Use of polypeptides in the preparation of a medicament for the treatment of streptococcal infection

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a polypeptide in preparation of a drug for resisting streptococcus infection. An amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The polypeptide is proved by experiments to significantly inhibit growth of Streptococcus suis and / or Streptococcus pneumoniae, and has no inhibiting effect on various gram-negative bacteria and other gram-positive bacteria tested, and shows highly specific narrow-spectrum antibacterial characteristics, so that the polypeptide has a good application prospect as a narrow-spectrum antibacterial peptide.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to the application of a polypeptide in the preparation of drugs against streptococcal infection. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides with broad-spectrum or narrow-spectrum antimicrobial activity, and have attracted widespread attention because they are less likely to induce drug resistance. However, while broad-spectrum antimicrobial peptides kill pathogens, they can also disrupt normal flora, increasing the risk of secondary infections.

[0003] Streptococcus suis ( 猪链球菌 Streptococcus pneumoniae (S. pneumoniae) is an important pathogen causing meningitis, arthritis, and septicemia in pigs, and can also infect humans through contact. Additionally, Streptococcus pneumoniae (S. pneumoniae) 肺炎链球菌 Infections can also lead to related diseases. Therefore, there is an urgent need to develop narrow-spectrum antimicrobial peptides that are highly specific and have minimal impact on normal flora. Summary of the Invention

[0004] The purpose of this application is to provide an application of a polypeptide in the preparation of drugs against streptococcal infections, in order to solve the technical problem of how to develop a narrow-spectrum antimicrobial peptide that has an inhibitory effect on either Streptococcus suis or Streptococcus pneumoniae.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] In some embodiments, the polypeptide is used to combat streptococcal infection, wherein the streptococcus is Streptococcus suis and / or Streptococcus pneumoniae.

[0007] In some embodiments, the effective inhibitory concentration of the polypeptide against the streptococcus is 150 μg / mL to 250 μg / mL.

[0008] Secondly, this application provides an application, namely the use of the polypeptide provided in the first aspect of this application in the preparation of an anti-streptococcal infection drug, wherein the streptococcus is Streptococcus suis and / or Streptococcus pneumoniae.

[0009] In some embodiments, the anti-streptococcal infection drug includes drugs for treating at least one of meningitis, arthritis, sepsis, lymph node abscess, and endocarditis caused by streptococcal infection in pigs; And / or, the anti-streptococcal infection drug includes a drug for treating at least one of the following diseases caused by Streptococcus pneumoniae infection: bacteremia, sepsis, pneumonia, meningitis, empyema, otitis media, arthritis, osteomyelitis, peritonitis, and endocarditis.

[0010] In some embodiments, the dosage form of the anti-streptococcal infection drug includes at least one of tablets, granules, capsules, suspensions, powder for injection, and injection solutions.

[0011] Thirdly, this application provides an anti-streptococcal infection drug, comprising the polypeptide provided in the first aspect of this application, wherein the streptococcus is Streptococcus suis and / or Streptococcus pneumoniae.

[0012] In some embodiments, the anti-streptococcal infection drug includes drugs for treating at least one of meningitis, arthritis, sepsis, lymph node abscess, and endocarditis caused by streptococcal infection in pigs; And / or, the anti-streptococcal infection drug includes a drug for treating at least one of the following diseases caused by Streptococcus pneumoniae infection: bacteremia, sepsis, pneumonia, meningitis, empyema, otitis media, arthritis, osteomyelitis, peritonitis, and endocarditis.

[0013] In some embodiments, the anti-streptococcal infection drug further includes a pharmaceutically acceptable carrier.

[0014] In some embodiments, the dosage form of the anti-streptococcal infection drug includes at least one of tablets, granules, capsules, suspensions, powder for injection, and injection solutions.

[0015] The first aspect of this application provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1. Experiments have demonstrated that this polypeptide significantly inhibits the growth of Streptococcus suis and / or Streptococcus pneumoniae, while showing no inhibitory effect on a variety of tested Gram-negative bacteria and other Gram-positive bacteria, exhibiting highly specific narrow-spectrum antibacterial properties. Therefore, the aforementioned polypeptide has excellent application prospects as a narrow-spectrum antimicrobial peptide.

[0016] The second aspect of this application provides that the polypeptide provided in the first aspect of this application exhibits highly specific narrow-spectrum antibacterial properties against Streptococcus suis and / or Streptococcus pneumoniae, and therefore can be used to prepare anti-streptococcal infection drugs against the above two streptococci.

[0017] The anti-streptococcal infection drug provided in the third aspect of this application includes the polypeptide provided in the first aspect of this application. This anti-streptococcal infection drug can effectively inhibit the growth of Streptococcus suis and / or Streptococcus pneumoniae, and has high specificity. It has the advantages of not easily disrupting the normal flora and not easily inducing drug resistance, and has good application prospects.

[0018] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a graph showing the effect of the antimicrobial peptides in the embodiments of this application on the growth of representative Gram-negative bacteria.

[0021] Figure 2 This is a graph showing the effect of the antimicrobial peptides in the embodiments of this application on the growth of representative Gram-positive bacteria. Detailed Implementation

[0022] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0023] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0024] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0025] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0026] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0027] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0028] The terms "first" and "second" are used only to describe purpose, to distinguish purposes such as substances from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

[0029] In a first aspect, embodiments of this application provide a polypeptide. Specifically, the amino acid sequence of the polypeptide in embodiments of this application is shown in SEQ ID NO.1.

[0030] SEQ ID NO. 1: SRVPRFYWKDRLLKMKMA.

[0031] The embodiments of this application demonstrate through experiments that the polypeptide significantly inhibits the growth of Streptococcus suis and / or Streptococcus pneumoniae, while having no inhibitory effect on a variety of tested Gram-negative bacteria and other Gram-positive bacteria, exhibiting highly specific narrow-spectrum antibacterial properties. Therefore, the above-mentioned polypeptide of the embodiments of this application has good application prospects as a narrow-spectrum antimicrobial peptide.

[0032] The peptides in the embodiments of this application can be prepared using methods commonly used in the field of peptide preparation. For example, they can be prepared using solid-phase chemical synthesis / transsalt modification expression methods.

[0033] In some embodiments, the polypeptide shown in SEQ ID NO. 1 can be synthesized using the Fmoc solid-phase synthesis method (i.e., 9-fluorophthaloylmethoxycarbonyl protected histidine-triphenylmethyl ester protected amino acid-solid-phase synthesis method). For example, using Rink Amide resin as a carrier, Fmoc-protected amino acids are sequentially coupled according to the amino acid sequence of SEQ ID NO. 1, followed by TFA cleavage, ether precipitation, reversed-phase high-performance liquid chromatography purification, and lyophilization. The polypeptide is then purified by HPLC to a purity of over 95%, and the molecular weight is consistent with the theoretical value as determined by mass spectrometry. After synthesis, it is lyophilized and stored for later use. The above preparation method is simple and suitable for industrial production.

[0034] Streptococcus ( 链球菌 Streptococci are a large group of Gram-positive cocci that reproduce by dividing in a single plane, forming chains of varying lengths or clusters, hence the name "streptococcus." Among them, Streptococcus pneumoniae (Streptococcus pneumoniae)链球菌 肺炎 (Streptococcus suis) is a classic diplococcus: lanceolate, arranged in pairs, with opposite wide ends, and specific identification characteristics: positive bile lysis test, most strains are sensitive to Optochin, and colonies exhibit umbilicated autolysis. 猪链球菌 The bacteria are arranged in short chains, with very few typical diplococcal morphologies. Identification features include: negative bile lysis test and the vast majority of strains being resistant to optoxin.

[0035] The polypeptides in this application embodiment exhibit a significant and uniform inhibitory effect on the growth of Streptococcus suis and / or Streptococcus pneumoniae. Therefore, in some embodiments, the polypeptides can be used to combat streptococcal infection, i.e., to combat Streptococcus suis and / or Streptococcus pneumoniae infection.

[0036] In some embodiments, the effective inhibitory concentration of the peptide in this application against *Streptococcus suis* and / or *Streptococcus pneumoniae* is 150 μg / mL to 250 μg / mL; exemplaryly, the effective inhibitory concentration of the peptide in this application can be 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, 250 μg / mL, etc. For example, in some embodiments of this application, the peptide significantly inhibits the growth of *Streptococcus suis* and *Streptococcus pneumoniae* at a concentration of 200 μg / mL.

[0037] Secondly, this application provides an application. Specifically, the application of the polypeptide provided in the first aspect of this application in the preparation of an anti-streptococcal infection drug, wherein the streptococcus is Streptococcus suis and / or Streptococcus pneumoniae.

[0038] The peptides provided in the embodiments of this application exhibit highly specific narrow-spectrum antibacterial properties against Streptococcus suis and / or Streptococcus pneumoniae, and therefore can be used to prepare anti-streptococcal infection drugs against the above two streptococci.

[0039] In some embodiments, the anti-streptococcal agent includes an agent for treating at least one of the following diseases caused by Streptococcus suis infection: meningitis, arthritis, sepsis, lymph node abscess, and endocarditis; and / or, the anti-streptococcal agent includes an agent for treating at least one of the following diseases caused by Streptococcus pneumoniae infection: bacteremia, sepsis, pneumonia, meningitis, empyema, otitis media, arthritis, osteomyelitis, peritonitis, and endocarditis.

[0040] After pigs are infected with Streptococcus suis, they generally develop the following typical diseases (common diseases in pig farms): (1) Acute septicemia: most common in weaned pigs and growing pigs, with a body temperature of 41~43℃, purple hemorrhage spots on the skin, ears, abdomen and limbs, difficulty breathing, and light red foam from the mouth and nose; the course of the disease is very short. (2) Meningitis (high incidence in piglets): most susceptible to weaned piglets, high fever, loss of appetite; typical neurological symptoms: circling, teeth grinding, chewing without food, lying down and paddling with limbs, convulsions, nystagmus, and hind limb paralysis. (3) Arthritis (most common in chronic cases): swollen and hot joints of the limbs, effusion and suppuration, lameness, reluctance to stand; long course of disease, pig growth stagnation and becoming stunted pigs, mostly from acute infection to chronic. (4) Suppurative lymphadenitis: swollen and hardened lymph nodes in the submandibular, cervical and pharyngeal regions, hot and painful, affecting eating and swallowing; in the later stage, abscesses rupture and yellowish-white pus flows out, etc. The incubation period for human infection with Streptococcus suis is generally several hours to 7 days, and the clinical manifestations are diverse: (1) Meningitis type (most common): The most typical manifestations are sudden high fever, severe headache, projectile vomiting, stiff neck, confusion, and convulsions. (2) Septicemia type: High fever, chills, which can progress to toxic shock syndrome, skin and mucous membrane ecchymosis, and high mortality rate. (3) Secondary local organ diseases in human infection: such as suppurative arthritis, endocarditis, etc.

[0041] Streptococcus pneumoniae can cause infections in multiple sites, which can be divided into two main categories: invasive severe cases and non-invasive mild cases. I. Non-invasive diseases (the most common, frequently seen in outpatient settings); such as community-acquired lobar pneumonia, acute otitis media, and sinusitis. II. Invasive pneumococcal infections; such as purulent meningitis, sepsis (septicemia), purulent arthritis, osteomyelitis, pericarditis, empyema, cellulitis, and deep wound abscesses.

[0042] The embodiments of this application provide an anti-streptococcal infection drug prepared based on the polypeptide shown in SEQ ID NO.1, which can be used to treat the above-mentioned symptoms caused by Streptococcus suis infection and can also be used to treat the above-mentioned symptoms caused by Streptococcus pneumoniae infection.

[0043] In some embodiments, the dosage form of the anti-streptococcal infection drug includes at least one of tablets, granules, capsules, suspensions, powder for injection, and injection solutions. That is, the dosage form of the anti-streptococcal infection drug prepared from the peptides provided in the embodiments of this application can be various, and can be selected and applied according to actual clinical needs.

[0044] Thirdly, embodiments of this application provide an anti-streptococcal infection drug. Specifically, the anti-streptococcal infection drug of this application includes the polypeptide provided in the first aspect of this application, and the anti-streptococcal infection drug targets Streptococcus suis and / or Streptococcus pneumoniae.

[0045] The anti-streptococcal infection drug provided in this application includes the polypeptide shown in SEQ ID NO.1. Therefore, this anti-streptococcal infection drug can effectively inhibit the growth of Streptococcus suis and / or Streptococcus pneumoniae. Moreover, it has high specificity and the advantages of not easily disrupting normal flora and not easily inducing drug resistance, and has good application prospects.

[0046] In some embodiments, the anti-streptococcal infection drug includes a drug for treating at least one of the following diseases caused by Streptococcus suis infection: meningitis, arthritis, septicemia, lymph node abscess, and endocarditis; and / or, the anti-streptococcal infection drug includes a drug for treating at least one of the following diseases caused by Streptococcus pneumoniae infection: bacteremia, septicemia, pneumonia, meningitis, empyema, otitis media, arthritis, osteomyelitis, peritonitis, and endocarditis. The anti-streptococcal infection drug of the present application embodiments can be used to treat the above-mentioned conditions caused by Streptococcus suis infection, and can also be used to treat the above-mentioned conditions caused by Streptococcus pneumoniae infection.

[0047] In some embodiments, the dosage form of anti-streptococcal drugs includes at least one of tablets, granules, capsules, suspensions, powder for injection, and injection solutions. Various dosage forms of anti-streptococcal drugs can be used, and the appropriate form is selected based on actual clinical needs.

[0048] In some embodiments, anti-streptococcal drugs may also include pharmaceutically acceptable carriers.

[0049] In this application embodiment, the polypeptide shown in SEQ ID NO.1 is used to prepare an anti-streptococcal infection drug (the streptococcus being Streptococcus suis and / or Streptococcus pneumoniae); the amount of the anti-streptococcal infection polypeptide used is an "effective amount," which refers to an amount that can produce function or activity in humans and / or animals and is acceptable to humans and / or animals. "Pharmaceutically acceptable carrier" refers to a carrier used for therapeutic administration, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc. In this application embodiment, various methods well known in the art can be used to administer the anti-streptococcal infection drug to mammals or humans. Including but not limited to: subcutaneous injection, intramuscular injection, percutaneous administration, local administration, implantation, sustained-release administration, etc.; the choice can be made according to the dosage form of the anti-streptococcal drug.

[0050] This application has undergone multiple experiments, and some of the experimental results are presented here for reference to further describe the application in detail. The following is a detailed description in conjunction with specific embodiments.

[0051] Example 1 Strains culture and growth curve determination: Several strains of Gram-negative bacteria and several strains of Gram-positive bacteria were selected. The specific strains are shown in Table 1 and Table 2.

[0052] Each strain was incubated overnight and then diluted to 1×10⁻⁶. 6 CFU / mL was added to each well of a 96-well plate, with a final concentration of 200 μg / mL of antimicrobial peptide (as shown in SEQ ID NO.1) added to each well as the experimental group. Simultaneously, no antimicrobial peptide was added to each strain as a control group. A shared negative control group was also provided using phosphate-buffered saline (PBS) supplemented with uncultured culture medium. The plates were incubated at 37°C with shaking, and OD values ​​were measured at 0h, 6h, 18h, 24h, 36h, and 48h. 600 The samples were continuously monitored for 48 hours. Each group had three replicates, and the average value was used to create a bar chart of growth data. For the four strains of *Streptococcus suis*, *Streptococcus pyogenes*, *Streptococcus pneumoniae*, and *Neisseria meningitidis*, the overnight culture medium used was Brain Heart Infusion Broth (BHI) with 10% goat serum; the other strains were cultured overnight using Luria-Bertani (LB) medium. Phosphate buffer was Solarbio's product number P1020, LB medium was Solarbio's product number L1010, BHI medium was Solarbio's product number LA0680, and goat serum was Solarbio's product number YZ-50012-8615.

[0053] Experimental results are as follows Figure 1 , Figure 2 As shown in the figure. The horizontal axis represents the experimental group treated with antimicrobial peptides, the control group without antimicrobial peptides, and the shared negative control group with PBS added to the culture medium for various strains. The vertical axis represents the OD values ​​at time points 0-6h-18h-24h-36h-48h. Figure 2 For example, in the figure, SS represents the control group in Streptococcus suis without the addition of antimicrobial peptides, SS+SR18 represents the experimental group in Streptococcus suis with the addition of the antimicrobial peptides of the present application, spn represents the control group in Streptococcus pneumoniae without the addition of antimicrobial peptides, spn+SR18 represents the experimental group in Streptococcus pneumoniae with the addition of the antimicrobial peptides of the present application, and other groups are similar, while Medium represents the common negative control group in which PBS is added to the culture medium without the cultured strain.

[0054] Figures 1-2Data show that at a high concentration of 200 μg / mL, the antimicrobial peptides provided in this application have no growth inhibitory effect on all Gram-negative bacteria listed in Table 1 (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Neisseria meningitidis, Acinetobacter baumannii); they also have no inhibitory effect on Gram-positive bacteria listed in Table 2 (Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes), but show significant inhibition on the growth of Streptococcus suis and Streptococcus pneumoniae, with OD values ​​within 6–48 h. 600 No sustained growth was observed. This indicates that the antimicrobial peptides in the embodiments of this application are narrow-spectrum antimicrobial peptides, specifically acting on Streptococcus suis and / or Streptococcus pneumoniae.

[0055] Table 1. Types of Gram-negative bacteria

[0056] Table 2. Types of Gram-positive bacteria

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any way. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. The use of a polypeptide in the preparation of an anti-streptococcal infection drug, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and the streptococcus is Streptococcus suis and / or Streptococcus pneumoniae.

2. The application as described in claim 1, characterized in that, The effective inhibitory concentration of the polypeptide against the streptococcus is 150 μg / mL to 250 μg / mL.

3. The application as described in claim 1, characterized in that, The anti-streptococcal infection drugs include drugs for treating at least one of the following diseases caused by Streptococcus suis infection: meningitis, arthritis, sepsis, lymph node abscess, and endocarditis; And / or, the anti-streptococcal infection drug includes a drug for treating at least one of the following diseases caused by Streptococcus pneumoniae infection: bacteremia, sepsis, pneumonia, meningitis, empyema, otitis media, arthritis, osteomyelitis, peritonitis, and endocarditis.

4. The application as described in claim 1, characterized in that, The anti-streptococcal infection drugs also include pharmaceutically acceptable carriers.

5. The application as described in any one of claims 1-4, characterized in that, The dosage forms of the anti-streptococcal infection drugs include at least one of tablets, granules, capsules, suspensions, powder for injection, and injection solutions.

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