Polypeptide RK12 capable of inhibiting activity of klebsiella pneumoniae and application of polypeptide RK12

Abstract

The invention provides a polypeptide RK12 with activity of inhibiting klebsiella pneumoniae and application of the polypeptide RK12. Belongs to the technical field of polypeptides and biological medicines. The amino acid sequence of the polypeptide RK12 provided by the invention is as shown in SEQ ID NO: 1. The polypeptide RK12 provided by the invention has the activity of inhibiting klebsiella pneumoniae, is good in antibacterial effect and high in stability, and has a relatively good application prospect in medicines for treating pneumonia caused by klebsiella pneumoniae infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide RK12 having the activity of inhibiting Klebsiella pneumoniae and an application thereof. Background Art

[0002] Pneumonia is an inflammation of the terminal airways, alveoli, and lung interstitium. It can be caused by a variety of causes, such as infection, physical and chemical factors, immune damage, etc. Generally, it can be divided into community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP / VAP) according to the different routes of acquisition, and can be divided into "typical" bacterial pathogens (such as Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, etc.) and "atypical" pathogens (Mycoplasma pneumoniae and Chlamydia pneumoniae, etc.) according to the different pathogens of infection.

[0003] Antimicrobial peptides are commonly found in animal immune cells, visceral mucosa, skin and other parts. Animal antimicrobial peptides mainly include mammalian antimicrobial peptides, amphibian antimicrobial peptides and insect antimicrobial peptides. Antimicrobial peptides found in amphibians, such as the peptide family and temporin family isolated from the skin of green frogs, have strong antibacterial effects on Escherichia coli, Staphylococcus aureus and Candida albicans. However, clinical problems such as pneumonia caused by Klebsiella pneumoniae still need to be solved.

[0004] It is reported that the polypeptide RK12 has anti-Propionibacterium acnes activity and anti-Candida albicans activity, but the present invention is the first to discover the anti-Klebsiella pneumoniae activity of the polypeptide RK12, and the polypeptide RK12 performs well in the inhibition zone experiment, MIC experiment, biofilm inhibition and clearance experiment, scanning electron microscopy and rat drug metabolism model on Klebsiella pneumoniae, and can be used in the preparation of pneumonia drugs against Klebsiella pneumoniae infection. Summary of the invention

[0005] In view of the above problems, the present invention provides a polypeptide RK12 having the activity of inhibiting Klebsiella pneumoniae and its application. The polypeptide RK12 provided by the present invention has the activity of inhibiting Klebsiella pneumoniae, good antibacterial effect and high stability, and has good application prospects in drugs for treating pneumonia caused by Klebsiella pneumoniae infection.

[0006] To achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a polypeptide RK12 having the activity of inhibiting Klebsiella pneumoniae, characterized in that the amino acid sequence of the polypeptide RK12 is RWWRFFKFWWKK, as shown in SEQ ID NO:1.

[0007] As a specific technical solution, the polypeptide RK12 contains 12 amino acids, a molecular weight of 1901.30, and an isoelectric point of 12.02.

[0008] In a second aspect, the present invention provides the use of the above-mentioned polypeptide RK12 in the preparation of a drug for treating pneumonia.

[0009] As a specific technical solution, the pneumonia is pneumonia caused by Klebsiella pneumoniae infection.

[0010] In a third aspect, the present invention provides a drug for treating pneumonia, comprising pharmaceutical excipients, characterized in that it also comprises the above-mentioned polypeptide RK12 or a pharmaceutically acceptable salt thereof.

[0011] As a specific technical solution, the dosage form of the drug is any one of an oral dosage form, an injection, an inhalation, and a sublingual dosage form.

[0012] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: (1) The in vitro experiments of the present invention have shown that the polypeptide RK12 has a strong activity of inhibiting Klebsiella pneumoniae, and its MIC value is 25 μg / mL. The inhibition of the growth of Klebsiella pneumoniae by polypeptide RK12 is concentration-dependent, and conforms to the S-shaped growth curve of bacteria at low concentrations, and completely inhibits bacterial growth at high concentrations. After the polypeptide RK12 of the present invention is administered by pulmonary spray, the drug is mainly concentrated in the lungs and thyroid gland, while the drug concentration in other tissues, especially in the liver and kidney tissues, is relatively low. Compared with the existing clinical drug polymyxin b, the polypeptide RK12 of the present invention has lower nephrotoxicity, can reduce the disease burden, and is conducive to restoring the weight of the treated subject.

[0013] (2) The polypeptide RK12 of the present invention has a small molecular weight, and whether it is artificially synthesized or expressed in a prokaryotic cell, the process is simple, it is easy to obtain, it has a stable structure, and it has strong inhibitory activity against bacteria. It is a safe, efficient, and reliable drug precursor molecule, which will have good application prospects in the prevention and treatment of pneumonia caused by Klebsiella pneumoniae infection and is of great significance in the development of drugs for the treatment of pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0015] Figure 1 This is a graph showing the results of determining the MIC value of polypeptide RK12 against Klebsiella pneumoniae.

[0016] Figure 2 This is a graph showing the effect of different concentrations of peptide RK12 on the growth curve of Klebsiella pneumoniae.

[0017] Figure 3 This is a diagram showing the inhibitory effect of peptide RK12 on bacterial biofilm formation of Klebsiella pneumoniae.

[0018] Figure 4 This is an electron micrograph of the bactericidal mechanism of peptide RK12 against Klebsiella pneumoniae.

[0019] Figure 5 This is the drug metabolism distribution diagram of various organs in rats after spray administration of peptide RK12. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. Example

[0021] Preparation of peptide RK12.

[0022] In this example, the polypeptide RK12 was synthesized by solid phase chemical synthesis, and its amino acid sequence is RWWRFFKFWWKK. The specific method is: S1. Weigh 0.2g of resin and place it in a dry and clean reaction tube. Add an appropriate amount of N, N-dimethylformamide (DMF) and activate for 30min. Weigh 1mmol of the first amino acid residue and 150mg of DMAP into the reaction tube. Use DMF as solvent to react for 3h. After the reaction is completed, wash with DMF 3-6 times, add appropriate pyridine and acetic anhydride in a volume ratio of 1:1, and react for 30min. After the reaction is completed, wash with DMF 3-6 times. Then use piperidine to elute the amino acid's protective group Fmoc, eluting twice for 15min each time, then wash with DMF 4 times and methanol 2 times.

[0023] S2. Weigh 3 mmol of the second amino acid and 3 mmol of HBTU in a reaction tube, add 0.5 mL of DIEA, react for 40 minutes, wash with DMF 3-6 times, add piperidine solution twice to elute the amino acid's protective group Fmoc, each time for 10 minutes, then wash with DMF 4 times and methanol 2 times.

[0024] S3. Repeat step S2 until the last amino acid residue. After the last amino acid is reacted, cut with trifluoroacetic acid for 2 hours, filter the reaction to obtain a trifluoroacetic acid solution of the polypeptide, precipitate with ether, centrifuge, and wash with ether 3-5 times to obtain a white solid. Desalt and freeze-dry by HPLC to obtain a polypeptide sample, and purify by high performance liquid chromatography (HPLC). Mass spectrometry is used to determine the purity, amino acid residue composition and molecular weight of the synthetic polypeptide, confirming that the synthetic product is the target polypeptide RK12, MW=1901.30, isoelectric point is 12.02, and purity is greater than 98%. Example 1

[0025] Determination of MIC value of peptide RK12 against Klebsiella pneumoniae.

[0026] The strain Klebsiella pneumoniae to be tested was inoculated onto an LB solid plate. After the colonies grew, a single colony was picked and transferred to LB liquid culture medium. Klebsiella pneumoniae was cultured at 37°C and 180 rpm for 3-5 h (logarithmic phase). The OD600 of the bacterial solution was measured using an ultraviolet spectrophotometer. According to the formula 1 OD (600 nm) = 1×10 9 CFU / mL method, the bacterial solution was diluted to 1×10 5 The concentration of CFU / mL. In the experiment, a sterile 96-well plate was used, and 100 μl of the diluted bacterial solution was added to each well. Then, 100 μl of the sample to be tested that was gradiently diluted with physiological saline was added to each well. The plate was mixed with a pipette and placed in a 37°C constant temperature incubator for overnight culture. Preparation of 96-well microplates: The concentrations of the peptide RK12 were 100μg / mL, 50μg / mL, 25μg / mL, 12.5μg / mL, and 6.25μg / mL. Physiological saline was used as a negative control, and polymyxin B was used as a positive control. After 24 hours of culture, the absorbance of the bacterial solution at 630 nm was measured with an enzyme-labeled instrument. The MIC value is the lowest drug concentration observed without any signs of bacterial growth. The average concentration of the sample in the well where no bacterial growth was detected and the adjacent wells was used as the MIC value. The test results were repeated 4 to 6 independent experiments. The results are as follows Figure 1 As shown, the MIC of peptide RK12 is 25 µg / mL. Example 2

[0027] Effect of peptide RK12 on the growth curve of Klebsiella pneumoniae.

[0028] The strain Klebsiella pneumoniae to be tested was inoculated onto an LB solid plate. After the colonies grew, a single colony was picked and transferred to LB liquid culture medium. Klebsiella pneumoniae was cultured at 37°C and 180 rpm for 3-5 h (logarithmic phase). The OD600 of the bacterial solution was measured using an ultraviolet spectrophotometer. According to the formula 1 OD (600 nm) = 1×109 CFU / mL method, the bacterial solution was diluted to 1×10 7 CFU / mL concentration was then incubated with different concentrations of peptide RK12, 0µg / mL, 5µg / mL, 10µg / mL, 20µg / mL, and 30µg / mL, and OD (630nm) was measured every hour for a total of 10 hours. The results are as follows Figure 2 shown.

[0029] Depend on Figure 2 It can be seen that the inhibition of Klebsiella pneumoniae growth by polypeptide RK12 is concentration-dependent, and conforms to the S-shaped growth curve of bacteria at low concentrations, while completely inhibiting bacterial growth at high concentrations. Example 3

[0030] Inhibitory effect of peptide RK12 on bacterial biofilm formation of Klebsiella pneumoniae.

[0031] The strain Klebsiella pneumoniae to be tested was inoculated onto an LB solid plate. After the colonies grew, a single colony was picked and transferred to LB liquid culture medium. Klebsiella pneumoniae was cultured at 37°C and 180 rpm for 3-5 h (logarithmic phase). The OD600 of the bacterial solution was measured using an ultraviolet spectrophotometer. According to the formula 1 OD (600 nm) = 1×10 9 CFU / mL method, the bacterial solution was diluted to 1×10 5 CFU / mL concentration, and then incubated with different concentrations of peptide RK12 of 5µg / mL, 30 µg / mL, and 50µg / mL for 48h, with 5µg / mL polymyxin B as the positive control. Then, the upper culture medium was carefully aspirated with a pipette, and then carefully rinsed three times with sterile PBS, fixed with 95% methanol for 20min, and then stained with 0.1% crystal violet for 20min, and then rinsed with PBS to remove excess crystal violet, and the biofilm was visible, and then dissolved with acetic acid and measured at OD (570nm). The results are as follows Figure 3 As shown (P < 0.05*, P < 0.01**, P < 0.005***).

[0032] Depend on Figure 3 It can be seen that the peptide RK12 at high concentrations (30 µg / mL, 50µg / mL) can effectively inhibit the formation of Klebsiella pneumoniae biofilm, and at low concentrations (5µg / mL) it has no effect on the formation of Klebsiella pneumoniae biofilm. However, for the already formed Klebsiella pneumoniae biofilm, both high and low concentrations of peptide RK12 have no significant effect. Example 4

[0033] The bactericidal mechanism of peptide RK12 against Klebsiella pneumoniae.

[0034] The strain Klebsiella pneumoniae to be tested was inoculated onto an LB solid plate. After the colonies grew, a single colony was picked and transferred to LB liquid culture medium. Klebsiella pneumoniae was cultured at 37°C and 180 rpm for 3-5 h (logarithmic phase). The OD600 of the bacterial solution was measured using an ultraviolet spectrophotometer. According to the formula 1 OD (600 nm) = 1×10 9 CFU / mL method, take 300µL of logarithmic bacteria, incubate with RK12 (20µg / mL) for 4h, then centrifuge at 1500g for 12min, discard the supernatant, add 1mL electron microscope fixative (2.5% glutaraldehyde), protect from light for four nights, centrifuge at 1500g for 12min, discard the supernatant, and dehydrate with 30%, 50%, 70%, 90%, and 100% ethanol for gradient dehydration. Take 20µL and place on silicon wafer, evaporate naturally overnight, then spray gold, and place on scanning electron microscope to scan the appearance of bacteria, the scale bar is 5 µm, 2 µm, and the determination is as follows Figure 4 shown.

[0035] Depend on Figure 4 It can be seen that the polypeptide RK12 can have a more obvious effect on Klebsiella pneumoniae. As shown in the figure, Klebsiella pneumoniae has holes, wrinkles, and breakage, that is, the polypeptide RK12 has a good bactericidal effect on Klebsiella pneumoniae. Example 5

[0036] Drug metabolism and distribution in various organs of rats after spray administration of peptide RK12.

[0037] The rats were fasted but not watered the day before the experiment after adaptive feeding. The animals were then divided into three groups (5min, 15min, 60min) with three rats in each group. After anesthesia, the spray needle was inserted into the trachea, and 1mg / kg of RK12 solution was sprayed and administered. The rats were fully shaken to establish the model. After spray administration, the rats were humanely dissected at 5min, 15min, and 60min, and plasma, lungs, hearts, livers, spleens, brains, kidneys, stomachs, small intestines (duodenum), large intestines (colon), bladders, thymus, thyroid glands, and testicular tissues were collected. 0.2 mL of blood was collected from the jugular vein or other suitable veins and placed in a labeled EDTA-K2 anticoagulant tube. After gently inverting the blood upside down to fully mix the anticoagulant (EDTA-K2) with the blood, it was immediately placed in wet ice and centrifuged as quickly as possible to separate the plasma. The centrifugation conditions were set at 4°C, 6800g, and 6 minutes. Tissue: The animals were anesthetized with isoflurane and the abdominal aorta was bled. The tissue was then taken. The tissue was washed, weighed, and stored below -20°C. The processed blood and tissue samples were then analyzed by LC-MS / MS. The results are shown in the figure below. Figure 5 shown.

[0038] Depend on Figure 5It can be seen that after administration via pulmonary spray, the drug is mainly concentrated in the lungs and thyroid gland, while the drug concentration in other tissues, especially in the liver and kidney tissues, is relatively low. That is, compared with the existing clinical drug polymyxin B, the polypeptide RK12 has lower nephrotoxicity. Combined with the in vitro antibacterial experiment, the polypeptide RK12 of the present invention not only has the ability to resist pneumonia caused by Klebsiella pneumoniae infection, but is also a safe and stable compound molecule.

[0039] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide RK12 having the activity of inhibiting Klebsiella pneumoniae, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. The polypeptide RK12 according to claim 1, characterized in that The polypeptide RK12 contains 12 amino acids, has a molecular weight of 1901.30, and an isoelectric point of 12.

02.

3. Use of the polypeptide according to claim 1 in preparing a drug for treating pneumonia.

4. The use according to claim 3, characterized in that The pneumonia is caused by Klebsiella pneumoniae infection.

5. A medicine for treating pneumonia, comprising a pharmaceutical excipient, characterized in that: Also included is the polypeptide RK12 according to claim 1 or a pharmaceutically acceptable salt thereof.

6. The drug according to claim 5, characterized in that The dosage form of the drug is any one of oral preparation, injection, inhalation and sublingual preparation.

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