactericidal plaques were also observed at 100 PFU. In addition, B-CAP_ColE1 formed very small bactericidal plaques with no proliferation. This may be because the bacteria around the bacteria injected with B-CAP_ColE1 DNA were bactericidal by ColE1.

[0273] Thus, the bactericidal activity of B-CAP_ColE1 on the plate was confirmed.

[0274] according to Fig.17 , the results of the investigation on the bactericidal power of B-CAP_ColE1 in liquid culture medium are described. The MC1061 strain and the T7-resistant strain, i.e., the MC1061R strain, were cultured in liquid medium, and B-CAP and B-CAP_ColE1 were added at MOIs of about 1, 0.1, 0.01, and 0.001 after 2 hours to study the proliferation of bacteria. Specifically, each bacterium in the proliferation phase was diluted and cultured, and B-CAP or B-CAP_ColE1 was added after 2 hours to make the MOIs of about 1, 0.1, 0.01, and 0.001.

[0275] Fig.17 Each sequence in the table shows the average of four independent experimental results. Fig.17 (a) in the figure is MC1061. Fig.17(b) is the result of MC1061R strain. When B-CAP was added to the E. coli strain MC1061 at MOI=1, the proliferation curve was slightly flat, but when it was added at MOI=0.1, 0.01, and 0.001, the proliferation curve did not change. When B-CAP was added at MOI=1, the proliferation curve became flat, which can be considered to be due to the fact that MC1061 was killed by endolysins encoded in the DNA of B-CAP. On the other hand, B-CAP_ColE1 inhibited the proliferation of MC1061 with an amount absolutely less than B-CAP.

[0276] The bactericidal effect of B-CAP_ColE1 at MOI = 0.001 was stronger than that of B-CAP at MOI = 1. B-CAP_ColE1 showed stronger antibacterial activity when the MOI was increased. 600 Therefore, it is suggested that B-CAP_ColE1 acts on bactericidal rather than bacteriostatic properties. In addition, since almost no bactericidal activity of B-CAP_ColE1 was confirmed for the ColE1-resistant E. coli MC1061R strain, it was shown that the bactericidal effect of B-CAP_ColE1 on the MC1061 strain is dependent on ColE1.

[0277] (Bactericidal effect of bactericidal B-CAP on drug-resistant Escherichia coli)

[0278] The drug resistance of Enterobacteriaceae including E. coli is a very serious problem at the global level. In fact, WHO classifies carbapenem-resistant Enterobacteriaceae (CRE) and ESBL-producing E. coli as CRITICAL priority, while CDC classifies CRE as Urgent Threads and ESBL-producing E. coli as Serious Threads.

[0279] Therefore, we investigated whether B-CAP_ColE1 was effective against clinical isolates of these drug-resistant E. coli.

[0280] according to Fig.18 , the bactericidal effect of B-CAP_ColE1 on the plate is explained. E. coli MC1061, ColE1-resistant MC1061R, carbapenem-resistant clinical isolates of E. coli, ESBL E. coli, T7-resistant clinical isolates, and propagation strain MC1061 (pKLC172) were planted on soft agar plates. Dilution series of B-CAP and B-CAP (ColE1) were prepared, spotted on the plates, and photographed after incubation at 37°C.

[0281] As a result, B-CAP_ColE1 effectively sterilized carbapenem-resistant E. coli clinical isolates, namely Ec89, Ec93, and Ec101. This bactericidal activity was roughly the same as that against MC1061, a laboratory strain. In addition, bactericidal plaques caused by B-CAP were confirmed, but the bactericidal plaques were thin, and it was judged to be about 1 / 1000 of the bactericidal power of B-CAP_ColE1. In addition, B-CAP_ColE1 also showed similarly strong bactericidal activity against multidrug-resistant bacteria, namely ESBL E. coli Ec320 and Ec353.

[0282] From this result, it was found that B-CAP_ColE1 also has bactericidal activity against drug-resistant Escherichia coli, which is a serious problem in clinical practice.

[0283] (Antibacterial therapeutic effect of bactericidal B-CAP on bacterially infected mice)

[0284] Next, in order to investigate the antibacterial therapeutic effect of B-CAP (ColE1), an antibacterial therapeutic test for bacterial infection was conducted using a mouse bacterial infection model.

[0285] according to Fig.19 , this antimicrobial treatment trial is described. Fig.19 (a) is the timing diagram. Fig.19 (b) shows the dosage. Here, 10 CFU of the bacterial suspension of clinically isolated carbapenem-resistant Escherichia coli Ec93 strain was inoculated into the peritoneal cavity of mice, and 1.5 hours later, B-CAP and B-CAP_ColE1 were directly administered into the peritoneal cavity at an MOI of 1 or 0.1, and the survival rate of the mice was investigated.

[0286] In addition, a group administered with phosphate-buffered saline (PBS) (indicated as "Saline" in the figure) was prepared as a negative control, and a group administered with proliferative T7 phage was prepared as a positive control.

[0287] exist Fig. 20 In the figure, the survival curve and the change in body weight up to 120 hours after administration are shown as the antibacterial therapeutic effect of B-CAP_ColE1. Fig. 20 (a) is the survival curve. Fig. 20 (b) is a curve of body weight change. Measurements were taken every 6 hours until 72 hours after infection, and survival and body weight were confirmed 96 hours and 120 hours later.

[0288] There was no difference between the B-CAP and PBS administration groups (Saline), and there was a significant difference between the B-CAP_ColE1 administration group and the PBS administration group (Saline). Here, the p value was calculated by the log-rank test (Mantel-Cox test). The results are expressed as statistical values ​​of three independent experiments using 6 mice per group. The result was p=0.0006.

[0289] All mice in the B-CAP administration group and the negative control PBS administration group (Saline) died 18 to 30 hours after infection. On the other hand, in the B-CAP_ColE1 administration group at MOI = 0.1, one mouse died 120 hours later, but all other mice survived.

[0290] In addition, the results of body weight measurement over time confirmed that the treatment success group had a peak every 42 hours and then gradually recovered. The group administered with proliferative T7 phage used as a positive control also recovered completely.

[0291] This result suggests that although B-CAP_ColE1 is non-proliferative, it can be used for antibacterial treatment of drug-resistant Escherichia coli and has a therapeutic effect even in small amounts.

[0292] (Summarize)

[0293] In this embodiment, by carrying bacteriocin, secretion gene and immunity gene in B-CAP at the same time, a large amount of bacteriocin is synthesized in the body of B-CAP infected bacteria, and B-CAP (ColE1) is constructed as an antibacterial agent that can secrete it outside the body. Moreover, even at MOI = 0.1, B-CAP_ColE1 shows a strong therapeutic effect on the mouse Escherichia coli peritoneal infection model. B-CAP is non-proliferative and ensures safety. At the same time, this preparation that obtains a therapeutic effect at MOI = 0.1 will become a very effective means for phage therapy, which has problems in safety and treatment accuracy in the past.

[0294] Industrial Applicability

[0295] According to the present invention, bacteria-targeting capsid particles (B-CAP) can be provided as therapeutic compositions, bactericides, foods, etc., and can be utilized industrially.

Claims

1. A bacteria-targeting capsid particle, characterized in that: The bacteria-targeted capsid particles are non-proliferative and have: bacteriophage capsid protein; and An element for bacteria-targeting capsid particles comprises a nucleic acid injection region of the genome of the bacteriophage, a replication region required for replicating nucleic acid, and a packaging region.

2. The bacteria-targeting capsid particle according to claim 1, characterized in that: The bacterial targeting capsid particle element contains a foreign gene.

3. The bacteria-targeting capsid particle according to claim 2, characterized in that: The exogenous gene comprises any one or any combination of bactericidal genes, biofilm degradation genes, antigen presentation genes and transfection genes.

4. The bacteria-targeting capsid particle according to claim 3, characterized in that: The bactericidal gene is used to produce a secretory bactericidal product that kills surrounding bacteria.

5. The bacteria-targeting capsid particle according to claim 4, characterized in that: The foreign gene contains a resistance factor that inhibits the bactericidal effect of targeted bacteria.

6. A therapeutic composition, characterized in that The therapeutic composition contains the bacteria-targeting capsid particle according to any one of claims 1 to 5.

7. A bactericide, characterized in that: The bactericide contains the bacteria-targeting capsid particle according to any one of claims 1 to 5.

8. A food, characterized in that The food contains the bacteria-targeting capsid particle according to any one of claims 1 to 5.

9. A method for eliminating bacteria, characterized in that: The method for eliminating bacteria utilizes the bacteria-targeting capsid particle according to any one of claims 1 to 5 to eliminate targeted bacteria.

10. The method for eliminating bacteria according to claim 9, characterized in that: The targeted bacteria are present in bacterial populations in humans, animals and / or the environment.

11. The method for eliminating bacteria according to claim 9, characterized in that: The targeted bacteria are present in food.

12. A sterilization method, characterized in that: The sterilization method uses the bacteria-targeted capsid particles according to any one of claims 1 to 5 to sterilize targeted bacteria.

13. A corrosion prevention method, characterized in that: The corrosion prevention method utilizes the bacteria-targeted capsid particles according to any one of claims 1 to 5 to sterilize targeted bacteria and prevent corrosion of articles.

14. A method of treating an animal, characterized in that: The animal treatment method comprises treating an animal using the bacteria-targeting capsid particle according to any one of claims 1 to 5.

15. A gene transfer method, characterized in that: The gene transfer method transfers the foreign gene contained in the bacteria-targeting capsid particle according to any one of claims 2 to 5 into a targeted bacterium.

16. A method for enhancing bacterial function, characterized in that: The method for adding function to bacteria comprises transferring the foreign gene contained in the bacteria-targeting capsid particle according to any one of claims 2 to 5 into a targeted bacterium to add function to the targeted bacterium.

17. A method for producing bacteria-targeted capsid particles, characterized in that: In the method for producing bacteria-targeted capsid particles, The capsid protein of the bacteriophage is prepared by synthesizing the capsid nucleic acid element which is fragmented from the genome of the bacteriophage and does not contain the packaging region, The element for bacteria-targeted capsid particles, which is separated from a portion of the genome of the bacteriophage other than the capsid nucleic acid element and includes a nucleic acid injection region, a replication region required for nucleic acid replication, and the packaging region, is packaged into the capsid protein. Production of non-proliferative, bacteria-targeted capsid particles.

18. The method for producing bacteria-targeted capsid particles according to claim 17, characterized in that: The capsid nucleic acid element and the bacteria-targeted capsid particle element are transformed into production bacteria for production, and the bacteria-targeted capsid particles are produced in the production bacteria.

19. The method for producing bacteria-targeting capsid particles according to claim 18, characterized in that: The capsid nucleic acid element is cloned via the chromosome or artificial chromosome used to prepare the bacteria.

20. The method for producing bacteria-targeting capsid particles according to claim 19, characterized in that: The bacterial targeting capsid particle element is transferred via the chromosome or plasmid.

21. A method for producing a nucleic acid for bacteria-targeting capsid particles, characterized in that: In the method for producing a nucleic acid for bacteria-targeted capsid particles, The capsid nucleic acid element that does not include the packaging region and synthesizes the capsid is prepared by isolating it from the phage genome. The bacteriophage genome is prepared by separating the elements for bacteria-targeted capsid particles including the nucleic acid injection region, the replication region required for nucleic acid replication, and the packaging region from the site other than the capsid nucleic acid element. Nucleic acids for constructing non-proliferative bacteria-targeted capsid particles.

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