CN107652359B - Antibacterial peptide KR-32 and application thereof - Google Patents

Abstract

The invention discloses an antibacterial peptide KR‑32 and its application. The full sequence of the antimicrobial peptide is: KFFKRLRRSVVKKFRSWLSRTAR RLENSAKKR, SEQ ID NO:1. The antimicrobial peptide KR-32 is an active polypeptide synthesized by artificial design. KR-32 contains 32 amino acid residues, has a molecular weight of 3896.7Da, an isoelectric point of 10.42, and a net charge of +13. The use of the antimicrobial peptide is used to prepare broad-spectrum antibacterial drugs for treating Gram-negative bacteria or Gram-positive bacteria infections. The use of the antibacterial peptide is used to prepare drugs for improving immune regulation. The use of the antimicrobial peptide is used to prepare a treatment or prevention preparation for piglets with weaning diarrhea. The antimicrobial peptide KR‑32 has the advantages of high net charge, broad antibacterial spectrum, strong bactericidal effect, and no bactericidal effect on probiotics. It also has the characteristics of low hemolytic activity and low cytotoxicity, and can play an immune regulation role to reduce inflammation. The antimicrobial peptide KR‑32 also has antiviral activity and can alleviate viral diarrhea in piglets caused by PEDV.

Description

Antimicrobial peptide KR-32 and its application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to antibacterial peptide KR-32 and its application.

Background technique

Antimicrobial peptides are a kind of polypeptide substances that widely exist in natural organisms, and their sources are very extensive. At present, 1700 have been found in organisms such as plants, insects, amphibians, mammals, marine organisms, bacteria, and viruses. A variety of antimicrobial peptides, antimicrobial peptides are important innate immune molecules, with broad-spectrum antibacterial activity, membrane-breaking bactericidal mechanism, and resistance to drug resistance. At the same time, antimicrobial peptides are considered to be the most potential alternatives to antibiotics, so the research and development of antimicrobial peptides will become an important breakthrough in finding alternatives to antibiotics, and the research on antimicrobial peptides in the treatment of piglet diarrhea provides important information for the practical application of antimicrobial peptides. reference value. At present, some antibacterial peptides with good antibacterial activity, such as C-BF and P-OG1, have certain cytotoxicity. How to enhance their antibacterial activity and reduce cytotoxicity has become one of the research hotspots, and the molecular improvement of antimicrobial peptides is a common research hotspot. s method.

Contents of the invention

In order to overcome the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide an antibacterial peptide KR-32 and its application.

An antibacterial peptide named KR-32, its full sequence is: KFFKRLRRSVVKKFRSWLSRTARRLENSAKKR, SEQ ID NO:1.

The antimicrobial peptide KR-32 is an active polypeptide synthesized by artificial design. KR-32 contains 32 amino acid residues, has a molecular weight of 3896.7Da, an isoelectric point of 10.42, and a net charge of +13.

The use of the antimicrobial peptide is used to prepare broad-spectrum antibacterial drugs for treating Gram-negative bacteria or Gram-positive bacteria infections.

The use of the antibacterial peptide is used to prepare drugs for improving immune regulation.

The use of the antimicrobial peptide is used to prepare a treatment or prevention preparation for piglets with weaning diarrhea.

Beneficial effects of the present invention:

The antimicrobial peptide KR-32 designed and synthesized in the present invention is an α-helical cationic polypeptide, which has the characteristics of short peptide chain length, good stability and convenient artificial synthesis. The antibacterial peptide KR-32 has the advantages of high net charge, broad antibacterial spectrum, strong bactericidal effect, and no bactericidal effect on probiotics. It also has the characteristics of low hemolytic activity and low cytotoxicity, and can exert immune regulation to reduce inflammation. The antibacterial peptide KR-32 also has antiviral activity, can inhibit the replication of PEDV virus in VERO cells, reduce the infectivity of the virus, delay the time of VERO cell lesions, and at the same time relieve piglet viral diarrhea caused by PEDV.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is a comparison chart of the hemolysis rate of porcine red blood cells by antimicrobial peptide KR-22 with different solubility, and the above results are the average value of 6 independent repeated experiments.

Figure 2 is a comparison chart of the cytotoxicity of the antimicrobial peptide KR-22 on porcine PMBCs, LDH represents lactate dehydrogenase, and the above results are the average values of 6 independent repeated experiments.

Figure 3 shows the proliferation rate of the antibacterial peptide KR-22 on porcine erythrocytes, and the above results are the average values of 6 independent repeated experiments.

Detailed ways

Embodiment 1, the preparation of antimicrobial peptide KR-32

Cathelicidin-BF (C-BF), a snake-derived antimicrobial peptide, has broad-spectrum antibacterial activity and has a good effect on Gram-negative bacteria, especially Escherichia coli that can cause diarrhea in piglets. The minimum inhibitory concentration (MIC) is 8 -64 μg/mL. The present invention uses C-BF as a template, and improves it through molecular design. The improvement method includes replacing template peptide amino acids, changing the length of the peptide chain (increasing the length of the peptide chain), changing the hydrophobicity of the molecule, and increasing the proportion of aromatic amino acids, etc., through Synthesize improved peptides by chemical synthesis.

The frog-derived antimicrobial peptide P-OG1, which is similar to the antibacterial peptide KR-22 of the present invention, was found by searching and comparing the NCBI protein database. At the same time, the peptides with strong antibacterial activity found in the literature Cecropin A and CP- 1. Combined with the template peptide C-BF for comparative detection.

The sequence related to the polypeptide is as follows:

1) The full sequence of KR-32 is: KFFKRLRRSVVKKFRSWLSRTARRLENSAKKR, SEQ ID NO: 1;

2) Bungarus fasciatus antimicrobial peptide (template peptide) C-BF:KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF, SEQ ID NO:2;

3) Rana grahami antimicrobial peptide P-OG1: GLWDTIKQAGKKFFLNVLDKIRCKVAGGCRT, SEQ ID NO: 3;

4) Antheraea yamamai Guerin-Meneville antimicrobial peptide Cecropin A(CA)KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK, SEQ ID NO: 4;

5) Wild boar (Sus scrofa) antimicrobial peptide CP-1: SWLSKTAKKLENSAKKRISEGIAAIIQGGPR, SEQ ID NO:5.

Example 2. Antibacterial test (MIC) of antimicrobial peptide KR-32

Inoculate the bacteria on the MH broth solid medium, cultivate until a single colony grows, pick a single colony and inoculate it in 3mL MH broth medium, put it into a shaker and culture until the bacterial solution is turbid, take 30μL of the bacterial suspension and transfer it to In 3mL of fresh MH broth medium, shake culture at constant temperature until OD600=0.5; take 10μL of bacterial suspension and transfer to 10mL of fresh MH broth medium, vortex and mix well, and control the concentration at 1×10 ⁵ —5×10 ⁵ CFU/mL for the determination of MIC; the antimicrobial peptides were serially diluted to a final concentration of 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 (μg/ mL), a total of 13 serial concentrations of peptide dilutions, the antibiotic (chlortetracycline) was serially diluted to a final concentration of 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 (μg /mL), a total of 11 serial concentrations of antibiotic dilutions; first add 90 μL of the prepared bacterial suspension to a sterile 96-well round bottom plate, and then add 10 μL of the corresponding concentration of peptide dilution or antibiotic dilution one by one, each antibacterial substance Do three repetitions. In addition, set the positive control well without adding antibacterial substances, only add 100 μL of bacterial suspension, and add 100 μL of LB medium to the negative control well; place the 96-well plate in a biochemical incubator at 37 ° C to keep it moist and culture for 18- After 24 hours of cultivation, observe whether there is bacterial precipitation at the bottom of each well. The minimum concentration without visible bacterial precipitation can be determined as the MIC of the antibacterial substance.

Note: MIC is the abbreviation of minimum inhibitory concentration, which refers to the minimum inhibitory concentration, which is an index for measuring the antibacterial activity of antibacterial drugs, and refers to the lowest value that can inhibit the growth of pathogenic bacteria in the medium after culturing bacteria in vitro for 18 to 24 hours drug concentration. The smaller the value, the better the antibacterial effect.

The test results are as follows: As shown in Table 1, P-OG1 has a certain antibacterial activity, and CA, C-BF, and CP-1 have better antibacterial activity, but the antibacterial activity is not as good as the antibacterial peptide KR-32. Chlortetracycline has a good antibacterial effect, but has a high killing effect on probiotics, while KR-32 has a weak inhibitory effect on probiotics. However, P-OG1 showed a dose-dependent effect, and the hemolysis rate and cytotoxicity increased with increasing concentration, and its safety was far lower than that of antibacterial peptide KR-32 (see Figure 3).

Example 3, hemolytic activity of antimicrobial peptide KR-32

After taking blood from the pig anterior vena cava, take 10mL of heparin-anticoagulated porcine anterior vena cava blood and 10mL of RPMI1640 medium and mix them evenly at a ratio of 1:1, and slowly drop 20mL of the diluted blood into 10mL of Ficoll-Paque lymphocyte separation medium , keep the interface clear, after all the blood is dripped into the separation solution, centrifuge at 1500rpm for 15min, after centrifugation, stratification occurs, the bottom layer is red blood cells, collect the bottom layer of red blood cells. Wash the erythrocytes with 1×PBS solution, resuspend the erythrocytes, centrifuge at 1000rpm for 10min without braking, discard the supernatant, and repeat three times until the supernatant is colorless. Prepare the red blood cell working solution for the determination of hemolysis rate by mixing the washed red blood cells and 1×PBS at a ratio of 1:89, that is, 1% red blood cell working solution.

Dilute the antibacterial peptide KR-32 on the 96-well plate according to the concentration gradient, 10uL per well, the final concentration is 512, 256, 128, 64, 32, 16, 8, 4 μg/mL, and add 90uL red blood cell working solution In the diluted antimicrobial peptide wells, at the same time set the positive control wells as 90uL red blood cell working solution with 1% Triton X-100 (final concentration 1%), and the negative control wells with 10uL 1×PBS, and each treatment had 3 replicates. After the 96-well plate was placed in a cell culture incubator for 24 hours, it was centrifuged at 1500 rpm for 20 minutes without braking. Carefully pipette the supernatant into a new flat-bottomed transparent 96-well culture plate, measure the absorbance at OD414nm and OD546nm with a microplate reader, and calculate the ΔOD value. The formula for calculating the hemolysis rate of red blood cells is: hemolysis rate (%)=100%*(ΔOD test-ΔOD negative control)/(ΔOD positive control-ΔOD negative control).

The hemolytic activity of antimicrobial peptide KR-32 on porcine red blood cells is shown in Figure 1.

As shown in Figure 1, when the concentration of antibacterial peptide KR-32 is 256 μg/mL, the hemolysis rate of antimicrobial peptide KR-32 is still lower than 20%, and the hemolysis rate of antimicrobial peptide KR-32 is the lowest among the three.

Embodiment 4, the cytotoxicity of antimicrobial peptide KR-32

Dilute the isolated and purified peripheral blood mononuclear cells (PBMCs) with medium RMPI1640 to ¹ ×106 cells/mL, inoculate 96-well plate at 90uL per well, and set background control (100μL RMPI1640 medium) and low-level control at the same time Wells (90 μL cell suspension + 10 μL PBS) and high-level control wells (90 μL cell suspension + 10 μL 20% TritonX-100). Incubate the 96-well plate at 37°C in a 5% CO ₂ incubator for 2 h; add 10 uL of KR-32, P-OG1 and C-BF at corresponding concentrations to make the final concentrations of antimicrobial peptides 512 μg/mL and 256 μg/mL, respectively. mL, 128 μg/mL, 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 6 replicates for each treatment. After culturing in the incubator for 24 hours, add 10 μL of 20% TritonX-100 to the positive control well, blow and mix with a pipette, and continue to culture for 15 minutes; after the culture, centrifuge at 1500 rpm for 10 minutes; after centrifugation, carefully draw 60uL of cells from each well Transfer the supernatant to the corresponding wells of a transparent flat-bottomed 96-well culture plate; add 30uL of diluted LDH detection reagent to each well, and incubate with shaking at room temperature for 30min in the dark, measure OD492nm and OD900nm with a microplate reader, and calculate the ΔOD value.

The LDH release rate was calculated according to the following formula: LDH release rate (%)=100%*(ΔOD test-ΔOD negative control)/(ΔOD negative control-ΔOD positive control).

The results of LDH release rate of antimicrobial peptides KR-32, P-OG1 and C-BF on porcine peripheral mononuclear cells are shown in Figure 2.

As shown in Figure 2, when the concentration is lower than 256 μg/mL, the LDH release rate of the antimicrobial peptide KR-32 pig peripheral mononuclear cells is less than 20%, and the results show that the cytotoxicity of the antimicrobial peptide KR-32 is low.

Example 5: Antimicrobial Peptide KR-32 Plays Immunomodulatory Effect to Treat Diarrhea in Weaned Piglets

A total of 18 weaned piglets with the same genetic background and similar body weight were randomly divided into 3 groups, 6 pigs in each group, injected intraperitoneally three times, once a day, normal saline (control group), 20 mg lactic acid Ciprofloxacin (antibiotic group), and 5 mg antimicrobial peptide KR-32 (antimicrobial peptide KR-32 group). Diarrhea rate and diarrhea index were counted every day. Diarrhea rate = [total number of pigs with diarrhea / (number of test pigs × test period)] × 100%. Scoring standard for diarrhea index: 0 points: normal; 1 point: soft stool; 2 points: moderate Degree of diarrhea; 3 points: severe diarrhea. The results are shown in Tables 2 and 3 below.

Note: Day 0 means before injection, and day 1 is day 1 after injection. Record diarrhea twice daily (morning and afternoon)

Note: According to the diarrhea index scoring standard, the feces of piglets in each column were scored, and the statistics were made once every morning and afternoon, and the average value was taken.

The feces of piglets in each pen were scored according to the diarrhea index scoring standard twice a day. (The scoring standard is: 0 points: normal; 1 point: soft stool; 2 points: moderate diarrhea; 3 points: severe diarrhea)

As shown in Tables 2 and 3, in terms of the antimicrobial peptide KR-32 group, the diarrhea rate of weaned piglets in the antimicrobial peptide KR-32 group was 25% and 16.7% lower than that of the control group and the antibiotic group, respectively. The diarrhea index of KR-32 was 1.55 and 0.67 lower than that of the control group and the antibiotic group, respectively, indicating that the antimicrobial peptide KR-32 had a significant alleviating effect on diarrhea in weaned piglets.

The effect of antimicrobial peptide KR-32 on serum cytokines of weaned piglets is shown in Table 4 below.

Note: Inflammatory cytokines (IL-6, IL-4, and TNF-α) refer to various cytokines involved in the inflammatory response. Within a certain range, the higher the value, the more serious the inflammation. During diarrhea, the piglets’ inflammation will increase accordingly.

In terms of cytokines, compared with the control group, the contents of pro-inflammatory factors TNF-α and IL-6 in the serum of weaned piglets in the antimicrobial peptide KR-32 group decreased by 13.5% ( P =0.073) and 13.6% ( P <0.05) respectively , but had no significant effect on the anti-inflammatory factor IL-4.

The above listed are only some specific embodiments of the present invention. It should be noted that the present invention is not limited to the above embodiments, and many other modifications can be made, including mutations in the antibacterial peptide KR-32 sequence, changing the number and types of amino acids, However, the functional properties of the core sequence are not affected. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

sequence listing

<110> Zhejiang University

<120> Antimicrobial peptide KR-32 and its application

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<213> Golden krait (Bungarus fasciatus)

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<213> Fingerless Frog (Rana grahami)

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Gly Leu Trp Asp Thr Ile Lys Gln Ala Gly Lys Lys Phe Phe Leu Asn

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Claims (5)

1. An antimicrobial peptide, characterized in that it is called KR-32, and its full sequence is: KFFKRLRRSVVKKFRSWLSRTARRLENSAKKR, SEQ ID NO:1. 2. The antimicrobial peptide according to claim 1, characterized in that: the antimicrobial peptide KR-32 is an artificially designed and synthesized active polypeptide, KR-32 contains 32 amino acid residues, a molecular weight of 3896.7Da, and an isoelectric point of 10.42, with a net charge of +13. 3. The use of the antimicrobial peptide according to claim 1 or 2, characterized in that it is used for the preparation of broad-spectrum antibacterial drugs for the treatment of Gram-negative or Gram-positive bacterial infections. 4. The use of the antimicrobial peptide according to claim 1 or 2, characterized in that it is used to prepare drugs for improving immune regulation. 5. The use of the antimicrobial peptide according to claim 1 or 2, characterized in that it is used to prepare a therapeutic preparation for weaned piglets with diarrhea.