Antibacterial protein composition for treating cow mastitis
By developing antibacterial protein SBL200 with specific lytic activity and using it for the treatment of streptococcal infection, the problem of poor treatment of streptococcal mastitis in the prior art was solved, and effective killing of streptococcal and significant therapeutic effects of dairy mastitis were achieved.
Patent Information
- Application Number
- CN202380055766.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-07-26
- Filing Date
- 2023-07-10
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art is difficult to effectively treat cow mastitis caused by streptococcal, especially due to antibiotic resistance problems, and the treatment effect is poor.
A specific antibacterial protein SBL200 is developed with the properties of lytic activity against Streptococcus and is included in the composition as an active ingredient for the treatment of cow mastitis caused by Streptococcus infection.
Through specific lysogenic activity, the antibacterial protein SBL200 can effectively kill streptococci, significantly improve the therapeutic effect of cow mastitis, and reduce the risk of antibiotic resistance.
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Abstract
Description
Technical Field
[0001] The present invention relates to an antimicrobial protein SBL200 having bacteriolytic activity against Streptococci, one of the pathogens of bovine mastitis, and a composition comprising the antimicrobial protein as an effective ingredient, and more particularly to an antimicrobial protein SBL200 specific for Streptococci, which has the ability to specifically lyse Streptococci and has an amino acid sequence represented by sequence number 1, and a composition for treating bovine mastitis comprising the antimicrobial protein SSL200 specific for Streptococci as an effective ingredient. Background Art
[0002] Cow mastitis is a disease caused by the invasion of bacteria widely distributed in nature into the cow's udder, causing inflammation. It has the highest incidence rate among cow diseases, and the increase in the number of cells and somatic cells in milk leads to a decline in milk quality. It is also a representative milk disease that causes economic losses such as reduced milk production and treatment costs. Even in the United States, a developed country, the economic losses caused by cow mastitis are very huge, resulting in economic losses of $200 per cow and up to $200 million per year.
[0003] The pathogenic factors of bovine mastitis are very complex. The factors that cause bovine mastitis can be roughly divided into three categories: pathogens, i.e. microorganisms, hosts, i.e. cows, and environmental factors that affect the two. Currently, there are more than 100 types of pathogens known to cause bovine mastitis, and they can cause infection through various pathways and present various disease forms.
[0004] Although the results of isolating the main pathogens of bovine mastitis vary slightly, streptococci account for about 10% of them. Although it cannot be said that the isolation rate is high, the frequency of antibiotic-resistant bacteria is high, so the treatment of bovine mastitis caused by streptococci is difficult. Therefore, it is necessary to develop a drug that can effectively treat bovine mastitis induced by streptococci.
[0005] As therapeutic agents for bovine mastitis, ointment preparations containing antibiotics such as cephalosporin antibiotics or injections containing herbal medicinal ingredients such as extracts of Phytolacca decandra are exemplified, but their effectiveness has been criticized due to problems such as minimal therapeutic effects and frequent emergence of drug-resistant bacteria. In view of the above, it is necessary to develop a therapeutic agent for bovine mastitis based on a new drug with better therapeutic effects and lower drug resistance characteristics. Summary of the invention
[0006] Therefore, as a solution to the problems caused by the use of existing antibiotics against harmful pathogenic bacteria, namely Streptococcus, the present inventors intend to provide an antimicrobial protein SBL200 that can specifically lyse Streptococcus, and provide a composition comprising the antimicrobial protein as an effective ingredient and useful for treating Streptococcus infection, and further provide a method for effectively treating bovine mastitis caused by Streptococcus using the composition.
[0007] Therefore, an object of the present invention is to provide an antimicrobial protein SBL200 having an amino acid sequence represented by SEQ ID NO: 1 and having antimicrobial activity capable of specifically lysing streptococci.
[0008] Another object of the present invention is to provide a method for effectively producing the antimicrobial protein SBL200 having an antimicrobial activity that can specifically lyse streptococci and having an amino acid sequence represented by sequence number 1. In this regard, Escherichia coli M-2 is provided as a production strain of the antimicrobial protein SBL200. The production strain, i.e., Escherichia coli M-2, was deposited in the Bioresource Center of the Korea Institute of Biotechnology on July 12, 2022 (deposit number KCTC15032BP).
[0009] Another object of the present invention is to provide a composition for treating mastitis in dairy cows induced by Streptococcus, comprising the antimicrobial protein SBL200 that can specifically lyse Streptococcus as an active ingredient.
[0010] In addition, another object of the present invention is to provide a method for treating streptococcal-induced bovine mastitis using a composition comprising the antimicrobial protein SBL200 as an active ingredient.
[0011] In order to achieve the above-mentioned objectives, the inventors of the present invention utilized the information of various antimicrobial proteins known to have antimicrobial activity against various bacterial species and maximized their own knowledge and expertise to produce various antimicrobial protein candidates in the form of recombinant proteins, and screened out antimicrobial proteins with excellent lytic activity by detecting their lytic activity against streptococci, and further developed a method for effectively producing the antimicrobial proteins, and finally developed a composition containing the antimicrobial proteins as active ingredients that can be used for the purpose of treating streptococcal-induced bovine mastitis, thereby completing the present invention.
[0012] Therefore, in one aspect of the present invention, the present invention provides an amino acid sequence of an antimicrobial protein SBL200 that can specifically lyse streptococci. Specifically, it is an amino acid sequence corresponding to sequence number 1. The antimicrobial protein SBL200 that can specifically lyse streptococci is composed of 291 amino acid residues and has a molecular weight of approximately 32.5 kDa.
[0013] It is obvious that the amino acid sequence of sequence number 1 can be partially deformed by relevant practitioners using known techniques. The deformation includes partial substitution of the amino acid sequence, partial addition of the amino acid sequence, and partial deletion of the amino acid sequence. However, it is preferable to apply the amino acid sequence of sequence number 1 disclosed in the present invention.
[0014] In addition, the present invention provides a strain, namely Escherichia coli M-2, which can be used for producing the antimicrobial protein SBL200 having the amino acid sequence of SEQ ID NO: 1. The strain, namely Escherichia coli M-2, is a strain that can produce SBL200 by transforming Escherichia coli with a plasmid having the nucleic acid sequence (6,106 bp) of SEQ ID NO: 2 produced by the present inventors.
[0015] In another aspect of the present invention, the present invention provides a composition that can be effectively used for treating streptococcal infection, comprising, as an active ingredient, an antimicrobial protein SBL200 characterized by the amino acid sequence of SEQ ID NO: 1 and having a specific bacteriolytic ability against streptococci.
[0016] The antimicrobial protein SBL200 characterized by the amino acid sequence of SEQ ID NO. 1 and having a specific bacteriolytic ability against streptococci contained in the composition of the present invention can specifically bacteriolyze streptococci as described above, and thus has the effect of treating various diseases induced by streptococci. In addition, the present invention can be implemented as an antibiotic, disinfectant, bactericide, and therapeutic agent, and is used to treat various diseases induced by streptococci.
[0017] Therefore, in another aspect of the present invention, the present invention provides a method for treating various diseases induced by Streptococcus by administering an antimicrobial protein SBL200 characterized by having an amino acid sequence represented by SEQ ID NO: 1 and specific to Streptococcus to a subject infected with tennis ball.
[0018] In the present specification, "diseases induced by streptococci" refers to diseases induced by streptococcal infection, such as bacteremia, endocarditis, abscesses, dental caries, pneumonia, meningitis, sinusitis, otitis media, pharyngitis, scarlet fever, suppurative skin diseases, cellulitis, necrotizing fasciitis, rheumatic fever, glomerulonephritis, neonatal infections (meningitis, pneumonia, bacteremia), urinary tract infections, chorioamnionitis, endometritis and wound infections, but is not limited to these.
[0019] In this specification, streptococci may be sensitive to existing antibiotics or resistant to existing antibiotics. In other words, whether or not the streptococci are resistant to existing antibiotics has no effect.
[0020] The term "treatment" or "treatment" as used in this specification refers to all actions that can inhibit infection induced by Streptococcus and alleviate the pathological state of diseases induced by Streptococcus.
[0021] In order to improve the efficiency in the application purpose as described above, an antibacterial substance that can provide antibacterial activity against other bacterial species may be added to the composition of the present invention.
[0022] The pharmaceutically acceptable carriers included in the composition of the present invention are substances commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, but are not limited thereto. In addition to the above ingredients, the composition of the present invention may additionally include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives.
[0023] The composition of the present invention can be administered orally or parenterally. In the case of parenterally, it can be administered by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration. In addition, it can also be used by coating or spraying on the diseased area, but is not limited to this.
[0024] The composition of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients in a method that can be easily implemented by a person having general knowledge of the technical field to which the present invention belongs, so as to be manufactured into a unit dosage form or a form to be loaded into a multi-dose container. In this case, the dosage form can be a solution, suspension or emulsion in an oily or aqueous medium, or a paste, powder, granule, tablet or capsule, and can also contain a dispersant or stabilizer.
[0025] In addition, the appropriate amount of coating, spraying or administration of the composition can vary depending on factors such as formulation method, administration method, age, body weight, sex, disease symptoms, diet, administration time, administration route, excretion rate and reaction sensitivity. Generally speaking, an experienced doctor or veterinarian can easily determine and prescribe an effective dosage according to the desired therapeutic effect.
[0026] The composition of the present invention can be implemented as an antibiotic, disinfectant, bactericide, and therapeutic agent.
[0027] According to the method for treating infection with Streptococcus using a composition containing the antimicrobial protein SBL200 having the amino acid sequence represented by SEQ ID NO: 1 as an effective ingredient, it is possible to effectively act on Streptococcus that is resistant to existing antibiotics or antimicrobial substances. In addition, the antimicrobial protein SBL200 specific to Streptococcus of the present invention does not affect other normal commensal bacteria except Streptococcus, thereby minimizing the side effects caused by using the composition containing the antimicrobial protein SBL200 as an effective ingredient. Existing antibiotics have the problem of inducing multiple side effects because they also have adverse effects on probiotics. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is an electrophoresis photograph illustrating the results of producing the antimicrobial protein SBL200, which is characterized by the amino acid sequence represented by SEQ ID NO: 1 and is specific to Streptococcus, in the form of a recombinant protein. Lane M is a protein size marker, and lane 1 is the chromatography eluate during secondary purification. DETAILED DESCRIPTION
[0029] Next, the present invention will be described in more detail with reference to examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.
[0030] Example 1: Preparation of antimicrobial protein SBL200 specific to Streptococcus
[0031] Next, the production of the antimicrobial protein SBL200 specific to Streptococcus will be described. In this example, Escherichia coli M-2, a strain produced by transforming Escherichia coli with a plasmid having the nucleic acid sequence of SEQ ID NO: 2 produced by the present inventors, was used as a production strain.
[0032] After adding 20 μl of Escherichia coli M-2 to 20 ml of LB medium (Tryptone 10 g / L, yeast extract 5 g / L and NaCl 10 g / L) containing 50 μg / ml kanamycin for inoculation, shaking culture was performed overnight at 37°C. The next day, the culture solution that had been cultured overnight was added to 100 mL of LB medium containing 50 μg / ml kanamycin at a volume ratio of 1 / 100. Culture was performed at a temperature of 37°C with a stirring speed of 200 rpm. When the cell concentration reached 0.3 to 0.35 based on the absorbance at 600 nm, the culture temperature was changed to 19°C. Then, after about 30 minutes, when the cell concentration reached 0.5 based on the absorbance at 600 nm, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce the expression of the antimicrobial protein SBL200 having the amino acid sequence represented by sequence number 1, and then the culture was continued for an additional 16 hours.
[0033] After the culture was completed, the cell culture solution was taken and the cell precipitate was recovered by centrifugation at 6,000 rpm at 4°C for 10 minutes, and the recovered cell precipitate was suspended in 10 ml of a buffer solution (20 mM Tris-HCl and 0.5 M NaCl, pH 7.5). The cell suspension prepared as described above was disrupted by ultrasonic disruption, which was repeated for 15 minutes under the condition of applying ultrasonic waves for 3 seconds and then stopping for 3 seconds, and was performed in an ice bath.
[0034] After the cells were pulverized, the supernatant obtained by centrifuging the cell lysate at 13,000 rpm at 4°C for 20 minutes was purified by a general affinity chromatography purification process.
[0035] Next, the purification process will be briefly described. In this example, 1 ml of HisTrap was used as the affinity resin. TMHP (GE Healthcare). The chromatographic operation was performed after the chromatography column was equilibrated in advance with buffer A (Buffer A, 20 mM Tris-HCl and 0.5 M NaCl, pH 7.5). After the sample was added to the chromatography column, it was washed with 5 column volumes (CV, Column Volume) of buffer A at a flow rate of 1 ml / min. After the washing was completed, the chromatographic operation was performed at a flow rate of 1 ml / min with a concentration gradient from 0% to 100% from buffer A to buffer B (buffer B, 20 mM Tris-HCl, 0.5 M NaCl and 1 M imidazole, pH 7.5). In the process, the desired antimicrobial protein SBL200 with the amino acid sequence represented by sequence number 1 was eluted. Figure 1 The figure shows the electrophoresis analysis results of the antibacterial protein SBL200 purified by the chromatography purification process.
[0036] The fractions containing the antimicrobial protein SBL200 at a high concentration were collected from the purified fractions obtained, and the fractions were dialyzed against a buffer solution (50 mM potassium phosphate, pH 7.5) to perform medium exchange, thereby obtaining an antimicrobial protein SBL200 solution having a purity of 90% or more.
[0037] The SBL200 protein solution was prepared by buffer-exchanging the SBL200 solution concentrated as described above with a buffer (buffer, 50 mM potassium phosphate, 5% (w / v) sorbitol, and 0.1% (w / v) poloxamer 188, pH 7.5).
[0038] Example 2: Preparation of a therapeutic agent for cow mastitis
[0039] The purified SBL200 solution was used to prepare an ointment formulation with the composition of 2 g / L allantoin, 0.6 g / L sodium benzoate, 3 g / L sodium propionate, 50 g / L propylene glycol and 3.6 ml / L SBL200 protein solution.
[0040] Specifically, manufacture is carried out in the manner described below. First, after adding 90L of sterile distilled water to the preparation tank, cool it to below 30°C. The following raw materials are added successively to allantoin, sodium proionate, sodium benzoate and propylene glycol under the mixing condition of 170rpm to dissolve. Next, the mixing rpm of the preparation tank is reduced to below 100. Next, the SBL200 protein solution manufactured in Example 1 is slowly added. Finally, after adjusting to a state close to the final amount using distilled water, stop injecting nitrogen. Next, the manufacture of the bovine mastitis therapeutic agent is terminated by adjusting to the final amount using pure water, and the manufactured product is refrigerated and stored.
[0041] Example 3: Antimicrobial activity detection of antimicrobial protein SBL200
[0042] The antibacterial activity of the streptococcus-specific antibacterial protein SBL200 produced in Example 1 was tested. The target strain information used in the antibacterial activity test is shown in Table 1.
[0043] Table 1
[0044] Antimicrobial activity test strains
[0045]
[0046]
[0047] In the antibacterial activity test, turbidity reduction assay and minimum inhibitory concentration (MIC) assay were used. Turbidity reduction assay was implemented as described below. The test strain was suspended in physiological saline so that its absorbance at 600nm reached about 0.7, and then 0.1ml of antimicrobial protein SBL200 solution (final concentration: 2.5μM) was added to 0.9ml of the suspension, and then the absorbance was measured at 600nm for 30 minutes. As a negative control group, a buffer (buffer, 50mM potassium phosphate, 5% (w / v) sorbitol and 0.1% (w / v) poloxamer 188, pH 7.5) that does not contain antimicrobial protein SBL200 was used instead of the antimicrobial protein SBL200 solution. The minimum inhibitory concentration (MIC) was performed and derived according to the general minimum inhibitory concentration (MIC) test method specified in the Clinical and Laboratory Standards Institute (CLSI) guidelines.
[0048] The results showed that the antimicrobial protein SBL200 exhibited bacteriolytic activity only against Streptococcus, and did not exhibit bacteriolytic activity against other test subject strains. The results are shown in Table 2.
[0049] Table 2 Antibacterial activity test results
[0050]
[0051] These results confirmed that the antibacterial activity of the antibacterial protein SBL200 of the present invention has very significant specificity against Streptococcus.
[0052] The above results confirm that the antimicrobial protein SBL200 specific to streptococci of the present invention can kill streptococci by lysing them. The above characteristics indicate that the composition containing the antimicrobial protein SBL200 specific to streptococci can be used for the purpose of killing streptococci when infected by streptococci, and can also be used for the purpose of treating streptococcal infection in the same manner as general antibiotics.
[0053] Example 4: Antimicrobial activity detection of antimicrobial protein SBL200 in milk
[0054] The antibacterial activity of the Streptococcus-specific antibacterial protein SBL200 produced in Example 1 was tested under milk conditions. 5 CFU / ml was inoculated with Streptococcus agalactiae (S. agalactiae) ATCC27956 (Bovine mastitis isolate), and after adding SBL200 protein sample at 50μg / ml, it was placed at room temperature for 30 minutes or 1 hour. Then 50μl of the sample was collected and diluted 100 times. After smearing 100μl of it on the plate culture medium, the number of bacteria in the milk was measured by the general plate counting method of counting the colonies generated after 1 day of culture. The results are shown in Table 3. In the following results, the case where the SBL200 protein sample was not added was the control group, and the control group was also measured for the number of bacteria by the general plate counting method in the same way as the treatment group.
[0055] Table 3
[0056] Results of bacterial count test in milk
[0057]
[0058] The above results confirm that the antimicrobial protein SBL200 can exert antimicrobial activity in the milk environment. The above results indicate that the antimicrobial protein SBL200 can be used as an active ingredient in a therapeutic agent for bovine mastitis.
[0059] Example 5: Detection of therapeutic effect on cow mastitis
[0060] The therapeutic effect of the antimicrobial protein SBL200 specific to streptococci on bovine mastitis was detected. Specifically, 6 cows suffering from bovine mastitis induced by streptococci were used as subjects, and the composition containing the antimicrobial protein SBL200 manufactured in Example 2 of the present invention was administered and tested whether it was effective in treating mastitis. The cows were divided into two groups with 3 cows in each group. The composition containing the antimicrobial protein SBL200 was injected into the treated group animals through the nipples every day, and the composition with the same ingredients except that the antimicrobial protein SBL200 was not included was injected into the control group animals. The injection volume each time was 5 ml. While treating for 10 days in the manner described above, the number of somatic cells contained in the raw milk collected from the cows before and after the treatment for 10 days was detected according to the general method. When mastitis occurs, in order to prevent the invasion of pathogens, the white blood cells will increase, and the white blood cell remains that died due to fighting with pathogens are called somatic cells. Its types include mammary epithelial cells and immune cells (leukocytes) such as neutrophils, lymphocytes and monocytes. As a somatic cell detection method, the most convenient direct microscopic monitoring method was adopted. Simply put, after staining by smearing and drying the raw milk sample within 1 cm2 of a slide, the number of somatic cells was directly checked under a microscope and multiplied by the microscope coefficient, thereby calculating the number of somatic cells in each 1 ml of raw milk. The results are shown below. The results in Table 4 below are the average values of the three cows in each test group, and the standard deviation is not provided because the value does not deviate much from the average value.
[0061] Table 4 Treatment effect of dairy cow mastitis
[0062]
[0063] In the table, the treatment group is the result in the group injected with the composition containing the antimicrobial protein SBL200; and the control group is the result in the group injected with the composition not containing the antimicrobial protein SBL200 but having the same remaining components.
[0064] The above results confirm that only when the composition containing the antimicrobial protein SBL200 of the present invention is injected, a significant level of mastitis treatment effect can be shown. The above results confirm that the composition containing the antimicrobial protein SBL200 of the present invention as an active ingredient can effectively treat cow mastitis. The above characteristics indicate that the composition containing the antimicrobial protein SBL200 of the present invention as an active ingredient can be used for the purpose of treating cow mastitis.
[0065] In the above content, specific parts of the present invention are described in detail, but people with general knowledge of the industry should understand that these specific descriptions are only preferred implementation examples, and the scope of the present invention is not limited thereby. Therefore, the substantive scope of the present invention should be defined by the attached claims and their equivalents.
[0066] Name of depository institution: KCTC
[0067] Accession number: KCTC15032BP
[0068] Collection date: 20220712
[0069]
Claims
1. An antibacterial protein SBL200 having antibacterial activity specific to Streptococcus and having an amino acid sequence represented by SEQ ID NO:
1.
2. The antimicrobial protein SBL 200 according to claim 1, characterized in that: The antimicrobial protein SBL200 consists of 291 amino acids and has a molecular weight of 32.5 kDa.
3. A composition for treating streptococcal infection, comprising the antimicrobial protein SBL 200 according to claim 1 as an active ingredient.
4. The composition for treating streptococcal infection according to claim 3, characterized in that: The composition is used as a therapeutic agent for cow mastitis.
5. A method for producing the antimicrobial protein SBL200 according to claim 1 using Escherichia coli M-2 (Deposit No. KCTC 15032BP) which is a production strain produced by transforming Escherichia coli with a plasmid represented by the nucleic acid sequence of SEQ ID NO: 2.