CN115043924A - Modified antibacterial peptide and application thereof - Google Patents
Modified antibacterial peptide and application thereof Download PDFInfo
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- CN115043924A CN115043924A CN202210465719.7A CN202210465719A CN115043924A CN 115043924 A CN115043924 A CN 115043924A CN 202210465719 A CN202210465719 A CN 202210465719A CN 115043924 A CN115043924 A CN 115043924A
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- antibacterial
- peptide
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- antibacterial peptide
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Abstract
The invention relates to a modified antibacterial peptide and application thereof, wherein the modified antibacterial peptide is obtained by modifying an antibacterial peptide with an amino acid sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The modified antibacterial peptide has broad-spectrum efficient antibacterial activity, extremely strong anti-inflammatory activity and anti-biofilm activity, has a synergistic antibacterial effect with the traditional antibiotics, has the beneficial characteristics of small molecular weight, simple structure, low hemolytic activity, high stability, simple preparation method and the like, and has a huge potential application prospect in the preparation of anti-inflammatory agents and antibacterial agents and the downstream fields of preservatives, animal feed additives, cosmetic additives and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a modified antibacterial peptide and application thereof.
Background
The antibacterial peptide is a polypeptide synthesized in a ribose body by gene coding, and different types of antibacterial peptides have the common characteristics of short peptide, strong cationic property, good thermal stability, small molecular weight, no drug shielding and low toxicity to eukaryotic cells. Today, antimicrobial peptides have been successfully isolated and classified in most organic organisms from prokaryotes to humans. Antibacterial peptides, which are generally active against bacteria, play an important role in the innate immunity of eukaryotes and are considered to be immune molecules that are effectively retained in the body of mammals during ancient evolution. Therefore, the antibacterial peptide serving as a polypeptide substance widely existing in organisms in the nature can be used as a first line of defense of organisms and can resist the invasion of pathogens. The antibacterial peptide has various biological activities of resisting bacteria, fungi and viruses, inhibiting and killing cancer cells and the like, and is not easy to generate drug resistance.
Currently, the application of antibacterial peptides in medicine has achieved satisfactory results, and many new drugs gradually enter the medicine market. For example, the antimicrobial peptide drug MAI278 developed by applying the amphibian antimicrobial peptide magainin is close to completing the phase III clinical test, and shows good killing effect on viruses and tumor cells. daptomycin is an anionic antimicrobial peptide developed by the company Cubit Pharmaceuticals and approved by the U.S. food and drug administration to market in 9 months 2003, and is useful for the treatment of skin infections and sepsis caused by gram-positive bacteria such as staphylococcus aureus. Li and the like make breakthrough progress on the research on the gene function of the murine Binlb antibacterial peptide. The gene is the first functional gene discovered at present and related to inflammation of the male reproductive system, and the epididymis is proved to have an immune system for the first time. MX-226(CPI-226) is an indolicidin antibacterial peptide separated from cattle, has completed phase IIIb clinical tests, can be used for locally preventing or reducing blood infection related to central venous catheters, and shows good application prospect. plectasin (plectasin) is an antibacterial peptide separated from ascomycete, has good bactericidal activity on streptococcus pneumoniae, can also inhibit growth of fungi and viruses, and has been studied in the early clinical stage in 2007. Besides, antibiotics, which are used as feed additives in animal production, play an important role in the development of animal husbandry, but the residues in animal bodies and animal products and the drug resistance problem caused by pathogenic bacteria have negative effects on the health and environment of human beings. Therefore, the antibacterial peptide is a substance which is most hopeful to replace the traditional antibiotics, and has good application prospect in the fields of medicine industry, food additives and the like. Therefore, the invention aims to modify the antimicrobial peptide Cm-CATH2 of the chelonian (Chelonia mydas) so as to provide a new antimicrobial peptide as a powerful candidate for a new generation of antimicrobial drugs.
Chinese patent CN201610585747.7 discloses an antimicrobial peptide Cm-CATH2, which is obtained by transforming Cm-CATH2 and has good antibacterial activity; chinese patent CN202011319757.9 discloses the application of antibacterial peptide Cm-CATH2 in preventing and controlling Fusarium septeminatum in grains; chinese patent CN202011319760.0 discloses an application of antibacterial peptide Cm-CATH2 in inhibiting vibrio parahaemolyticus in marine products. The patents show that the antibacterial peptide Cm-CATH2 has a good inhibition effect on bacteria, but the research on structural modification and anti-inflammatory effect is not involved. Therefore, the invention is expected to provide an antibacterial peptide Cm-CATH2 modified body with antibacterial and anti-inflammatory effects at the same time, so as to be used as a potential antibacterial anti-inflammatory drug.
Disclosure of Invention
In order to solve the technical problems, the invention firstly shortens the peptide chain of the antibacterial peptide shown in SEQ ID NO.1 to obtain a series of shortened peptides by two-step structural modification, then researches the antibacterial, anti-inflammatory and hemolytic activities of the shortened peptides to screen out the antibacterial peptides with better antibacterial activity, anti-inflammatory activity and lower hemolytic activity, and then replaces Arg and Lys in the antibacterial peptides with non-natural D-type amino acids (D-Arg and D-Lys) to obtain the modified antibacterial peptide.
The first purpose of the invention is to provide a modified antibacterial peptide, which is obtained by modifying the antibacterial peptide with an amino acid sequence shown as SEQ ID NO.1, wherein the modification comprises the shearing removal of 8 amino acids at the N terminal of the antibacterial peptide with an amino acid sequence shown as SEQ ID NO. 1.
Wherein the amino acid sequence shown in SEQ ID NO.1 is:
Arg 1 Arg 2 Ser 3 Arg 4 Phe 5 Gly 6 Arg 7 Phe 8 Phe 9 Lys 10 Lys 11 Val 12 Arg 13 Lys 14 Gln 15 Leu 16 Gly 17 Arg 18 Val 19 Lys 20 Arg 21 His 22 Ser 23 Arg 24 Ile 25 Thr 26 Val 27 Gly 28 Gly 29 Arg 30 Met 31 Arg 32 Phe 33 。
further, the amino acid sequence of the modified antibacterial peptide is shown as SEQ ID NO.2, and the specific sequence is as follows:
Phe 1 Lys 2 Lys 3 Val 4 Arg 5 Lys 6 Gln 7 Leu 8 Gly 9 Arg 10 Val 11 Lys 12 Arg 13 His 14 Ser 15 Arg 16 Ile 17 Thr 18 Val 19 Gly 20 Gly 21 Arg 22 Met 23 Arg 24 Phe 25 。
furthermore, the modification also comprises replacing arginine Arg in the amino acid sequence shown in SEQ ID NO.2 with non-natural D-amino acid D-Arg, and replacing lysine Lys with non-natural D-amino acid D-Lys, and the rest are natural L-amino acids.
Further, the molecular weight of the modified antibacterial peptide is 3026.67 Da.
Further, the preparation method of the modified antibacterial peptide comprises the following steps: according to the amino acid sequence of the modified antibacterial peptide, a polypeptide solid phase synthesis method is adopted for chemical synthesis to obtain a complete sequence, and HPLC reverse phase column chromatography is used for desalting to obtain the modified antibacterial peptide.
The second purpose of the invention is to provide the application of the modified antibacterial peptide in preparing antibacterial agents.
Further, the antibacterial agent is used for inhibiting gram-positive bacteria or gram-negative bacteria.
Further, the gram-positive bacteria include, but are not limited to, staphylococcus aureus (e.g., staphylococcus aureus CMCC26003, staphylococcus aureus ATCC43300, staphylococcus aureus 31, staphylococcus aureus 08032706), enterococcus faecalis (e.g., ATCC 29212), enterococcus faecium, clostridium perfringens (e.g., ATCC 13124), staphylococcus epidermidis, and the like.
Further, the gram-negative bacteria include, but are not limited to, Escherichia coli (e.g., Escherichia coli ATCC25922, Escherichia coli CMCC 44102), Pseudomonas aeruginosa (e.g., Pseudomonas aeruginosa CMCC 10104), Acinetobacter baumannii (e.g., Acinetobacter baumannii ATCC19606, Acinetobacter baumannii 2, Acinetobacter baumannii 6, Acinetobacter baumannii 16), Shigella flexneri (e.g., Shigella flexneri ATCC 12022), Salmonella typhimurium (e.g., Salmonella typhimurium ATCC 14028), Vibrio alginolyticus, Vibrio harveyi, and the like.
The third purpose of the invention is to provide the application of the modified antibacterial peptide in preparing anti-inflammatory agents.
Further, the anti-inflammatory agent is used for inhibiting tumor necrosis factor (TNF-alpha) or interleukin 6 (IL-6).
The fourth purpose of the invention is to provide the application of the modified antibacterial peptide in preparing anti-biofilm drugs.
Further, the anti-biofilm agent is used for removing a biofilm or inhibiting the formation of a biofilm.
The fifth purpose of the invention is to provide an antibacterial drug composition, which comprises the modified antibacterial peptide and antibiotics.
Further, the antibiotic may be meropenem, polymyxin B, ampicillin, vancomycin, or the like.
By the scheme, the invention at least has the following advantages:
(1) the invention carries out two-step reconstruction on the sea turtle antibacterial peptide Cm-CATH2, firstly, the peptide chain of the sea turtle antibacterial peptide Cm-CATH2 is shortened, and then on the basis of the obtained optimal antibacterial peptide, Arg and Lys in the peptide are replaced by non-natural D-type amino acids (D-Arg and D-Lys) to obtain the further reconstructed antibacterial peptide which contains 25 amino acid residues and has the molecular weight of 3026.67Da, and both the antibacterial peptide and the D-Arg have broad-spectrum efficient antibacterial activity and extremely strong anti-inflammatory activity.
(2) The modified antibacterial peptide has the beneficial characteristics of simple structure, low hemolytic activity, high stability, simple preparation method and the like, can be applied to the fields of medicines, cosmetics, food preservation, breeding industry and the like, and greatly expands downstream application.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.
FIG. 1 shows the anti-inflammatory activity assay results of the engineered antimicrobial peptides dNCCM 2 and NCM 4;
FIG. 2 shows the results of the stability assays for the engineered antibacterial peptides dNCCM 2 and NCM4 protease;
FIG. 3 is a graph showing the peak area changes of the stability tests of the engineered antibacterial peptides dNCCM 2 and NCM4 protease;
FIG. 4 shows the removal activity of engineered antibacterial peptides dNCCM 2 and NCM4 on bacterial biofilms; wherein a is a.baumann ni ATCC19606 and B is s.aureus CMCC 26003; represents P <0.05, represents P <0.01, represents P < 0.001;
FIG. 5 shows the inhibitory effect of engineered antibacterial peptides dNCCM 2 and NCM4 on bacterial biofilm formation; wherein a is a.baumann ni ATCC19606 and B is s.aureus CMCC 26003; represents P <0.05, represents P <0.01, represents P < 0.001.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1
Chemical synthesis of modified antibacterial peptide dNCM2
The chafer antibacterial peptide Cm-CATH2 is a polypeptide encoded by gene, contains 33 amino acid residues, has molecular weight of 4089.9Da and isoelectric point of 12.96. The total sequence of the tortoise green antimicrobial peptide Cm-CATH2 is as follows: arg 1 Arg 2 Ser 3 Arg 4 Phe 5 Gly 6 Arg 7 Phe 8 Phe 9 Lys 10 Lys 11 Val 12 Arg 13 Lys 14 Gln 15 Leu 16 Gly 17 Arg 18 Val 19 Lys 20 Arg 21 His 22 Ser 23 Arg 24 Ile 25 Thr 26 Val 27 Gly 28 Gly 29 Arg 30 Met 31 Arg 32 Phe 33 . According to the amino acid sequence of the antimicrobial peptide Cm-CATH2 of the green sea turtle, a modified body NCM4 (the whole sequence is Phe: 4) is designed and obtained by a molecular modification method 1 Lys 2 Lys 3 Val 4 Arg 5 Lys 6 Gln 7 Leu 8 Gly 9 Arg 10 Val 11 Lys 12 Arg 13 His 14 Ser 15 Arg 16 Ile 17 Thr 18 Val 19 Gly 20 Gly 21 Arg 22 Met 23 Arg 24 Phe 25 ) Then, the NCM4 is further subjected to structural modification of amino acid substitution, Arg and Lys in the NCM4 are substituted by unnatural D-type amino acids (D-Arg and D-Lys) to obtain the antibacterial peptide dNCM2, and the antibacterial peptide dNCM2 is chemically synthesized by a polypeptide solid phase synthesis method, and the specific preparation method isThe method comprises the following steps:
(1) preparation method of dNCM 2: the entire sequence of dNCM2 was synthesized using an automatic polypeptide synthesizer (433A, Applied Biosystems) based on the amino acid sequence of dNCM2, and desalted by HPLC reverse phase column chromatography.
(2) The molecular weight determination was carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).
(3) Purified dNCM2 was purified by HPLC to verify its purity.
The measurement results are as follows:
dNCM2 is a modified body of the antimicrobial peptide Cm-CATH2 of the green sea turtle. dNCM2 is a linear polypeptide containing 25 amino acid residues and having a molecular weight of 3026.67 Da. The complete sequence of dNCM2 is: phe (Phe) 1 Arg 2 Arg 3 Val 4 Arg 5 Arg 6 Gln 7 Leu 8 Gly 9 Arg 10 Val 11 Leu 12 Arg 13 His 14 Ser 15 Arg 16 Ile 17 Thr 18 Val 19 Gln 20 Gln 21 Arg 22 Met 23 Arg 24 Phe 25 Wherein all lysine and arginine are unnatural D-type amino acids, and the rest are natural L-type amino acids.
Example 2
Pharmacological experiments of modified antibacterial peptides dNCCM 2 and NCM 4:
1. determination of antibacterial activity of modified antibacterial peptide dNCCM 2 and NCM 4:
(1) respectively picking the test strains preserved on the inclined plane, uniformly coating the test strains on a MH solid culture medium (Beijing Sorleibao science and technology Co., Ltd.), placing a sterilized filter paper sheet with the diameter of 0.5Cm on the surface of the culture medium, dropwise adding 10 microliter of antibacterial peptide dNCM2, NCM4 or Cm-CATH2 sample solution dissolved in sterilized deionized water and carrying out inverted culture at 37 ℃ for 18-20 hours, and observing whether the inhibition zone is formed or not. If the sample has antibacterial activity, clear and transparent inhibition zones can be formed around the filter paper sheet, and the larger the inhibition zone is, the stronger the antibacterial activity of the sample is.
(2) Determination of Minimum Inhibitory Concentration (Minimum Inhibitory Concentration) of modified antibacterial peptide dNCCM 2 and NCM4 (2-fold dilution method):
and selecting the strains with the inhibition zones in the previous step to perform an MIC determination experiment. The test strains were inoculated into MH liquid medium (Beijing Solebao Tech Co., Ltd.), shake-cultured at 37 ℃ to logarithmic phase, and then the culture broth cultured to logarithmic phase was diluted to 2X 10 with fresh MH liquid medium 5 cfu/ml is ready for use.
Mu.l MH liquid culture medium is added into each hole of a sterile 96-hole plate in advance, then 100 mu.l of antibacterial peptide dNCCM 2, NCM4 or Cm-CATH2 sample solution which is diluted to a certain concentration by using the MH liquid culture medium and filtered by a 0.22 mu m-hole filter membrane is added into a first hole, 100 mu.l of the antibacterial peptide dNCCM 2, NCM4 or Cm-CATH2 sample solution is uniformly mixed, added into a second hole after being uniformly mixed, diluted in multiple proportions in sequence (see table 1), 100 mu.l of the antibacterial peptide is sucked out from a second hole and discarded, and a 10 th hole is a control tube.
TABLE 1 dilution method
The tubes were mixed well and incubated at 37 ℃ for 18 hours with slow shaking, and the light absorption was measured at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible.
TABLE 2 engineered antimicrobial peptides dNCM2 and NCM4 antimicrobial Activity
MIC: the minimum inhibitory concentration, and the result is the average value of three independent repeated experiments.
As can be seen from Table 2, the unmodified antimicrobial peptide Cm-CATH2 and the modified antimicrobial peptides NCM4 and dNCM2 have extremely strong antibacterial activity on both gram-positive bacteria and gram-negative bacteria, wherein the antibacterial activity comprises part of clinically isolated pathogenic bacteria, the MIC value of the tested bacteria of NCM4 is less than or equal to 18.75 mug/ml, and the MIC value of the tested bacteria of dNCM2 is in the range of 9.38-75 mug/ml.
2. Hemolytic activity assay of engineered antibacterial peptide dNCM2 and NCM 4:
mixing collected rabbit blood with Ashi solution for anticoagulation, washing with normal saline for 2 times, and resuspending into 10 7 -10 8 cell/ml suspension. Mixing the diluted erythrocyte suspension with dNCCM 2, NCM4 or Cm-CATH2 sample dissolved in normal saline, keeping temperature at 37 deg.C for 30min, centrifuging at 1000rpm for 5min, and measuring absorbance of the supernatant at 540 nm. The negative control was physiological saline, the positive control was Triton X-100, and the percentage of hemolysis was calculated according to the following equation: percent of hemolysis H% ═ A Sample (I) -A Negative control /A Positive control ×100％。
The results show a sample concentration of 100. mu.g/ml, a percentage hemolysis of dNCM2 of 1.29%, a percentage hemolysis of NCM4 of 1.51%, and a percentage hemolysis of Cm-CATH2 of 4.1%. The dNCCM 2 and NCM4 both have lower hemolytic activity and are not easy to cause rupture and lysis of the red blood cells of the mammals. Especially in the range of antibacterial activity, the safety is high.
3. Determination of anti-inflammatory activity of modified antibacterial peptide dNCCM 2 and NCM 4:
macrophages in abdominal cavities of C57 mice of 6-8 weeks old are extracted, cultured overnight in 1640 medium containing 10% serum, changed into 1640 medium containing 2% serum the next day, cells are stimulated by Escherichia coli LPS (Sigma, USA) with the final concentration of 100ng/mL, polypeptides dNCCM 2 or NCM4 with the final concentration of 20 mug/mL are added, a blank control group without the polypeptides and LPS and a positive control group with only LPS are added for incubation for 16h, supernatant is taken, and the content of proinflammatory factors IL-6 and TNF-alpha in the supernatant is detected by an ELISA kit (R & D, USA). Each done in triplicate.
The result is shown in figure 1, dNCM2 can obviously inhibit expression of proinflammatory factors IL-6 and TNF-alpha induced by LPS in mouse peritoneal macrophages, and the result shows that dNCM2 has extremely strong anti-inflammatory activity which is equivalent to NCM 4.
4. Experimental study on stability of modified antibacterial peptide dNCM2 and NCM 4:
(1) enzyme stability testing: 0.25% pancreatin for cell digestion was mixed with the polypeptide sample in a molar ratio of 1: 200, incubating at 37 ℃, sampling 50 mu L at 0, 6, 12 and 24 hours respectively, then diluting the sampled product by 1 time with a polypeptide solvent, filtering with a 0.22 mu m filter membrane, taking 20 mu L, and determining the residual quantity of the polypeptide sample by using reverse high performance liquid chromatography. Wherein phase A was subjected to gradient elution with pure water containing 0.1% trifluoroacetic acid (TFA) and phase B was subjected to gradient elution with acetonitrile containing 0.1% TFA to obtain elution peaks and integrated areas at different time points after polypeptide samples dNCCM 2 or NCM4 were mixed with pancreatin, and then plotted with software Origin 2018.
The results are shown in fig. 2 and 3, antibacterial peptide dNCM2 has strong enzyme stability, the peak height and peak area of dNCM2 are still not changed greatly after 24h of enzyme action, and antibacterial peptides NCM4 and Cm-CATH 26 h are degraded significantly, which shows that the enzyme stability of dNCM2 is significantly better than that of NCM4 and Cm-CATH 2.
(2) Salt tolerance, heat tolerance and heat stability testing
Salt tolerance: escherichia coli ATCC25922 was cultured with MH broth (Qingdao Haibo Biotechnology Co., Ltd.) at 37 ℃ for 12 hours, and then diluted to 10 with fresh MH broth containing 0, 50, 100, 150, 200 and 400mM sodium chloride, respectively 6 CFU/ml. Different concentration gradients of dNCM2, NCM4 and Cm-CATH2 samples were prepared with MH liquid medium containing the corresponding sodium chloride concentrations. The effect of sodium chloride on the antibacterial activity of dCM 2, NCM4 and Cm-CATH2 was determined by determining the MIC values of dCM 2, NCM4 and Cm-CATH2 for E.coli ATCC25922 using a 2-fold dilution method.
Heat resistance: the effect of heating at different temperatures on the antibacterial activity of the samples was determined by dissolving dNCCM 2, NCM4 or Cm-CATH2 in sterile deionized water (2 mg/ml), incubating at 4, 20, 37, 50, 70 and 90 ℃ for 1 hour, and then determining the MIC values of the samples against E.coli ATCC25922 using a 2-fold dilution method.
Thermal stability: dNCCM 2, NCM4 or Cm-CATH2 were dissolved in sterile deionized water (2 mg/ml) and incubated at 37 ℃ for 0-96 hours. Samples were taken at 0, 6, 12, 24, 48, 72 and 96 hours to determine MIC values for E.coli ATCC25922, respectively, and the samples were heat-stable.
As shown in Table 3, dNCCM 2, NCM4 and Cm-CATH2 have strong salt tolerance. The antibacterial activity of dNCCM 2, NCM4 and Cm-CATH2 is kept unchanged under the condition that the concentration of the physiological salt of the human body is lower than or equal to that of the physiological salt of the human body (less than or equal to 150mM NaCl). The antibacterial activity of dNCM2 and NCM4 is only slightly reduced with the increase of the salt concentration after the salt concentration is higher than the physiological salt concentration of a human body, particularly dNCM2, and the antibacterial activity is also kept unchanged under the condition of 200mM NaCl.
TABLE 3 engineered antimicrobial peptides dNCM2 and NCM4 salt tolerance
As shown in table 4, dNCM2 has strong heat resistance. The antibacterial activity of the dNCM2 solution did not change after standing at 90 ℃ for 1 hour. In contrast, NCM4 and Cm-CATH2 were slightly less heat resistant, and their MIC values were all increased after 1 hour of heating at high temperature.
TABLE 4 Heat tolerance of engineered antimicrobial peptides dNCCM 2 and NCM4
Many conventional antibiotics, such as cephalosporin antibiotics, are very unstable in solution and lose activity within a few hours, which greatly limits their use. In contrast, dNCM2 has good thermal stability. The antibacterial activity of dNCM2 solution did not change after 96 hours at 37 ℃ (see Table 5). In contrast, NCM4 and Cm-CATH2 were slightly less thermally stable and had elevated MIC values after 96 hours at 37 ℃.
TABLE 5 thermal stability of the engineered antimicrobial peptides dNCM2
Example 3
Modified antibacterial peptides dNCCM 2 and NCM4 biological membrane clearing and inhibiting activity test
1. Biofilm removal Activity assay
Taking out the preserved strain from a refrigerator at minus 80 ℃, rapidly melting the strain in water bath at 37 ℃, dipping a little liquid by using an inoculating loop, carrying out streaking on the strain in a Z shape by dividing four regions on an LB solid culture medium, and carrying out constant-temperature culture at 37 ℃ from the last tail end until a single bacterial colony grows out in each streaking; picking a single colony in a sterile liquid MHB culture medium, and carrying out shaking culture at 37 ℃ and 180rpm until the logarithmic phase; detecting the concentration of bacterial liquid, diluting to 2 × 10 7 CFU/mL. 200. mu.L of the above-mentioned bacterial suspension was added to a sterile 96-well plate, and cultured at 37 ℃ for 48 hours to allow biofilm formation. Aspirate the bacteria from each well and wash three times with PBS. 200. mu.L of the diluted polypeptide sample was added to each well so that the final concentration of the polypeptide sample was 0.5 XMIC, 1 XMIC, 2 XMIC, 4 XMIC and 8 XMIC, and incubated at 37 ℃ for 24 hours. Adding crystal violet dye solution (0.1%) into each well, dyeing for 30min, sucking out the dye solution, washing with sterile PBS for three times, and air drying in an ultra-clean bench. Adding 100 μ L of anhydrous ethanol into each well, standing for 20min, and dissolving crystal violet. The OD value was measured at an ultraviolet wavelength of 560nm, and the three were set up in parallel. The percentage of Biofilm formation (Biofilm Retention%, BR%) was calculated using the following formula: BR (%) - [ 100% × (F)% ] 0 -F peptide )/F 0 ]In this experiment, PBS was selected as a negative control, and the measured value was regarded as the maximum biofilm residual amount. Biofilm Retention%, BR% being the percentage of Biofilm survival, F 0 Absorbance of PBS-treated group, F peptide Absorbance values for the polypeptide treatment groups.
2. Biofilm inhibition activity assay
Taking out the preserved strain from a refrigerator at minus 80 ℃, rapidly melting in water bath at 37 ℃, dipping a little liquid, drawing a line in a Z shape on an LB solid culture medium, and culturing at constant temperature of 37 ℃ until a single colony grows out; picking a single colony in a sterile liquid MHB culture medium, and carrying out shaking culture at 37 ℃ and 180rpm until the logarithmic phase is reached; detecting the concentration of bacterial liquid, diluting to 2 × 10 7 CFU/mL. To a sterile 96-well plate was added 190. mu.L of the above-described bacterial suspension, and diluted polypeptide samples were added to each well, 3 replicates per concentration set. The polypeptide samples were allowed to reach final concentrations of 0.5 × MIC, 1 × MIC, 2 × MIC, 4 × MIC and 8 × MIC, and incubated at 37 ℃ for 48 hours. The bacterial solution was aspirated from each well and washed three times with PBS,the plate is placed on a superclean bench for ventilation and drying. Adding crystal violet dye solution (0.1%) into each well, dyeing for 30min, sucking out the dye solution, washing with sterile PBS for three times, and air drying in an ultra-clean bench. Adding 100 μ L of anhydrous ethanol into each well, standing for 20min, and dissolving crystal violet. The OD value was measured at an ultraviolet wavelength of 560 nm. The percentage of Biofilm Formation (Biofilm Formation%, BF%) was calculated using the following equation: BF (%) - [ 100% × (F) 0 -F peptide )/F 0 ]Wherein PBS (F) 0 ) As a negative control, the measured value was regarded as the maximum biofilm formation amount. Biofilm Formation%, BF% being the percentage of Biofilm Formation, F peptide Absorbance values for the polypeptide treatment groups.
As shown in fig. 4 and 5, both engineered polypeptides dNCM2 and NCM4 were effective in clearing biofilms produced by acinetobacter baumannii (fig. 4A) and staphylococcus aureus (fig. 4B), and there was a concentration dependence, and dNCM2 performed better than NCM4 in clearing biofilms produced by acinetobacter baumannii. Both the engineered polypeptides dNCM2 and NCM4 were able to inhibit the production of biofilms by acinetobacter baumannii (fig. 5A) and staphylococcus aureus (fig. 5B) at high concentrations. Compared with NCM4, the dNCM2 can better inhibit Staphylococcus aureus from producing a biofilm, and can better inhibit Acinetobacter baumannii from producing a biofilm.
Example 4
Determination of synergistic antibacterial action of modified antibacterial peptides dNCCM 2 and NCM4 and antibiotics
The polypeptide was prepared at a concentration of 20X 8MIC in sterile water, and sequentially diluted in multiple aliquots to 20X 1/64MIC, plus solvent to 11 concentrations for use. Antibiotic (Meropenem, polymyxin B, ampicillin and vancomycin) medicines are weighed, dissolved into a solution of 2mg/mL by sterile water, diluted in multiple times in sequence to obtain the concentration of 4MIC-1/16MIC, and added with a solvent to obtain 8 concentrations for later use. Diluting the bacterial liquid to 5X 10 5 CFU/mL, spare.
Chessboard method detection, adding 90 mul diluted bacterial liquid into 96-well plate; polypeptides were added to the bacteria at 5 μ L per well, one concentration per column, for a total of 11 columns: antibiotics were added to the bacteria at 5. mu.L per well, one concentration per row, for 8 rows; find MICA, MICB, A, B calculate FIC, FIC ═ FMICA + FMICB ═ A/MICA + B/MICB (A, B represent the concentration at the optimum concentration point of the combination of the two drugs, MICA, MICB represent MIC when the two drugs are used singly), FIC < 0.5 has synergistic effect, FIC > 4 has antagonistic effect, 0.5 < FIC < 4 has additive effect. The dosing regimen is shown in the table below.
Acinetobacter baumannii (A. baumannii ATCC19606) and methicillin-resistant staphylococcus aureus (S.aureus ATCC43300) are selected as test bacteria, and the synergistic antibacterial action of several antibiotics and the modified antibacterial peptide dNCM2 is detected. Wherein, the meropenem and the polymyxin B can cooperatively play a role in resisting acinetobacter baumannii, and the FICI indexes are 0.25 and 0.375 respectively. Ampicillin and vancomycin can cooperate with modified antibiotic peptide dNCM2 to resist the action of methicillin-resistant Staphylococcus aureus, and FICI indexes are 0.375 and 0.1875, respectively.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Sequence listing
<110> Suzhou university
<120> modified antibacterial peptide and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> PRT
<213> (Artificial sequence)
<400> 1
Arg Arg Ser Arg Phe Gly Arg Phe Phe Lys Lys Val Arg Lys Gln Leu
1 5 10 15
Gly Arg Val Lys Arg His Ser Arg Ile Thr Val Gly Gly Arg Met Arg
20 25 30
Phe
<210> 2
<211> 25
<212> PRT
<213> (Artificial sequence)
<400> 2
Phe Lys Lys Val Arg Lys Gln Leu Gly Arg Val Lys Arg His Ser Arg
1 5 10 15
Ile Thr Val Gly Gly Arg Met Arg Phe
20 25
Claims (10)
1. A modified antimicrobial peptide, comprising: the amino acid sequence of the modified antibacterial peptide is shown in SEQ ID NO. 2.
2. The engineered antimicrobial peptide of claim 1, wherein: arginine in the amino acid sequence shown in SEQ ID NO.2 is D-type arginine, and lysine is D-type lysine.
3. Use of the engineered antimicrobial peptide of claim 1 or 2 in the preparation of an antimicrobial agent.
4. Use according to claim 3, characterized in that: the antibacterial agent is used for inhibiting gram-positive bacteria or gram-negative bacteria.
5. Use according to claim 4, characterized in that: the gram-positive bacteria comprise staphylococcus aureus, enterococcus faecalis, enterococcus faecium, clostridium perfringens or staphylococcus epidermidis.
6. Use according to claim 4, characterized in that: the gram-negative bacteria comprise escherichia coli, pseudomonas aeruginosa, acinetobacter baumannii, shigella flexneri, salmonella typhimurium, vibrio alginolyticus or vibrio harveyi.
7. Use of the engineered antibacterial peptide of claim 1 or 2 in the preparation of an anti-inflammatory agent.
8. Use of the engineered antimicrobial peptide of claim 1 or 2 in the preparation of an anti-biofilm agent.
9. An antibacterial pharmaceutical composition, characterized in that: the antibacterial pharmaceutical composition comprising the engineered antibacterial peptide of claim 1 or 2 and an antibiotic.
10. The antibacterial pharmaceutical composition according to claim 9, characterized in that: the antibiotic is selected from one of meropenem, polymyxin B, ampicillin and vancomycin.
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|---|---|---|---|---|
| CN106188265A (en) * | 2016-07-22 | 2016-12-07 | 大连理工大学 | A kind of antimicrobial peptide Cm CATH2 and gene, preparation method and application |
| CN112625107A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM8 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN113735956A (en) * | 2021-07-20 | 2021-12-03 | 苏州市第九人民医院 | Antibacterial peptide CCM7WC, and preparation method and application thereof |
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| CN106188265A (en) * | 2016-07-22 | 2016-12-07 | 大连理工大学 | A kind of antimicrobial peptide Cm CATH2 and gene, preparation method and application |
| CN112625107A (en) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | Modified antibacterial peptide C-CM8 of tortoise green antibacterial peptide, and preparation method and application thereof |
| CN113735956A (en) * | 2021-07-20 | 2021-12-03 | 苏州市第九人民医院 | Antibacterial peptide CCM7WC, and preparation method and application thereof |
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| JIANGUANG LU ET AL: "- and Unnatural Amino Acid Substituted Antimicrobial Peptides With Improved Proteolytic Resistance and Their Proteolytic Degradation Characteristics", FRONTIERS IN MICROBIOLOGY, vol. 11, pages 2 * |
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| CN117138024A (en) * | 2023-08-18 | 2023-12-01 | 苏州大学 | Vancomycin-antibacterial peptide conjugate and application thereof |
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