KR20090021475A - Bacteria lysates prepared by bacteriolytic lysin enzyme, and vaccine composition for preventing animal disease comprising the same - Google Patents

Abstract

Provided is vaccine compositions for bacterial infectious diseases, including bacteria lysates which is safer than live vaccines or attenuated vaccines and prepared using lysis protein in a mild condition. A bacteria lysates is prepared by working of a bacteriolytic lysin enzyme capable of specifically destroying a peptidoglycan structure in a bacillus cell membrane. The bacteriolytic lysin enzyme has an amino acid sequence of the sequence number 1. A vaccine composition for bacterial infectious diseases comprises the bacteria lysates as an active ingredient as well as an antibody formed by administrating the bacteria lysates to an animal.

Description

Bacteria lysates prepared by bacteriolytic lysin enzyme, and vaccine composition for preventing animal disease comprising the same

The present invention relates to a vaccine composition for preventing infectious diseases of animals caused by bacteria, and more specifically, bacteriophage-derived lytic proteins that specifically destroy peptidoglycan structures present in bacterial cell membranes. The present invention relates to a vaccine composition for preventing disease comprising bacterial lysate (whole cell lysate; whole cell homogenate) killed by the action of a lysin enzyme (bacteriolytic protein; lytic protein). Bacterial lysates killed by the action of lysine, the lysing protein of the present invention, are not converted into toxic strains and are safer to use than antigen-promoting live vaccines or attenuated bacterial vaccines. Efficacy, also called efficacy, is similar to live vaccine. Therefore, the bacterial lysate disclosed in the present invention is a composition having excellent protection against bacteria, and may be usefully used as a vaccine or feed additive for preventing infectious diseases of animals caused by bacteria.

Vaccines are antigens used to actively immunize animals for the prevention of infectious diseases, or biological preparations containing the antigen as an active ingredient, were proposed by the French microbiologist L. Pasteur. In particular, vaccines as biological agents refer to immunogens which are administered to animals for the purpose of preventing infectious diseases, thereby immunizing the living body. In general, when a vaccine is administered, the antibody is produced in the living body, and immunization is obtained. Once the generated antibody remains in the living body for a relatively long time, even if an infection caused by the causative agent of the disease occurs, it can be prevented and consequently prevent the disease. It can be. Such vaccines are widely used in most animals including humans, livestock, and the like.

In general, vaccine vaccines include killed vaccines used for killing bacteria, inactivated vaccines, live vaccines using live bacteria, and attenuated strains. There are various types such as attenuated vaccines, bacterial toxoids or derivatives thereof. In addition, due to the rapid development of molecular biology and biotechnology, new types of vaccine preparation technologies such as DNA vaccines using specific genes of pathogens have been developed through genetic recombination methods.

An important point in the development of effective vaccines is that the vaccine must have sufficient antigenicity to induce immunity. This requires that the formation of antibodies against disease-causing agents be facilitated to properly induce immune responses in vivo. Therefore, it is desirable to be developed in a form similar to a possible disease agent. That is, the antigen site should be preserved as much as possible in the preparation of the vaccine. For this reason, live vaccines that use live bacteria without any treatment can have the best effect as vaccines. However, in the case of live vaccines because the disease causing agent causing disease is used alive, it is converted to a toxic strain in the future and has a risk of causing the disease, which is used only in very few infectious diseases. As an alternative to overcome these drawbacks, attenuated vaccines that have attenuated live bacteria have been developed and used.However, in this case, antigenicity is weakened compared to live vaccines, which makes them less effective as vaccines and is still safe. There is no problem. Although the bacteriophage vaccine developed due to safety in use can solve the above problems in terms of safety, the antigenicity is much lower than that of live vaccine or attenuated vaccine. Accordingly, there has been a demand for the development of an ideal new vaccine formulation that is safe to use, such as vaccinated vaccines, and superior in antigenicity as a live vaccine.

The low antigenicity of the vaccinia vaccine is believed to be due to the method of producing the dead poison vaccine. In the conventional vaccine production, bacteria are produced by heating bacteria at 56 to 60 ° C. for 30 to 60 minutes, adding chemicals such as formalin, phenol, and mazonin, or killing bacteria by irradiation of ultraviolet rays. In this method, antigenic sites related to antigenicity are denatured by physical and chemical stimuli applied during the manufacturing process, resulting in a vaccine with significantly reduced antigenicity compared to live vaccines. Therefore, there is a demand for the development of a mild manufacturing method that causes as little denaturation of the antigenic site as possible.

On the other hand, the use of bacterial lysate, a form of vaccinated vaccine, as a vaccine is not common, but some cases have been reported (FEMS Immunology & Medical Microbiology 50: 249-256, 2007; J Med Microbiol 52: 217-222, 2003; US Pat. No. 6787146). In these cases, freeze-thawing, osmotic bursting, fine grinding, sonication, the use of polytrons, as a method of crushing bacteria, The use of a blender, the use of a tissue homogenizer, and the method of mixing them are used. Since these methods are mostly methods of crushing bacteria by giving a stimulus, the antigenic sites of the obtained bacterial crush products can be physically or chemically denatured, resulting in weakening of antigenicity of the vaccine. In addition, these methods do not provide standardized bacterial lysates because they randomly disintegrate the bacteria without any form. Therefore, in order to obtain the maximal efficacy of the bactericidal vaccine, it is necessary to disintegrate bacteria under mild conditions, and to have a uniform bacterium in which the bacterial disintegration pattern is constant.

Bacteriophages are a type of virus that infects bacteria, often called abbreviations. Bacteriophages are simple organisms in which the protein envelope is wrapped around the core of a genetic material consisting of nucleic acids. Nucleic acids are composed of DNA or RNA, either single or double chains. The bacteriophage was discovered in 1915 by a British bacteriologist Twort, in a study of the transparent melting of micrococcus colonies by something. Also, in 1917, French bacteriologist d'Herelle discovered that some of the filtrates of foreign patients had a function of dissolving Shigella disentriae , and through this research, they independently discovered bacteriophages and consumed them. In the sense, they named it bacteriophage. Bacteriophages are essential for survival and all bacteria are known to contain specific bacteriophages.

The bacteriophage penetrates the host, completes the replication process, and then expresses a group of enzymes necessary to degrade the cell wall of the host bacterium. These enzymes attack the peptidoglycan layer of the cell wall, responsible for the rigidity and mechanical strength of the cell wall, destroying the cell wall. That is, the bacteriophage lysate protein is a protein that acts to destroy the cell wall of the host when the bacteriophage comes out from the host. The bacteriophage lysate protein is also commonly called lysine. In particular, in the case of lysine enzymes, a case (Nature 418: 884-889, 2002) used to kill anthrax that can be used as a biochemical weapon in bioterrorism has been reported, and lysine enzyme that can specifically kill bacteria and The research on the action is getting active.

In conclusion, the development of a safe vaccine formulation that can effectively prevent the infectious diseases caused by bacteria through the development of the conventionally requested technology is a very serious problem. In particular, the development of vaccine formulations that can effectively induce immune responses in vivo, such as live vaccines, but which are not converted to toxic strains in the future, has always been required, and the development of related technologies has a social and economic ripple effect. Can be said to be very large.

Accordingly, the present inventors have a vaccine preparation for preventing animal infectious diseases caused by bacteria, and have the maximum similarity of antigenicity as possible to live vaccines, and are safe for use in the prevention and treatment of bacterial infectious diseases, such as safe vaccines. To develop an effective vaccine.

In particular, the bacteriophage-derived lysine protein lysine enzyme specifically destroys the peptidoglycan structure in the bacterial cell membrane, and as a result of efforts to develop a vaccine composition utilizing this characteristic, it is not converted into a toxic strain. Therefore, it was possible to develop a vaccine composition manufacturing technology that is safe in use and excellent in antigenicity, and Staphylococcus aureus , which is the causative agent of various infectious diseases, is selected as a model bacterium and the vaccine composition of the present invention can be usefully used for preventing bacterial infection. The present invention has been completed by revealing that there is.

Accordingly, an object of the present invention is to provide a bacterial lysate obtained by killing bacteria by destroying the bacterial cell membrane peptidoglycan structure using lysine enzyme, a bacteriophage-derived lysine protein.

It is also an object of the present invention to provide a vaccine composition comprising the bacterial lysate as an active ingredient to be used as a pharmaceutical composition for the prevention or treatment of infectious diseases in animals.

In addition, another object of the present invention to provide a feed additive comprising the bacterial lysate as an active ingredient.

In addition, another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of animal infectious diseases, including the antibody produced after administration of the bacterial lysate to the animal as an active ingredient.

 In order to achieve the above object, the present inventors devised an entirely new method of producing a new vaccine by deviating from the existing method of producing the vaccine. In order to reduce the denaturation of the antigenic site due to the radical conditions that frequently occur in the conventional manufacturing method, the present inventors tried to devise a more mild method for producing a bacteriophage vaccine, and as a result, it was made possible by using a lysate protein derived from bacteriophage.

The present invention provides a bacterial lysate prepared by specifically killing bacteria using a lysine enzyme, a lytic protein derived from bacteriophage that destroys the peptidoglycan structure of the bacterial cell membrane.

The present invention also provides a vaccine composition and feed additive comprising the bacterial lysate as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating an infectious disease of an animal comprising the antibody produced after administration of the bacterial lysate to the animal as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present inventors have isolated a novel bacteriophage that can specifically kill Staphylococcus aureus, a model target bacterium of the present invention, and the bacteriophage screened as described on June 14, 2006 (Agricultural Biotechnology Research Institute (Accession Number KACC 97001P)). And on July 18, 2007, it was deposited again with the Korea Institute of Biotechnology and Biotechnology Center (Accession No. KCTC 11153BP), and applied for a patent (Korean Patent Application No. 2006-55461). In addition, the present inventors have disclosed a novel antimicrobial protein derived from bacteriophages capable of specifically killing Staphylococcus aureus by using the genetic information of the bacteriophage that can specifically kill Staphylococcus aureus. There is also a bar (Korean Patent Application No. 2006-73562).

Research on bacteriophage and lysine enzymes has been actively conducted around the world, and it is recognized as a new material that can solve the problems related to existing antibiotics due to the problem of using antibiotics. It is actively underway recently.

However, the research related to such lysine enzyme is mainly the study as an alternative to antibiotics, or the efforts to remove specific bacteria by using lysine enzyme itself having bacterial killing ability as an injection or a therapeutic agent, mainly the present invention As described above, there has been no report on the use of a bacterial lysate prepared by utilizing the lysine lysing ability of the lysine enzyme as a pharmaceutical composition for preventing or treating an infectious disease such as a vaccine composition.

The inventors notice that lysine, a lytic protein derived from bacteriophage, specifically destroys only the peptidoglycan structure of the bacterial cell wall and the outer cell wall of the bacterial cell wall. We have developed a vaccine composition containing the killed bacterial lysate as an active ingredient, and the bacterial lysates contained in this vaccine composition are completely destroyed and cannot be converted into toxic strains. While the advantage is that it will be impossible to cause disease by converting into a toxic strain in the future, the antigen site provides a form that best matches the original form of the live vaccine.

The present inventors tried to find the most suitable vaccine form in the bacteriophage vaccine, and among the vaccines, the vaccine form based on the bacterial lysate was used as a starting point of the present invention. Conventional methods used for bacterial disruption in the production of vaccines based on bacterial disruption are rather severe conditions, so the antigenic sites of the obtained bacterial disruption may be physically or chemically denatured, and these methods may be randomized without randomly morphizing bacteria. It does not provide bacterial lysate in its normal form, so it is noted that it eventually leads to a weakening of antigenicity of the vaccine, and strives to devise a method capable of crushing bacteria under mild conditions and having a uniform bacterial morphology with constant bacterial morphology. The present invention was completed. In the present invention, the bacteriophage-derived lytic protein is used to obtain bacterial lysates. Bacterial disruption by bacteriophage-derived lytic proteins is a relatively favorable condition compared to conventional methods due to biological reactions.Because lytic proteins do not randomly destroy bacteria, they regularly cut certain sites and destroy bacteria. In comparison, it provides a relatively constant aspect of the bacterial lysate.

Although they have the same potential antigen, the actual vaccine effect can vary greatly depending on their implementation (The Journal of Immunology 178: 48.16, 2007). This is because the response of the actual immune system is different depending on the embodiment. Therefore, minute differences in vaccine preparation can be seen as significant differences in the efficacy of the vaccine.

The present invention discloses the use of bacterial lysates. However, the bacterial lysate can be further processed and used for the same purpose. It is common in the art to use only certain parts or components for vaccine purposes, such as the use of fractions by size, use as subunit vaccines (J Med Microbiol 52: 217-222, 2003). . Therefore, although it is not described in detail, it will be obvious that it is included in the scope of the present invention. Commonly available fractionation methods include centrifugation, filtration, dialysis, electrophoresis, affinity chromatography, and ion exchange chromatography. chromatography, size exclusion chromatography, ammonium sulfate precipitation, and the like. For reference, bacteriophage-derived lytic proteins derived from bacteriophage may be removed from the bacterial lysate, but this incurs an additional purification cost and also eliminates the killing effect of the bacterium caused by the lytic protein contained in the bacterial lysate at the beginning of the vaccine composition. Done. It is therefore entirely a matter of subjective judgment.

In the present invention, a vaccine composition using a bacterial lysate obtained by the action of lysine enzyme specifically killing Staphylococcus aureus is disclosed as an applicable example of the present invention. Staphylococcus aureus has been selected as a representative of pathogenic bacteria and Shigella disentriae ), Bacillus subtilis ), Pseudomonas A variety of pathogenic bacteria, including aeruginos a), coagulase negative Staphylococcus, Streptococcus, Coliform bacteria, E. coli, dysentery, typhoid, cholera, and enterococci It can be applied to the invention. Of course, when the present invention is applied to each bacterium, an appropriate lysate protein capable of specifically disrupting each bacterium should be applied. Staphylococcus aureus is a Gram positive bacterium that is a pathogenic microorganism that causes purulent, abscess formation, various purulent infections, and sepsis. In addition, Staphylococcus aureus is the main causative agent of mastopathy, which is in the cow's body and invades the breast through the papilla and continues to survive in the white blood cell even if it is devoured by the white blood cell in the milk, producing biofilm. In this case, once formed, membranes of various sizes encapsulated with fibrous tissues are formed in the mammary gland, and once infected, it is impossible to infiltrate the drug, resulting in a chronic infection pattern. Vaccines known to prevent infectious diseases of Staphylococcus aureus include glycoconjugate vaccines including conjugates and immunocarriers of polysaccharides or glycopeptide surface antigens obtained from Staphylococcus aureus, binding proteins of Staphylococcus aureus and these fragments. Multi-component vaccines, combinations of antibodies against Staphylococcus aureus, DPNAG (deacetylated poly N-acetylated glucosamine) polysaccharide vaccines, and mutagenic toxin vaccines that have modified certain amino acid sequences of toxins from Staphylococcus aureus have been developed. Until now, no effective vaccine composition has been obtained.

As an immunologically effective dose of the vaccine composition used in the present invention, a dose suitable for eliciting an immune response may be used. The specific dose will depend on the age, body weight and clinical symptoms of the animal to be treated and the method of administration. Such titrations are usually readily determined and prescribed by an experienced physician.

The composition of the vaccine composition of the present invention is a pharmaceutical composition using physiological saline or phosphate buffered saline or ethanol polyols such as glycerol or propylene glycol, according to a method which can be easily carried out by those skilled in the art. Or by physiologically acceptable vehicle. Small amounts of surfactant may also be included to enhance the stability of the vaccine composition of the present invention.

Vaccine compositions of the present invention include vegetable oils or emulsions thereof; Surfactants such as hexadecylamine, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N, N-dioctadecyl-N'-N'bis (2-hydroxy Ethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; Polyamines such as pyran, dextransulfate, poly IC, carbopol; Peptides such as muramyl dipeptide, dimethylglycine, tuftsin; Immunostimulatory complexes; Lipopolysaccharides such as MPLR (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Montana); And additional adjuvants such as mineral gels. In addition, the bacterial lysate as an active ingredient of the vaccine composition of the present invention is liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolide, Or ISCOMS (immunostimulatory complex), and supplementary active ingredients can also be used. In addition, the bacterial lysate of the present invention may be administered with a bacterial toxin and its attenuated derivatives.

Vaccine compositions of the present invention include, but are not limited to, parenteral, intraarterial, intradermal, transdermal (by use of sustained release polymers, etc.), intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal routes of administration. Can be administered to the animal by a variety of routes. The application amount of the bacterial lysate used in such a vaccine composition may vary slightly depending on the nature of the bacterial lysate produced. Adjustment and manipulation of the set dose range used with conventional carrier antigens for application to the vaccine compositions of the present invention are within the ability of those skilled in the art. Vaccine compositions of the present invention are intended for use in immature and adult animals.

As used herein, the term 'treatment' includes (i) the prevention of infectious diseases caused by bacteria; (Ii) inhibiting infectious diseases caused by bacteria; And (iii) alleviation of an infectious disease caused by bacteria.

The present invention also provides a feed additive containing the bacterial lysate as an active ingredient.

The feed additive of the present invention may be in the form of a dry or liquid formulation, and one or more enzyme preparations may be added. Enzyme preparations can be added either dry or liquid. Enzyme preparations include lipolytic enzymes such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, starches and glycogen. amylase, an enzyme that hydrolyzes alpha-1,4-glycoside bonds, and phosphatase, an enzyme that hydrolyzes organic phosphate esters. Carboxymethylcellulase that breaks down cellulose, xylanase that breaks down xylose, and maltase that hydrolyzes maltose into two molecules of glucose (glucose). maltase, and sugar-producing enzymes such as invertase, which hydrolyzes saccharose to form a glucose-fructose mixture. It can be used.

In addition, other non-pathogenic microorganisms may be added to the feed additive including the bacterial lysate in the present invention. Microorganisms that may be added include Lactobacillus, which has physiological activity and organic degradability under anaerobic conditions such as Bacillus subtilis , Bacillus subtilis , which can produce proteolytic enzymes, lipolytic enzymes and sugar converting enzymes, and bovine stomach. Strains ( Lactobacillus sp.), Filamentous fungi such as Aspergillus oryzae , which have been shown to increase body weight, increase milk yield and increase digestive absorption of feed (J Animal Sci 43: 910-926, 1976) and yeasts such as Saccharomyces cerevisiae (J Anim Sci 56: 735-739, 1983).

In addition, feedstocks such as peanuts, peas, sugar beets, pulp, grain by-products, animal viscera flour and fishmeal flour, including various grains and soy protein, may be processed or processed.

The present invention also provides a composition comprising an antibody produced as an active ingredient after administration of a bacterial lysate prepared through the action of lysine to the animal. The composition containing the antibody produced by administration of the bacterial lysate as an active ingredient can be used as a pharmaceutical composition for the prevention or treatment of infectious diseases caused by bacterial infection.

Since the initial development of smallpox vaccines by Edward Jennner et al., The initial vaccine composition has been used to immunize an animal by using the infectious bacteria themselves or in the form of dead bacteria, or by using the infectious agent itself or a part thereof. Later, a vaccine composition, such as serum, containing an immune component such as an antibody produced from an immunized animal was prepared and developed in such a manner as to immunize the non-infected animal.

This is because serum from animals immunized by an infectious bacterium contains an immune substance such as an antibody against the bacterium, and thus could be used as an effective vaccine. However, this is mainly the administration of the bacteria itself to the animal, if the immune component such as the produced antibody as an active ingredient, the present invention is not the bacteria themselves, but the bacterial lysates produced by the action of lysine enzyme derived from bacteriophage animal After immunization, the composition containing the produced antibody as an active ingredient was used to prevent and treat bacterial infectious diseases. Although the effect is not the same, there have been cases of obtaining antibodies by administering bacterial lysates prepared by methods different from the present invention to animals in similar attempts (Clin Vaccine Immunol 14: 518-526, 2007; J Med microbiol 52: 217-). 222, 2003). However, as described above, since there is a big difference in the antigenicity of the bacterial lysates prepared according to the method applied to the bacterial crushing, it will be distinguished from the present invention.

The present invention provides a bacterial lysate prepared under mild conditions using a lytic protein derived from bacteriophage. In addition, the method for producing bacterial lysate disclosed in the present invention is based on the inherent function of the bacteriophage-derived lytic protein capable of selective cleavage of specific sites. to provide.

Vaccine composition comprising a bacterial lysate prepared by killing bacteria specifically using lysine enzyme, a lysine protein derived from bacteriophage disclosed in the present invention as an active ingredient, the bacteria are completely destroyed and not converted into toxic bacteria. It is safer to use than vaccines and live attenuated vaccines, and the antigenicity for inducing immunity is similar to that of live vaccines.

The vaccine composition may be usefully used as a vaccine or feed additive effective in preventing infectious diseases of animals caused by bacteria.

In addition, the composition comprising the antibody obtained by administering the bacterial lysate produced by the action of the lysine enzyme lysine protein derived from the bacteriophage as an active ingredient, the composition of the bacterial lysate solution prepared by the administration of conventional inactivated bacteria or a general method The protective effect against bacterial infection is better than that of the composition containing the antibody produced by administration as an active ingredient. Such a composition can be utilized as an agent for preventing and treating infectious diseases of animals caused by bacteria.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention, the content and scope of the present invention is not limited by the following examples. The following examples show the effectiveness of the present invention for a combination of Staphylococcus aureus and bacteriophage-derived lysate protein, but the present invention is not limited thereto and can be applied to a combination of various bacteria having a cell wall and the lysate protein thereof. Will be too natural for those skilled in the art. There have been several reports of effective combinations of bacteria and bacteriophage-derived lytic proteins specific to that bacterium (Nature 418: 884-889, 2002; PNAS 98: 4107-4110, 2001; Journal of Bacteriology 182: 5823-5831 , 2000). As in this report, bacteriophages effective for each bacterium are usually present in the bacterium, and lysate proteins derived from the bacteriophage are present. Even if the bacteriophage is not found to date, researchers are convinced that it must exist somewhere in nature (Nature 418: 884-889, 2002).

Example 1 Killing Effect of Bacterial Cause of Animal Infectious Disease by Lysine Enzyme Action

In this example, it was confirmed whether lysine, a lytic protein derived from bacteriophage, specifically killed the bacteria causing the infectious disease of animals, and the killing effect was verified against Staphylococcus aureus selected as a model bacterium.

The present inventors cloned the gene of lysine, a lytic protein, from the genome of Staphylococcus aureus-specific bacteriophage, and prepared lysine enzyme in the form of recombinant protein to confirm that it had antibacterial activity such as killing effect on Staphylococcus aureus. Has applied for a patent (Korean Patent Application No. 2006-73562). In this embodiment, this is referred to as reference. The amino acid sequence of the lytic protein derived from Staphylococcus aureus specific bacteriophage used in this Example is shown by SEQ ID NO: 1. Of course, the present invention is not limited to Staphylococcus aureus, it is also not limited to the lytic protein specified by SEQ ID NO: 1.

 In accordance with the invention disclosed by the present inventors (Korean Patent Application No. 2006-73562), a production strain of lysed protein derived from bacteriophage (deposited on July 18, 2007, to the Korea Institute of Bioscience and Biotechnology, Deposit No. KCTC 11151BP Lysed protein was produced and separated and purified, and then the killing effect on bacteria was investigated. The test method was Tryptic Soy Broth (TSB) medium (casein digest, 17 g / l; soybean digest, 3 g / l; dextrose, 2.5 g / l; NaCl, 5 g / l; dipotassium phosphate, 2.5 g / l) 1 ml of Staphylococcus aureus culture medium having an absorbance of about 1 at 600 nm incubated in ℓ) was plated and dried on a plate medium, and 5 µl of protein solution containing lysed protein was dropped, followed by incubation overnight at 37 ° C incubator. The degree of lysis of cocci was investigated. A total of 60 bacteria, including Staphylococcus aureus and other bacteria, were used for the bactericidal effect of lysine enzyme. These bacteria are owned by the Culture Collection of Antimicrobial Resistant Microbes (Seoul Women's University Science Hall 429, 126, Gongneung 2-dong, Nowon-gu, Seoul). The list of subject strains used is shown in FIG. 1.

As a result, after treatment with lysine, a bacteriophage-derived lysine protein, a certain time passes, the death of Staphylococcus aureus occurs, resulting in a transparent lytic plaque. 2 shows the size of the lysate formed. From these results, it was confirmed that bacteria causing infectious diseases were specifically killed by the action of lysine, a lytic protein derived from bacteriophage.

Example 2: Survival time of mice after intraperitoneal challenge

The present inventors first measured survival time after infection by intraperitoneal injection of about 3 × 10 ⁸ cfu Staphylococcus aureus into 10 BALB / c (5 weeks old, 16.5 g body weight, female) mice. To explain this, the target bacteria for investigating the efficacy of the vaccine composition of the present invention include SA3 (antibiotic resistant strain bank strain No. CCARM 3095, methicillin-resistant Staphylococcus (Methicillin-Resistant Staphylococcus) aureus ; MRSA); Clinical isolates from humans). For the preparation of the bacterial solution to be administered, the bacteria were recovered by centrifuging the bacterial culture solution at 4 ° C. at 14,000 rpm for 5 minutes, and the recovered bacteria were washed twice with physiological phosphate saline (PBS) and finally, the bacteria were added to the physiological phosphate saline solution. The mice were suspended and injected intraperitoneally.

The results for this are shown in FIG. 3. In FIG. 3 each data point represents one mouse. After infection with Staphylococcus aureus SA3, infection was observed every 6 hours for a total of 5 days, followed by every 12 hours for 9 days. As a result, the median survival time of mice infected with Staphylococcus aureus was about 55 hours, and mice intraperitoneally treated with physiological phosphate were survived within the observation period.

Example 3: Active Immunization of Mice Using Bacterial Lymphates Prepared by Lysine Enzyme Activity and Test of Bacterial Infection Protection

In this example, it was examined whether the bacterial lysate in the form of killing bacteria by the action of lysine, a lytic protein derived from bacteriophage, had a protective ability against bacterial infection. This is meaningful to find out whether the bacterial lysates specifically killed by lysine enzyme can be used as a vaccine composition for preventing bacterial infectious diseases.

In order to investigate whether the bacterial lysate disclosed in the present invention has a protective ability against bacterial infection as a vaccine composition against bacterial infection, four groups (10 mice per group) consisting of BALB / c mice having the same conditions as in Example 2 were examined. Bacterial lysates due to bacterial death induced by saline and Staphylococcus aureus-specific lysine enzymes were immunized intraperitoneally. First, the vaccine composition used for immunization was prepared as follows. Lysase enzyme, a lytic protein derived from bacteriophage, was added to 1 ml of bacterial suspension of about 3 * 10 ⁸ cfu to 20 ㎍, followed by standing at 37 ° C for 1 hour to cause bacterial death (Fig. 4). Using the obtained bacterial lysate, a vaccine composition was prepared according to a conventional method. For reference, colonies of Staphylococcus aureus were not formed when the prepared bacterial lysate was plated on TSB medium and then incubated at 37 ° C. overnight. This indicates no live bacteria present in the bacterial lysate. The vaccine composition thus prepared was intraperitoneally administered to 10 BALB / c mice per experiment group at 200 μl per time. In the control group, physiological phosphate saline was administered in the same and the same manner instead of the vaccine composition. The experimental group administered with the vaccine composition consisted of a group that received only one initial administration, one additional administration of bacterial lysate that became the antigen 7 days after the first administration, and two additional administrations every 7 days. For both experimental and control groups, on day 7 after the last dose, 1 * 10 ⁷ cfu levels of SA3 bacterial suspension were intraperitoneally administered. Three days after the bacterial suspension was administered, blood was collected from the rats, and 500 μl of the cells was diluted in 10, and then plated in Mueller-Hinton agar medium, which was incubated at 37 ° C. overnight, and finally the number of living bacteria Was investigated. As a result, all the vaccine compositions were protected against bacterial infection as compared to the control group, and when the number of live cells was compared, the vaccine was boosted only once every 7 days after the initial vaccine composition was administered. Compared with the administration of the composition, the bacterial infection protection ability was increased by an average of 100 times, and it was confirmed that 10000 times increased when boosted twice at the interval of 7 days after the initial vaccine composition administration (FIG. 5).

 The results show that the bacterial lysate prepared by the action of lysine enzyme, a bacteriophage-derived lysine, has a protective effect against bacterial infection, which includes the bacterial lysate disclosed in the present invention as an active ingredient. It can be said that the vaccine composition can be used as a pharmaceutical composition for preventing infectious diseases of animals caused by bacteria.

Example  4: Bacterial debris  Analysis of the composition using the antibody produced by administration as an active ingredient

Typically, administration of the vaccine composition to an animal results in the production of antibodies. Therefore, the present inventors administered to the animal a bacterial lysate obtained by the action of lysine enzyme, a lytic protein derived from bacteriophage, and then Staphylococcus aureus bacteria by the anti-lysine enzyme action of the composition comprising the antibody produced therefrom as an active ingredient. In order to confirm the lysate serum antibody (hereinafter referred to as anti-SA3) titer, the degree of antibody titer of the hyperimmune serum obtained by administering the bacterial lysate prepared in Example 3 to the animal was measured using a standard enzyme-related immunosorbent method (ELISA). ) Was investigated.

In detail, first, the bacterial lysates were prepared by the same method as described in Example 3 to obtain superimmune serum, and then immunized by intraperitoneal injection into mice in the same manner as in Example 3. That is, it consists of a control group using physiological phosphate saline, and a group administered only once for the first time, a group administered with an additional dose of bacterial lysate, which becomes an antigen after 7 days after the first dose, and a group administered twice additionally at 7 day intervals. In each experimental group, serum was collected from mice by orbital posterior bleeding on the 7th day after the last immunization, and the incidence of specific anti-SA3 antibody titers was measured. That is, 100 μl of Staphylococcus aureus bacterial lysate prepared as in Example 3 was added to each well of a 96-well microplate, and then allowed to stand overnight at 4 ° C., and then included Triton X-100 (Triton X 100). Washed twice with PBS and 5% fetal bovine serum (BSA) was added and allowed to stand for 30 minutes to 1 hour at room temperature. It was washed twice more in the same manner as before, dried in air, and then incubated at 37 ° C. for 1 hour by the addition of serum containing anti-SA3 antibody diluted in a series of dilutions. After washing, a secondary antibody conjugate (both anti-mouse IgG; HRP-conjugated) was added to each well in dilution, then incubated for 1 hour at 37 ° C. and washed once again. To each well, 1.5 ml of urea peroxide solution dissolved in sodium acetate / citric acid buffer and 0.2 ml of 0.6% TMB solution dissolved in DMSO were added to measure the degree of coloring. After the reaction for about 10 minutes in the light blocked state, the addition was terminated by adding 4N HSO and the absorbance was measured at 450 nm with an ELISA reader. the results are as follow. The figures in Table 1 indicate the degree of antibody production in mice and are expressed as mean logarithm.

 (A) (B)  (C) (D) Physiological saline solution Serum from mice intraperitoneally injected with bacterial lysate Serum from mice intraperitoneally injected with bacterial lysate Serum from mice injected three times intraperitoneally with bacterial lysate <0.5 (± 0.37) 3.01 (± 0.28) 4.67 (± 0.41) 5.88 (± 0.31)

In Table 1 above,

(A): mean log value (± SD) of ELISA titers measured with serum samples from mice injected intraperitoneally with phosphate phosphate;

(B): mean log value (± SD) of ELISA titers measured with serum samples of mice 7 days after intraperitoneal injection of Staphylococcus aureus bacterial lysates obtained by lysine enzyme action;

(C): Average logarithm of ELISA titers measured by serum samples of mice inoculated once more intraperitoneally with Staphylococcus aureus bacterial lysates obtained by lysine enzyme action after 7 days and then after 7 days Value (± SD);

(D): Mean average of ELISA titers measured by serum samples of mice inoculated twice a day at 7 days after intraperitoneal injection of Staphylococcus aureus bacterial lysate obtained by lysine enzyme action. Log value (± SD)

Example 5 Investigation of Protective Capability of Staphylococcus Aureus Test Infection of Immune Serum with Antibody Produced by Administration of Staphylococcus Aureus Bacterial Fragment

In general, when a vaccine composition is administered to an animal, an antibody is produced, and the serum containing the antibody or purified antibody separated from the serum may be used as a vaccine composition, which has been used for the development of traditional vaccines such as smallpox vaccines. .

Therefore, the present inventors administered the bacterial lysate obtained by the action of lysine enzyme, a bacteriophage-derived lysine enzyme, to the animal, and then confirmed the protective induction ability against bacterial infection of the composition comprising the antibody produced therefrom as an active ingredient. The protective effect of Staphylococcus aureus was tested by passively immunizing the group of mice immunized with the superimmune serum obtained by administering the bacterial lysate to the animals. This is summarized as follows. First, a bacterial liquid prepared by inactivating the bacterial lysate prepared in Example 3, a staphylococcus aureus equivalent to the yellow staphylococcus aureus used in the preparation of the bacterial lysate for 1 hour inactivation at 60 ° C. to obtain the superimmune serum, and Bacterial lysate containing the same amount of Staphylococcus aureus was pulverized by general ultrasonic pulverization in 10 BALB / c mice (5 weeks, 16.5 g, female) at a frequency of 200 μl once a week for 5 weeks. Each was intraperitoneally administered. In the control group, physiological phosphate saline was administered in the same manner. On the day after the final administration, the rats' hearts were punctured and bled to obtain blood and serum from them. In order to investigate the passive immunity effect by the obtained immune serum, each obtained serum was diluted four-fold with physiological phosphate saline and intraperitoneally administered to 10 BALB / c mice in each experimental group. After one hour of administration, the results were observed every 12 hours after infection with Staphylococcus aureus 1 * 10 ⁷ cfu. Each data point in FIG. 6 represents one mouse. The median survival time was about 3 days in the group of mice treated with no serum prior to challenge and the group of serum prepared from blood taken from physiological saline-only mice. Serum prepared from blood obtained from mice treated with a bacterial solution prepared by inactivating the suspension containing Staphylococcus aureus at 60 ° C. for 1 hour, and a bacterial crush solution obtained by crushing the suspension containing Staphylococcus aureus by a general ultrasonic grinding method. The median survival time of the group of mice immunized with was about 7-10 days. This survival was significantly longer than the group of mice receiving serum prepared from blood collected from mice receiving no or only phosphate saline. On the other hand, in the case of the mouse group receiving the immune serum obtained by inactivation of the inactivated bacteria, or in the mouse group receiving the immune serum obtained by the administration of the bacterial crush solution in which the suspension containing Staphylococcus aureus was crushed by a general ultrasonic grinding method. The median survival time was about 7 to 8 days, and the median survival time was about 9 to 10 days in the mouse group receiving the immune serum obtained by administration of the bacterial lysate prepared in Example 3.

 The results show that the composition comprising the antibody produced by the administration of the bacterial lysate produced by lysine enzyme as an active ingredient has a passive immune effect, which is produced by the administration of the bacterial lysate disclosed in the present invention. It can be said that the composition containing the antibody as an active ingredient can be used as a pharmaceutical composition for preventing infectious diseases of animals by bacteria.

In addition, a composition comprising an antibody produced by administration of a bacterium inactivated by a composition containing an antibody produced by administration of a bacterial lysate produced by the action of lysine enzyme, a lytic protein derived from bacteriophages, as an active ingredient or yellow. It can be shown that the protective effect against bacterial infection is superior to the composition comprising the antibody produced by the administration of the bacterial crushing liquid suspended in the suspension containing staphylococci by a general ultrasonic grinding method. From this, it can be said that the antibody generating ability is superior to the administration of bacteria inactivated by the administration of bacterial lysine, a lytic protein derived from bacteriophage, or the administration of bacterial lysate pulverized by general ultrasonic grinding. In other words, the antigenicity (immunogenicity) is higher.

It can be seen that the antibody produced by administration of the bacterial lysate disclosed in the present invention can be used not only for the purpose of prophylaxis but also for the purpose of treatment.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a list of bacteria targeted for the bactericidal effect of lytic proteins.

Figure 2 shows the results of investigation of the bactericidal effect of the lytic protein, the size of the lysate generated by the lytic protein treatment is shown as the diameter. A represents the diameter of the lytic plaque.

Figure 3 shows the survival time of mice after intraperitoneal challenge, "SA3" is a case of administration of Staphylococcus aureus SA3 and "PBS" shows a case of physiological saline solution.

Figure 4 is a photograph showing the bacterial lysate prepared by lytic protein treatment, the left side is a bacterial lysate produced by adding the lysate protein to the suspension of Staphylococcus aureus for 1 hour, the right side is a yellow grapes not treated It is a suspension of cocci.

5 is a result of investigation of the bacterial infection protection ability using the bacterial lysate prepared by lysine enzyme action, the control group was administered physiological phosphate saline instead of the vaccine composition, Experimental group 1 was administered only once the vaccine composition, experimental group 2 is the case where the vaccine composition is first administered 7 days after the first administration, and experimental group 3 is the case where the vaccine composition is initially administered and then twice additionally every 7 days after the initial administration of the vaccine composition.

FIG. 6 is a result of investigation of the protective ability against S. aureus test infection of serum using the antibody produced by administration of Staphylococcus aureus bacterial lysate, and "no treatment" is the case of test infection without any treatment. , “PBS” is the case obtained from serum from physiological saline-administered mice, “Inactivation” is the case obtained from serum from mice treated with inactivated bacteria, and “Bacterial lysate 1” is ultrasonic crushing. Serum was obtained from the mice to which the bacterial crushed solution was administered by the method and examined, and "bacterial lysate 2" was obtained from the mice to which the bacteria were administered according to the present invention.

<110> INTRON BIOTECHNOLOGY CO., LTD <120> Bacteria lysates prepared by bacteriolytic lysin enzyme, and          vaccine composition for preventing animal disease comprising the          same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 495 <212> PRT <213> Antimicrobial Protein <400> 1 Met Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala   1 5 10 15 Lys Gly Thr Val Asp Ser Pro Tyr Arg Ile Lys Lys Ala Thr Ser Tyr              20 25 30 Asp Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly          35 40 45 Tyr Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp      50 55 60 Leu Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile  65 70 75 80 Lys Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser                  85 90 95 Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr             100 105 110 Gln Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser         115 120 125 Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys     130 135 140 Pro Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu 145 150 155 160 Ile Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys                 165 170 175 Ser Ala Ser Lys Thr Pro Ala Pro Lys Lys Lys Ala Thr Leu Lys Val             180 185 190 Ser Lys Asn His Ile Asn Tyr Thr Met Asp Lys Arg Gly Lys Lys Pro         195 200 205 Glu Gly Met Val Ile His Asn Asp Ala Gly Arg Ser Ser Gly Gln Gln     210 215 220 Tyr Glu Asn Ser Leu Ala Asn Ala Gly Tyr Ala Arg Tyr Ala Asn Gly 225 230 235 240 Ile Ala His Tyr Tyr Gly Ser Glu Gly Tyr Val Trp Glu Ala Ile Asp                 245 250 255 Ala Lys Asn Gln Ile Ala Trp His Thr Gly Asp Gly Thr Gly Ala Asn             260 265 270 Ser Gly Asn Phe Arg Phe Ala Gly Ile Glu Val Cys Gln Ser Met Ser         275 280 285 Ala Ser Asp Ala Gln Phe Leu Lys Asn Glu Gln Ala Val Phe Gln Phe     290 295 300 Thr Ala Glu Lys Phe Lys Glu Trp Gly Leu Thr Pro Asn Arg Lys Thr 305 310 315 320 Val Arg Leu His Met Glu Phe Val Pro Thr Ala Cys Pro His Arg Ser                 325 330 335 Met Val Leu His Thr Gly Phe Asn Pro Val Thr Gln Gly Arg Pro Ser             340 345 350 Gln Ala Ile Met Asn Lys Leu Lys Asp Tyr Phe Ile Lys Gln Ile Lys         355 360 365 Asn Tyr Met Asp Lys Gly Thr Ser Ser Ser Thr Val Val Lys Asp Gly     370 375 380 Lys Thr Ser Ser Ala Ser Thr Pro Ala Thr Arg Pro Val Thr Gly Ser 385 390 395 400 Trp Lys Lys Asn Gln Tyr Gly Thr Trp Tyr Lys Pro Glu Asn Ala Thr                 405 410 415 Phe Val Asn Gly Asn Gln Pro Ile Val Thr Arg Ile Gly Ser Pro Phe             420 425 430 Leu Asn Ala Pro Val Gly Gly Asn Leu Pro Ala Gly Ala Thr Ile Val         435 440 445 Tyr Asp Glu Val Cys Ile Gln Ala Gly His Ile Trp Ile Gly Tyr Asn     450 455 460 Ala Tyr Asn Gly Asn Arg Val Tyr Cys Pro Val Arg Thr Cys Gln Gly 465 470 475 480 Val Pro Pro Asn His Ile Pro Gly Val Ala Trp Gly Val Phe Lys                 485 490 495  

Claims (5)

A bacterial lysate prepared by specifically killing bacteria by the action of a lysine enzyme, a bacteriophage-derived lytic protein that specifically destroys the peptidoglycan structure of the bacterial cell membrane. According to claim 1, wherein the bacteriophage-derived lysed protein is bacterial lysate, characterized in that having the amino acid sequence of SEQ ID NO: 1. A vaccine composition for preventing bacterial infectious diseases comprising the bacterial lysate of claim 1 or 2 as an active ingredient. A feed additive comprising the bacterial lysate of claim 1 or 2 as an active ingredient. A composition for preventing and treating bacterial infectious diseases comprising the antibody formed by administering the bacterial lysate of claim 1 or 2 as an active ingredient.

Images