CN108264539B - Antibacterial peptide RL-18 and application thereof - Google Patents
Antibacterial peptide RL-18 and application thereof Download PDFInfo
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Abstract
The invention discloses an antibacterial peptide RL-18, the amino acid sequence of which is shown as SEQ ID NO.1, the molecular weight of the antibacterial peptide RL-18 is 2139.615Da, and the isoelectric point is 12.4. The antibacterial peptide RL-18 is synthesized by adopting an automatic polypeptide synthesizer according to a conventional polypeptide solid-phase synthesis method. The antibacterial peptide RL-18 disclosed by the invention has high-efficiency antibacterial activity on gram-positive bacteria (staphylococcus aureus), gram-negative bacteria (escherichia coli and salmonella) and fungi (candida albicans), is quick in antibacterial action, has low hemolysis, good salt tolerance and heat resistance, and does not generate drug resistance. Therefore, the antibacterial peptide RL-18 has wide application prospects in the aspects of preparing anti-infective drugs, developing animal husbandry, preventing and treating human diseases, washing cosmetics and the like.
Description
Technical Field
The invention belongs to the technical field of antibacterial peptide research, and particularly relates to an antibacterial peptide RL-18 and application thereof.
Background
Since the discovery of penicillin by fleming, Antibiotics (Antibiotics) drugs are a class of drugs commonly used by human beings for treating infectious diseases, which significantly improves the quality of life of human beings and saves the lives of countless people and animals. However, the drug resistance of pathogenic microorganisms caused by the improper use (such as mass use and abuse) of antibiotics is continuously generated, and the harmful events of antibiotic residues are frequent, so that great challenges are brought to clinical disease diagnosis, the human health is seriously threatened, and the animal husbandry development is influenced. The search for new antibacterial drugs becomes an important way to solve the problems of antibiotic resistance and residues. Antimicrobial peptides are a very promising alternative to antibiotics. Antimicrobial peptides (AMPs) are small molecular polypeptides having a variety of biological activities including antibacterial, antifungal, antiviral, antiparasitic, antitumor, immunomodulatory, and endotoxin neutralizing effects. The antibacterial peptide can be encoded by host genes, or can be artificially modified and designed according to the characteristics of the antibacterial peptide. Due to the characteristics of small molecular weight, good thermal stability, good water solubility, wide antibacterial spectrum, difficult formation of drug resistance and the like, the antibacterial peptide is considered as the best substitute of antibiotics.
The research of antibacterial peptides has been paid much attention and has become a hotspot in the field of biological pharmacy. An authoritative antimicrobial peptide database APD3(http:// APs. unmac. edu/AP/main. php) has recorded 2878 antimicrobial peptide sequences (truncated to 2017.12.22). Currently, many antimicrobial peptides have been prepared as pharmaceuticals. The sources of the antibacterial peptide mainly comprise organism separation identification and artificial design and modification. The antibacterial peptide from natural sources can be obtained by organism separation and identification, but the yield is low and the activity is not ideal. The artificial design and modification of the antibacterial peptide are rapid and effective ways for obtaining the antibacterial peptide, and become an important content for developing the antibacterial peptide. Therefore, obtaining an antibacterial peptide with a simple structure, high antibacterial activity and easy preparation is an urgent need in the development and research of antibacterial peptides. The invention discloses an antibacterial peptide RL-18 according to the relationship between the physicochemical properties (mainly ionic property, hydrophobicity, secondary structure and the like) and the structure-activity (mainly antibacterial activity) of the antibacterial peptide.
Disclosure of Invention
The invention aims to provide an antibacterial peptide RL-18 and application thereof. The antibacterial peptide RL-18 has good antibacterial activity on gram-positive bacteria (staphylococcus aureus), gram-negative bacteria (escherichia coli and salmonella) and fungi (candida albicans), has quick antibacterial action, and has low hemolytic property, heat resistance and salt tolerance. Therefore, the antibacterial peptide RL-18 has wide application prospects in the aspects of preparing anti-infective drugs, developing animal husbandry, preventing and treating human diseases, preserving foods, detergents, cosmetics and the like.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides an antibacterial peptide RL-18, wherein the amino acid sequence of the antibacterial peptide RL-18 is as follows:
arginine-phenylalanine-lysine-leucine-serine-histidine-serine-leucine-lysine-threonine-leucine-alanine-serine-arginine-leucine (SEQ ID No. 1).
According to the antibacterial peptide RL-18, the antibacterial peptide RL-18 has 18 amino acid residues, the molecular weight of the antibacterial peptide RL-18 is 2139.615Da, and the isoelectric point of the antibacterial peptide RL-18 is 12.4.
The antibacterial peptide RL-18 can be used for preparing medicines for treating gram-positive bacteria, gram-negative bacteria or/and fungal infection. The medicine comprises the antibacterial peptide RL-18 and is mixed with one or more than one pharmaceutically acceptable carriers and/or additives.
The antibacterial peptide RL-18 can also be used for preparing feed additives, disinfectants, preservatives, detergents or cosmetic additives.
The antibacterial peptide RL-18 is synthesized by adopting an automatic polypeptide synthesizer according to a conventional polypeptide solid phase chemical synthesis method, the synthesis direction is from a C end to an N end, and the detailed synthesis steps are as follows:
a. swelling resin: adding Fmoc-Leu-Wang resin into a reactor of an automatic polypeptide synthesizer, adding dimethyl formamide DMF (dimethyl formamide) for swelling, wherein the amount of the DMF added into 1g of the Fmoc-Leu-Wang resin is about 12mL (the DMF completely immerses the resin), the swelling time is 10-30 min, and the swelling can be repeated for 1-2 times;
b. deprotection: after the resin is swelled, 20% of piperidine (20% of piperidine is prepared by dissolving piperidine in dimethylformamide DMF) in a storage tank is added into a reactor, the resin is immersed (about 10mL of 20% of piperidine is required to be added into 1g of Fmoc-Leu-Wang resin), the swelled resin is deprotected for about 30min, and then the dimethylformamide DMF is added for washing;
c. condensation reaction: then adding a second amino acid arginine Fmoc-Arg according to the sequence of the amino acid sequence of the antibacterial peptide RL-18, and adding a condensing agent HCTU (6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate) and a catalyst NMM (N-methylmorpholine) or DIEA (N, N diisopropylethylamine) for condensation reaction; after the reaction is finished, adding dimethyl formamide DMF for repeated washing to remove unreacted amino acid; the molar ratio of the arginine to the Fmoc-Leu-Wang resinin resin is 5: 1; the molar ratio of the condensing agent HCTU to the leucine Fmoc-Leu is 1: 1; the molar ratio between the catalyst NMM or DIEA and leucine is 4: 1;
deprotecting and washing the product obtained after washing according to the same method of the step b, sequentially adding Fmoc-Ser, Fmoc-Ala, Fmoc-Leu, Fmoc-Thr, Fmoc-Lys, Fmoc-Leu, Fmoc-Ser, Fmoc-His, Fmoc-Ser, Fmoc-Leu, Fmoc-Lys, Fmoc-Phe, Fmoc-Arg and Fmoc-Arg according to the same method of the step c to perform condensation reaction and washing, and finally performing condensation reaction and washing to obtain a polypeptide product; carrying out deprotection and washing on products obtained after each condensation reaction and washing according to the method in the step b, and then adding the next amino acid to carry out condensation reaction and washing (in each condensation reaction, the molar ratio of the added amino acid to Fmoc-Leu-Wang resin is 5: 1; the molar ratio of the condensing agent HCTU to each added amino acid is 1: 1; and the molar ratio of the catalyst NMM or DIEA to each added amino acid is 4: 1);
d. deprotection and cleavage of the polypeptide product: adding 20% piperidine to the polypeptide product from step c and submerging the polypeptide product (about 10mL of 20% piperidine was used to deprotect 1g of the resin) to deprotect the polypeptide product; after deprotection, sequentially adding dimethylformamide DMF and dichloromethane DCM for repeated washing; after washing, the polypeptide is cleaved from the resin by addition of trifluoroacetic acid, TFA (1g of resin is cleaved with TFA in a volume of about 20 mL); filtering the cracked product (by using a sand core funnel) to obtain a filtrate;
e. and (3) precipitating and synthesizing the polypeptide: washing the obtained filtrate with cold ether, centrifuging to precipitate and synthesize polypeptide, wherein the process comprises the following steps: d, adding cold diethyl ether (preserved at 4 ℃ in advance) with the volume 3 times of that of the filtrate obtained in the step d, sealing, reversing, uniformly mixing, carrying out ice bath for 10min, centrifuging at 4000r/m for 10min, removing supernatant, and recovering precipitate; re-suspending the precipitate with cold ether, repeating the steps for three times, and drying the obtained precipitate for 12-16 h to obtain a crude synthetic peptide product;
f. purification of crude synthetic peptide: and (3) performing column purification on the synthetic peptide crude product by adopting a semi-preparative high performance liquid chromatograph P2000, and collecting fractions with the purity of more than or equal to 95% to obtain the purified antibacterial peptide RL-18 with the purity of more than or equal to 95%.
A semi-preparative high performance liquid chromatograph P2000 is provided by Beijing Wawawa Innovation technologies, Inc., a C18 reverse phase column (4.6 x 250mm) is adopted as a chromatographic column, 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid is dissolved in 100mL acetonitrile solution) is adopted as a mobile phase A, 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid is dissolved in 100mL deionized water) is adopted as a mobile phase B, the proportion of the mobile phase A is gradually adjusted to 52% from 27% within 25min after sample loading by using the processing column, the proportion of the mobile phase B is gradually adjusted to 48% from 73%, the proportion of the mobile phase A is changed to 100% and the proportion of the mobile phase B is changed to 0% during 25.1min, separation is carried out for 30min at the flow rate of 1mL/min, the detection wavelength is 220nm, and fractions with the purity of more than or equal to 95% are collected (as shown in figure 1);
the confirmation and identification of the antibacterial peptide RL-18 of the invention are as follows: the molecular weight of the antibacterial peptide of the obtained product is determined by adopting a liquid chromatography-mass spectrometer (Waters micromass ZQ-2000), and the determination is as follows: RL-18 has a molecular weight of 2139.615Da (as shown in FIG. 2). High performance liquid chromatography conditions: the chromatographic column is a C18 reverse phase column (4.6 × 250mm), the mobile phase A is 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid is dissolved in 100mL acetonitrile solution), the mobile phase B is 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid is dissolved in 100mL deionized water), the proportion of the mobile phase A is gradually adjusted from 25% to 50% within 25min after loading, the proportion of the mobile phase B is gradually adjusted from 75% to 50%, the proportion of the mobile phase A is changed to 100% and the proportion of the mobile phase B is changed to 0% within 25.1min, separation is carried out at the flow rate of 1mL/min for 30min, and the detection wavelength is 220 nm. Mass spectrometry conditions: and adopting a positive ionization mode, wherein the capillary voltage is 3.00KV, the outlet voltage of the capillary is 50V, the fragmentation voltage is 5V, the flow rate of the drying gas is 1.5L/min, the temperature of the drying gas is 350 ℃, and the scanning range is 400-1900 m/z.
The invention has the following positive beneficial effects:
the antibacterial peptide RL-18 consists of 18 amino acids, has antibacterial activity on gram-positive bacteria (staphylococcus aureus), gram-negative bacteria (escherichia coli and salmonella) and fungi (candida albicans), has quick antibacterial action and strong antibacterial activity, has low hemolysis, and has good heat resistance and salt tolerance and no drug resistance. Therefore, the antibacterial peptide RL-18 has wide application prospects in the aspects of preparing anti-infective medicaments, developing animal husbandry and treating human diseases.
Drawings
FIG. 1 is a high performance liquid chromatogram of the antimicrobial peptide RL-18 of the invention.
FIG. 2 is a mass spectrum of the antimicrobial peptide RL-18 of the invention.
FIG. 3 shows the results of isoelectric point analysis of antimicrobial peptide RL-18.
FIG. 4 shows the result of the determination of the minimum inhibitory concentration of antimicrobial peptide RL-18 on Escherichia coli CVCC 2059.
FIG. 5 shows the result of the determination of the minimum inhibitory concentration of antibacterial peptide RL-18 to Salmonella CVCC 3376.
FIG. 6 shows the result of the determination of the minimum inhibitory concentration of antimicrobial peptide RL-18 against Staphylococcus aureus ATCC 6538.
FIG. 7 shows the results of the determination of the minimum inhibitory concentration of the antimicrobial peptide RL-18 against Candida albicans ATCC 10231.
FIG. 8 shows the results of the determination of the minimum inhibitory concentration of antibacterial peptide RL-18 against the clinical drug-resistant strains of Escherichia coli.
FIG. 9 shows the results of measurement of the hemolysis rate of antibacterial peptide RL-18.
FIG. 10 shows the results of measuring the heat resistance of antibacterial peptide RL-18.
FIG. 11 shows the results of the salt tolerance assay of antibacterial peptide RL-18.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1:
an antibacterial peptide RL-18, the amino acid sequence of which is as follows:
arginine-phenylalanine-lysine-leucine-serine-histidine-serine-leucine-lysine-threonine-leucine-alanine-serine-arginine-leucine (SEQ ID No. 1).
The antibacterial peptide RL-18 contains 18 amino acid residues, and has a molecular weight of 2139.615Da and an isoelectric point of 12.4. The antibacterial peptide RL-18 is positively charged and can attract negative charges on the surface of bacteria.
The antibacterial peptide RL-18 is synthesized by an automatic polypeptide synthesizer according to a conventional polypeptide solid phase chemical synthesis method, and the finally synthesized antibacterial peptide RL-18 is purified and analyzed by a semi-preparative high performance liquid chromatograph, wherein the purity of the antibacterial peptide RL-18 is more than or equal to 95%.
The identification of the antibacterial peptide RL-18 product obtained by the invention comprises the following steps:
(1) determination of molecular weight: the molecular weight of the obtained antibacterial peptide RL-18 is determined by an analytical liquid chromatography mass spectrometer (Waters micromass ZQ-2000), and the molecular weight is determined to be 2139.615Da, and the result is shown in figure 2.
The specific detection conditions are as follows: chromatographic conditions are as follows: the chromatographic column is a C18 reverse phase column (4.6 × 250mm), the mobile phase A is 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid is dissolved in 100mL acetonitrile solution), the mobile phase B is 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid is dissolved in 100mL deionized water), the proportion of the mobile phase A is gradually adjusted from 25% to 50% within 25min after loading, the proportion of the mobile phase B is gradually adjusted from 75% to 50%, the proportion of the mobile phase A is changed to 100% and the proportion of the mobile phase B is changed to 0% within 25.1min, separation is carried out at the flow rate of 1mL/min for 30min, and the detection wavelength is 220 nm. Measurement conditions of mass spectrometry: and adopting a positive ionization mode, wherein the capillary voltage is 3.00KV, the outlet voltage of the capillary is 50V, the fragmentation voltage is 5V, the flow rate of the drying gas is 1.5L/min, the temperature of the drying gas is 350 ℃, and the scanning range is 400-1900 m/z.
(2) Amino acid sequence structure determination: and (3) determining the amino acid sequence structure of the antibacterial peptide RL-18 by adopting an automatic amino acid sequencer. The antibacterial peptide RL-18 is determined to contain 18 amino acid residues, and the amino acid sequence is as follows: arginine-phenylalanine-lysine-leucine-serine-histidine-serine-leucine-lysine-threonine-leucine-alanine-serine-arginine-leucine.
(3) Isoelectric point analysis:
opening EditSeq in DNAStar software, opening file, clicking new protein in new, inputting the amino acid sequence of antibacterial peptide RL-18, and storing the amino acid sequence as a pro format document. And then opening a Protean in DNAStar software, opening a file, clicking "open", selecting the document just stored in the pro format, clicking "open", and selecting "transformation cut" in "Analysis", so that the isoelectric point data can be obtained, wherein the result is shown in figure 3. The isoelectric point of the antibacterial peptide RL-18 is 12.4.
Example 2: application implementation of antibacterial peptide RL-18 of the invention
1. Antibacterial activity analysis of antibacterial peptide RL-18
The antibacterial activity of the antibacterial peptide RL-18 is determined by adopting an agar plate diffusion method, and tested microorganism strains are as follows: escherichia coli, salmonella, staphylococcus aureus and candida albicans, Escherichia coli CVCC2059, staphylococcus aureus ATCC6538 and candida albicans ATCC10231 were purchased from Nanjing fecal diagnosis Biotech Co., Ltd, salmonella CVCC3776 was purchased from China veterinary microbial strain preservation management center, and Escherichia coli clinical drug-resistant strains (isolated from diarrhea piglets and identified by susceptibility test and 16S rRNA sequencing).
The above test microorganism (1X 10)6CFU/mL) was diluted in 50 ℃ basal medium (TSB 0.3g, ultrapure agarose 0.1g, ultrapure water to 10mL, pH 7.4) respectively, the plates were poured out and sterilized, coagulated, punched with a punch (hole diameter about 3mm), bottom-sealed with alcohol lamp by slight heating,mu.L of antimicrobial peptide (1mg/mL) was added to each well using a pipette, and each plate included a positive control and a negative control. Positive control: gentamicin against gram-negative and gram-positive bacteria, fluconazole against fungi; negative control: PBS buffer. The plate was allowed to stand for 1h to allow the test solution to diffuse into the agarose. Then, an upper layer of overlay medium (TSB 0.3g, ultrapure agarose 0.1g, ultrapure water to 10ml, pH 7.4, 50 ℃ C. or so) was added thereto. The plates were incubated at 37 ℃ overnight in an inverted manner, and then the diameter of the transparent circle around the well was recorded, and the measurement was repeated three times for each strain, and the average value was calculated.
TABLE 1 antibacterial peptide RL-18 analysis results of antibacterial activity
Note: a shows that the strain is resistant to gentamicin, and the used positive control drug is not gentamicin, but Bac5 antibacterial peptide, and the patent application number: 201710643182.8.
the results of the antibacterial activity test in Table 1 show that the antibacterial peptide RL-18 of the invention has higher antibacterial activity on escherichia coli, salmonella, staphylococcus aureus and candida albicans, and also has higher antibacterial activity on clinical drug-resistant strains of escherichia coli. Therefore, the antibacterial peptide RL-18 of the invention is expected to have good application prospect in the aspect of preparing medicaments for treating animal and human bacterial diseases, and meanwhile, according to the general characteristics of the known antibacterial peptide, the antibacterial peptide RL-18 has good application prospect in the aspects of feed additives, disinfectants, preservatives, detergents or cosmetic additives and the like.
2. Minimum Inhibitory Concentration (MIC) assay for antimicrobial peptide RL-18
The tested microbial strains were: escherichia coli, salmonella, staphylococcus aureus and candida albicans, Escherichia coli CVCC2059, staphylococcus aureus ATCC6538 and candida albicans ATCC10231 are purchased from Nanjing fecal diagnosis Biotech limited, Salmonella CVCC3776 is purchased from China veterinary microbial strain preservation management center, and Escherichia coli clinical drug-resistant strains (separated from diarrhea piglets and identified by drug susceptibility test and 16S rRNA sequencing).
The tested strain is enlarged and cultured in TSB liquid culture medium, and OD of the bacterial liquid is measured600The value is obtained. When OD value is 0.6-0.8, centrifuging the bacterial liquid at 6000r/min for 10min, discarding supernatant, collecting thallus precipitate, re-suspending thallus precipitate with PBS buffer solution (0.01M) of the same volume, and diluting bacterial suspension with MH culture medium to 2 × 106CFU/mL. The antibacterial peptide RL-18 sample is diluted by deionized water to make the concentration of the antibacterial peptide RL-18 sample be 0.78-1200 mug/mL. In a 96-well cell culture plate, 50. mu.L of antibacterial peptide RL-18 with different concentrations and 50. mu.L of diluted bacterial solution are added to each well, three wells are repeated for each concentration, the mixture is cultured at 37 ℃ for 16-18h, then the solution in each well is mixed, and the OD is measured600Values were given as their MICs at the concentration of antimicrobial peptide RL-18 corresponding to the sudden change in OD values, and the results are shown in FIGS. 4, 5, 6, 7 and 8.
As can be seen from FIGS. 4, 5, 6, 7 and 8, the Minimum Inhibitory Concentration (MIC) of antibacterial peptide RL-18 against Escherichia coli CVCC2059 was 12.5. mu.g/mL, the Minimum Inhibitory Concentration (MIC) against Salmonella CVCC3776 was 12.5. mu.g/mL, the Minimum Inhibitory Concentration (MIC) against Staphylococcus aureus ATCC6538 was 400. mu.g/mL, the Minimum Inhibitory Concentration (MIC) against Candida albicans ATCC10231 was 200. mu.g/mL, and the Minimum Inhibitory Concentration (MIC) against clinical drug-resistant strains of Escherichia coli was 50. mu.g/mL. The result shows that the minimum inhibitory concentration of the antibacterial peptide RL-18 to common bacteria reaches microgram level, and the minimum inhibitory concentration to clinical drug-resistant strains of escherichia coli also reaches microgram level, so that the antibacterial peptide RL-18 disclosed by the invention has extremely strong inhibitory activity.
3. Hemolytic analysis of antimicrobial peptide RL-18
Fresh chicken blood is taken, anticoagulated by 3.8% sodium citrate, the anticoagulated blood is centrifuged for 10min at 3000r/min, and is washed and precipitated for 3 times by Phosphate Buffer Solution (PBS) until the supernatant is colorless and transparent, and then 1% erythrocyte is prepared by PBS. The concentration of antimicrobial peptide RL-18 was adjusted to 0.39-400. mu.g/mL with PBS and an equal volume of the red blood cell suspension was added. PBS is used as a negative control, 1% Tritonx-100 is used as a positive control, the reaction is carried out for 1h at 37 ℃, centrifugation is carried out for 10min at 1500r/min, supernatant is sequentially added into a 96-well plate, and the OD value at 540nm is measured by a microplate reader. Then, the hemolysis rate of the antimicrobial peptide RL-18 was calculated according to the calculation formula of hemolysis rate (%) (detection well OD value-negative well OD value)/(positive well OD value-negative well OD value) × 100%, and the results thereof are shown in fig. 9.
As can be seen from FIG. 9, the hemolysis rate of the antibacterial peptide RL-18 is lower than 50% under the condition of Minimum Inhibitory Concentration (MIC), which indicates that the antibacterial peptide RL-18 has better safety and great clinical application prospect.
4. Analysis of Heat resistance of antibacterial peptide RL-18
Respectively subjecting 1mg/mL antibacterial peptide RL-18 solution to 0 deg.C, 25 deg.C, 50 deg.C, 75 deg.C, and 100 deg.C for 30 min; then, the antibacterial activity of the antibacterial peptide RL-18 solutions treated at the five different temperatures was analyzed by the antibacterial activity analysis method in example 2 (the antibacterial activity was analyzed by the agar plate diffusion method described above), and the tested microorganism strain was Escherichia coli CVCC2059, and the results are shown in FIG. 10.
As can be seen from FIG. 10, the antibacterial activity of the antibacterial peptide RL-18 against Escherichia coli gradually decreased with the increase of temperature, but the antibacterial peptide RL-18 still had better antibacterial activity after high temperature treatment (70 ℃ and 100 ℃). Therefore, the antibacterial peptide RL-18 has better heat resistance.
5. Analysis of salt tolerance stability of antibacterial peptide RL-18
10 parts of 1mg/mL antibacterial peptide RL-18 solution are subpackaged, the number is 1-10, sodium chloride is added into the 1-5 antibacterial peptide RL-18 solution respectively, and the final concentration of the sodium chloride in the 1-5 antibacterial peptide RL-18 solution is 50, 100, 150, 200 and 250mM respectively; adding sodium potassium chloride into the antibacterial peptide RL-18 solution with the serial number of No. 6-10 to enable the final concentration of the potassium chloride in the antibacterial peptide RL-18 solution with the serial number of No. 6-10 to be 50mM, 100 mM, 150 mM, 200 mM and 250mM respectively, and placing the antibacterial peptide RL-18 solution with the serial number of No. 1-10 for 30min at room temperature; and then, determining the bacteriostatic activity of the antibacterial peptide RL-18 with the serial number of 1-10 according to the agar plate diffusion method, wherein the tested microorganism is Escherichia coli CVCC 2059.
As can be seen from FIG. 11, the antibacterial activity of the antibacterial peptide RL-18 against Escherichia coli is reduced with the increase of the salt ion concentration, but the antibacterial peptide RL-18 still has better antibacterial activity at high concentration (higher than physiological concentration). This shows that the antibacterial peptide RL-18 has better salt tolerance.
In conclusion, the antibacterial peptide RL-18 product has low hemolytic property, wide antibacterial spectrum, good heat resistance and salt tolerance, high antibacterial effect on gram-negative bacteria, gram-positive bacteria and fungi, and particularly good antibacterial activity on clinical drug-resistant bacteria. Therefore, the antibacterial peptide RL-18 of the product can be better applied to the preparation of medicines for treating diseases caused by gram-positive bacteria, gram-negative bacteria and fungi, and can also be used for preparing feed additives, disinfectants, preservatives, detergents or cosmetic additives.
Sequence listing
<110> institute of science and technology of Henan
<120> antibacterial peptide RL-18 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Arg Arg Phe Lys Leu Leu Ser His Ser Leu Leu Lys Thr Leu Ala Ser
1 5 10 15
Arg Leu
Claims (4)
1. An antibacterial peptide RL-18, wherein the amino acid sequence of the antibacterial peptide RL-18 is as follows:
arginine-phenylalanine-lysine-leucine-serine-histidine-serine-leucine-lysine-threonine-leucine-alanine-serine-arginine-leucine.
2. Use of the antimicrobial peptide RL-18 according to claim 1, wherein the use is: application of antibacterial peptide RL-18 in preparation of medicines for treating gram-positive bacteria, gram-negative bacteria or/and fungal infection is provided.
3. Use according to claim 2, wherein the medicament comprises the antibacterial peptide RL-18 in admixture with one or more pharmaceutically acceptable carriers and/or additives.
4. Use of the antimicrobial peptide RL-18 according to claim 1 for the preparation of a feed additive, disinfectant, preservative, detergent or cosmetic additive.
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