CN103435686B - Anti-drug resistance bacteriological infection peptide C bf-14 and uses thereof - Google Patents
Anti-drug resistance bacteriological infection peptide C bf-14 and uses thereof Download PDFInfo
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- CN103435686B CN103435686B CN201310413721.0A CN201310413721A CN103435686B CN 103435686 B CN103435686 B CN 103435686B CN 201310413721 A CN201310413721 A CN 201310413721A CN 103435686 B CN103435686 B CN 103435686B
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Abstract
The present invention relates to biomedical sector, be specifically related to a kind of antimicrobial polypeptide.Its amount of activated amino acid sites, according to the antimicrobial polypeptide of Cathelicidin family, suddenlys change by the present invention, adopts solid-state chemical reaction method method to obtain antimicrobial polypeptide penicillin resistance bacterium to high inhibit activities.Results of pharmacodynamic test shows, antimicrobial polypeptide of the present invention has good antibacterial activity in vitro to drug tolerant bacteria, can be used for the disease for the treatment of drug resistant bacterial infections aspect.
Description
Technical field
The present invention relates to field of biomedicine technology, be specifically related to a kind of antimicrobial polypeptide and application thereof, antimicrobial polypeptide of the present invention has the purposes of anti-drug resistance bacteriological infection.
Background technology
Along with antibiotic extensive application, particularly aphalangia requisition medicine, select antimicrobial drug for subsequent use, over-treatment and frequently change dressings irrelevantly, cause the resistant rate of bacterium more and more higher, drug-resistant intensity is more and more serious.The existence of spontaneous medicament-resistant mutation and the continuous action of antibiotic selective pressure, and the evolution of the adaptive capacity to environment of pathogenic bacteria and human body microenvironment ecologic change urges, and is the basis of resistant organism and multi-drug resistant bacteria generation clinically.Multi-drug resistant bacteria, broadly comprises extensive resistant organism or superbacteria, and its emergence and development brings huge challenge to clinical diagnosis and treatment.
Antibacterial peptide is the very potential new antimicrobial agent source of one, and due to Antibacterial Mechanism and various biological activity of its uniqueness, from research and development so far, its Study and appliance has become the focus in bio-pharmaceuticals and clinical medicine domain.There is the advantages such as molecular weight is little, has a broad antifungal spectrum, Antibacterial Mechanism uniqueness, not easily generation resistance compared with conventional antibiotic.Cationic antibacterial peptide is broadly defined as and is less than 50 amino acid whose polypeptide being rich in positive ion, and has the hydrophobic residue of half or more.The contained positive charge of polypeptide derives from the positive charge entrained by Methionin and arginine.Most of antibacterial peptide tool cationic and amphiphilic, because of length, charge number, aminoacid sequence is different with the position of secondary structure and the characteristic of polypeptide is different.Polypeptide is divided into four classes according to its secondary structure, is α spiral, β lamella, random coil and linear peptides respectively.This peptide species has broad-spectrum bactericidal action, all effective to gram-positive microorganism, Gram-negative bacteria and fungi.Some antibacterial peptides also have other physiologically actives, antiviral, anticancer, promote wound healing, activate immunity response etc.These characteristics of cationic polypeptide make it become the strong candidate of new medicine.The research of the mechanism of action of cationic antibacterial peptide is also more and more extensive.The mechanism of action of antibacterial peptide has a variety of, and some can make cell membrane permeate, and some acts on cell walls, also has macromolecular synthesis in some T suppression cell.
But at present, there are three large bottlenecks in the development of antibacterial peptide, that is: the production cost of antibacterial peptide is high; The toxicity of antibacterial peptide is larger; Antibacterial peptide is to the poor stability of proteasome.Existing many scholar's research show the structure can improving original antibacterial peptide from design, and retain its avtive spot, reduction peptide chain, to reduce the production cost of antibacterial peptide.When carrying out antibacterial peptide design, need to consider the factor such as toxicity and stability, itself and original natural antibacterial peptide being consistent in toxicity and stability, by planning as a whole the factors such as electric charge, hydrophobicity, secondary structure, making its low toxicity efficient stable more.
Summary of the invention
The present invention is according to the antimicrobial polypeptide of Cathelicidin family, its amount of activated amino acid sites is suddenlyd change, adopt solid-state chemical reaction method method to obtain antimicrobial polypeptide penicillin resistance bacterium to high inhibit activities, antimicrobial polypeptide of the present invention, aminoacid sequence is as shown in SEQ ID:NO:1.Be called for short Cbf-14, total order is classified as: arg-leu-leucine-arginine-Methionin-phenylalanine-phenylalanine-arginine-lysine-leucine-lysine-lysine-Serine-Valine.
Cbf-14 is by Cbf-K
16c terminal amino acid carry out brachymemma, add α spiral tumor-necrosis factor glycoproteins, what adopt solid-state chemical reaction method method to obtain to have a high inhibit activities to bacterium and persister thereof has 14 amino acid whose antibacterial peptide mutant simultaneously.After transformation, it contains 7 positive charges, and molecular weight is 1819.33, and iso-electric point is 12.31.Solid phase synthesis cost reduces 2/3, and toxicity does not have considerable change, therefore significantly solves the problem that the production cost of antibacterial peptide is high, is conducive to the application of antibacterial peptide in the infection for the treatment of drug-resistant bacteria.
Adopt solid-state chemical reaction method method can obtain antimicrobial polypeptide of the present invention.
Antibacterial activity in vitro research shows, peptide C bf-14 of the present invention has the effect killing drug tolerant bacteria significantly, can be used to the medicine preparing anti-drug resistance bacteriological infection.The preferred streptococcus aureus of described drug tolerant bacteria, staphylococcus epidermidis, escherichia coli, Pseudomonas aeruginosa and Klebsiella Pneumoniae.
Antibacterial peptide also likely acts on higher organisms and comprises human body cell while sterilization, because the mode of action of antibacterial peptide is all make cell generation seepage dead at perforate cell membranes.So using antibacterial peptide can make red corpuscle generation seepage and antibacterial peptide can make mouse primary splenocyte occur dead as whether a virose standard.If antibacterial Toplink makes the oxyphorase generation seepage in red corpuscle, just can by detecting OD
540measure its toxicity size.Therefore the present invention also have detected antibacterial peptide Cbf-14 of the present invention to the hemolytic activity of sheep red blood cell (SRBC).Experiment shows, the hemolysis rate of antibacterial peptide of the present invention is very low.The present invention simultaneously finds within the scope of experimental concentration, and antibacterial peptide of the present invention does not have toxic action to mouse boosting cell.Result confirms, the toxicity of antibacterial peptide of the present invention is minimum.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates of antibacterial peptide Cbf-14 of the present invention
Fig. 2 is the Mass collection of illustrative plates of Cbf-14
Fig. 3 is the circular dichroism spectrum of Cbf-14
Fig. 4 is the killing curve of Cbf-14 to the streptococcus aureus of Penicillin-resistant
Fig. 5 is the killing curve of Cbf-14 to the staphylococcus epidermidis of Penicillin-resistant
Fig. 6 is the killing curve of Cbf-14 to the escherichia coli of Penicillin-resistant
Fig. 7 is the killing curve of Cbf-14 to the Pseudomonas aeruginosa of Penicillin-resistant
Fig. 8 is the killing curve of Cbf-14 to the Klebsiella Pneumoniae of Penicillin-resistant
Fig. 9 is the haemolysis curve of Cbf-14 to sheep red blood cell (SRBC)
Figure 10 is the toxicity of Cbf-14 to mouse primary splenocyte
Embodiment
Embodiment 1
The synthesis of antimicrobial polypeptide Cbf-14 and structural identification:
Solid phase synthesis process synthetic polypeptide of the present invention routinely.Specific experiment step is as follows:
The peptide sequence of the Cbf-14 of preparation is:
H-Arg-Leu-Leu-Arg-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-OH
The synthesis of polypeptide: the synthesis of polypeptide holds N end to carry out one by one from C.Fmoc-Val-Wang Resin methylene dichloride is soaked 15 minutes, treats that resin expands, pump methylene dichloride; Add hexahydropyridine/DMF solution that volume ratio is 1:4, use nitrogen to agitate, react 2 times, the time is 5 minutes and 15 minutes, and reaction terminates rear DMF washing resin 9 times.The resin that takes a morsel adds each 2-3 of toner ABC and drips (A liquid: triketohydrindene hydrate/ethanol solution; B liquid: pyridine; C liquid: phenol/ethanol solution) at 100 DEG C, be total to heat 3 minutes, solution and color of resin become blueness, to remove amido protecting.Add Fmoc-Val-OH and HOBT; dissolve with appropriate DMF; add DIC and Collidine; nitrogen is agitated, and reacts 1 hour, and reaction terminates rear DMF washing resin 6 times; repeat condensation reaction; connect each Fmoc protected amino acid successively, complete the synthesis of linear sequence, drain after resin methylene dichloride and ether are soaked.Add TFA, in constant-temperature table, react 2h, shaking speed 110r/min, temperature 25 DEG C.Elimination resin, adds anhydrous diethyl ether, obtains solid with after centrifuge in filtrate, adds anhydrous diethyl ether washing, more centrifugal, repeats post-drying for several times and can obtain Cbf-14 crude product polypeptide.
Purifying: take a certain amount of crude product, adds appropriate acetonitrile, ultrasonicly goes out macrobead magazine to clarification strainer.Cross preparative liquid chromatography instrument, sample is collected in segmentation simultaneously.Do gradient analysis with analytical chromatograph, required purity sample will be reached and retain.Then lyophilize process is carried out.Polypeptide after purifying is through mass spectrum and high performance liquid chromatography qualification.
As can be seen from accompanying drawing 1, by HPLC, polypeptide of the present invention, determines that its purity is greater than 98.33%.
Can determine that its molecular weight is respectively 1819.33 by accompanying drawing 2, its molecular weight and theoretical value 1819.30 are coincide.
Use MOS450 circular dichroism spectrometer under room temperature, measure the secondary structure of antibacterial peptide Cbf-14.Antibacterial peptide Cbf-14 concentration is 200 μ g/ml, and solution environmental is respectively Sodium dodecylbenzene sulfonate (SDS) and the trifluoroethanol (TFE) of pure water and 25mM.Sweep limit is the quartz container of 190 ~ 250nm, 0.1cm path length, slit 1nm, and sweep velocity is 100nm/min, and each sample continuous sweep 3 times, averages.The results are shown in Table 1 and accompanying drawing 3.
The secondary structure of table 1Cbf-14
As can be seen from accompanying drawing 3 and table 1, Cbf-14 all exists in random coil structure in pure water.The SDS of 25mM exists in micella peptide aggregation in water, can the lipid bilayer of analog cell film.Cbf-14 has two smallest peaks at 208nm and 222nm place, and this is typical alpha-helix spectrogram.Cbf-14 is dissolved in TFE solution, also occurs part α-helixstructure, but its principal mode is beta sheet structure.The above results illustrates the conformation that Cbf-14 is unstable in aqueous, but when their combine or close to cytolemma, during hydrophobic environment in contact membranes, can be induced into α-helixstructure and beta sheet structure.The alpha-helix of Cbf-14 in SDS is the highest, is secondly in TFE.
Embodiment 2
Peptide C bf-14 of the present invention is to the drug effect of penicillin resistance bacterium:
(1) preparation of inoculum
Experiment is connected to nutrient agar slopes with bacterium from glycerine pipe, and after 37 DEG C of overnight incubation, then picking is inoculated in 2ml nutrient broth medium a little, cultivates 8h, is diluted to 10 with aseptic MH cultured solution of broth for 37 DEG C
5the bacterial suspension of about CFU/ml.
(2) configuration of medicine
Accurately take Cbf-14 and penicillin is configured to the drug solution that concentration is 1024 μ g/ml, 0.22 μm of film sterile filtration, packing, it is for subsequent use to put-70 DEG C of preservations.
(3) mensuration of minimal inhibitory concentration (MIC)
With aseptic MH cultured solution of broth become 1ml to contain medicine stoste doubling dilution solution that concentration is 512,256,128,64,32,16,8,4,2,1,0.5,0.25 μ g/ml.The above-mentioned bacterium liquid getting 1ml adds in the pastille substratum prepared, and now, each test tube drug concentration is respectively 256,128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/ml.Separately establish not that pastille group is in contrast.Put in 37 DEG C of incubators and cultivate 16 ~ 20h, observe antibacterial effect, and record MIC value.The results are shown in Table 2:
Table 2Cbf-14 is to the MIC of Penicillin-resistant bacterium and standard bacteria
As seen from Table 2, drug-resistant bacteria selected by the present invention all reveals very strong resistance to penicillin, 256 μ g/ml are all greater than to the MIC of streptococcus aureus, staphylococcus epidermidis, escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, and the MIC value of Cbf-14 to streptococcus aureus, staphylococcus epidermidis persister only have 16 μ g/ml.Be 32 μ g/ml to the MIC of escherichia coli and Pseudomonas aeruginosa.Result proves, Cbf-14 has the activity of certain resistance to penicillin resistant organism.
(4) mensuration of minimal bactericidal concentration (MBC)
Successively the above-mentioned each pipe culture having no bacterial growth being drawn 0.1ml is respectively added in aseptic plate, mix with nutrient agar, put 37 DEG C and cultivate 18h again, the minimum dilution drug level that on plate, bacterium colony is less than 5 is minimum bactericidal concentration MBC.The results are shown in Table 3:
Table 3Cbf-14 is to the MBC of Penicillin-resistant bacterium and standard bacteria
As can be seen from Table 3, the MBC of antibacterial peptide of the present invention to Penicillin-resistant bacterium is about four times of MIC value, and Cbf-14 is 16 μ g/ml to the MBC of streptococcus aureus, is then 64 μ g/ml to the MBC of staphylococcus epidermidis and escherichia coli.Cbf-14 to the MBC value of Pseudomonas aeruginosa and Klebsiella Pneumoniae at 128 μ g/ml.Penicillin does not then kill bacterium (MBC>256 μ g/ml) within the scope of experimental concentration.
(5) antibacterial peptide is to the drafting of the killing curve of Penicillin-resistant bacterium
Adopt the drug level of the MIC of 4 times, will prepare about 1 × 10
6the bacteria suspension of CFU/ml and medicament mixed, make its final concentration be 10
5about CFU/ml.When 0min, 10min, 30min, 1h, 2h, 4h and 8h, draw culture serial dilution, carry out live bacterial count, each extent of dilution does three parallel laboratory tests, averages.With bacterial concentration logarithm for ordinate zou, incubation time is X-coordinate, draws killing curve.Do blank and positive control simultaneously.The results are shown in accompanying drawing 1-5.
The determination of drug concentration Cbf-14 of this experiment employing 4 × MIC is to the killing curve of streptococcus aureus, staphylococcus epidermidis, escherichia coli, Pseudomonas aeruginosa and Klebsiella Pneumoniae.
Have good germicidal action as can be seen from accompanying drawing 4, Cbf-14 to streptococcus aureus, and effect rapidly.All thalline can be killed in 10min.
After making Resistant Staphylococcus epidermidis have dropped about 3 lgCFU, 4h in 60min as can be seen from accompanying drawing 5, Cbf-14, bacterium is killed completely.As can be seen here, the germicidal action of Cbf-14 to streptococcus aureus and staphylococcus epidermidis is strong and quick-acting.
Comparatively mild as can be seen from the sterilization effect effect of the right escherichia coli of accompanying drawing 6, Cbf-14, after 4 hours, killing curve tends to balance substantially, can substantially kill all microorganisms.
As can be seen from accompanying drawing 7, Cbf-14 to the sterilization effect of Pseudomonas aeruginosa and Cbf-14 similar to colibacillary effect, penicillin resistant Pseudomonas aeruginosa can be killed completely at 6h.
Mild to Klebsiella Pneumoniae effect as can be seen from accompanying drawing 8, Cbf-14, can substantially kill penicillin resistant Klebsiella Pneumoniae at 6h.
Antibacterial activity in vitro research shows, peptide C bf-14 has the effect killing drug tolerant bacteria significantly, can be used to the drug effect preparing anti-drug resistance bacteriological infection.Described drug tolerant bacteria excellent finger streptococcus aureus, staphylococcus epidermidis, escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae.
Embodiment 3
The toxicity of polypeptide antibacterial peptide Cbf-14 of the present invention, comprise hemolytic measure and to eukaryotic cell toxicity test.
The hemolytic of polypeptide antibacterial peptide Cbf-14 of the present invention measures:
With refrigerated centrifuge (4 DEG C) by the centrifugal 10min of fresh sheep whole blood 3000rpm, the sheep red blood cell (SRBC) obtained PBS is washed three times, resuspended in the volume ratio by 10%.By the medicine of resuspended sheep red blood cell (SRBC) equal-volume mixing gradient dilution, the final concentration of Cbf-14 is made to be 512,256,128,64,32,16,8,4 and 2,1 μ g/ml, and establish PBS group as negative control, 0.1%Triton X-100 group as positive control, each gradient establish 6 parallel.All mixtures are put 37 DEG C of incubators and hatch 1h.Use the centrifugal 10min of refrigerated centrifuge (4 DEG C) 3000rpm afterwards again, get supernatant and 540nm wavelength surveys light absorption value.The results are shown in accompanying drawing 6.
Hemolysis rate=[(A540
cbf-14-A540
pBS)/(A540
0.1%Triton x-100-A540
pBS)] × 100.
Sheep Blood cell is used to the hemolytic detecting antibacterial peptide Cbf-14, and the hemolytic of antibacterial peptide is considered to the main criterion of polypeptide to Mammalian blood cells toxicity.Very low to the hemolytic of Sheep Blood cell as can be seen from accompanying drawing 9, Cbf-14, also only have 1% at the hemolysis rate of tested maximum concentration 512 μ g/ml.Positive control 0.1%Triton X-100 hemolysis rate just reaches 100%.Result shows, Cbf-14 does not produce toxic action to Mammalian blood cells in trial stretch.
Antibacterial peptide Cbf-14 of the present invention is to the toxicity test of splenocyte:
Get the spleen of healthy ICR mouse, grind to form cell dispersion through 300 object screen clothes.With RPIM-1640(containing 10% calf serum) substratum is by resuspended for cell one-tenth 5 × 10
7individual/ml, is placed in aseptic 96 orifice plates.Add the Cbf-14 medicine of serial dilution simultaneously, make its final concentration be 50,100,200,400,800,1600 μ g/ml, not drug containing group and ConA are set simultaneously and make negative control and positive control respectively, each medicine gradient establish three parallel.Put CO
2incubator is cultivated and is hatched 48 hours.Then MTT (5 μ g/ml) is added, then after hatching 4 hours, through the centrifugal 10min of plate centrifuge 3000rpm.Supernatant discarded, adds DMSO and is dissolved in 490nm survey light absorption value, draw cell toxicant curve.The results are shown in accompanying drawing 10.
As can be seen from accompanying drawing 10: along with the concentration of antibacterial peptide Cbf-14 increases, light absorption value only has slight fluctuating, do not have significance to increase or reduce.Illustrate that the splenocyte survival number through antibacterial peptide Cbf-14 process 48h significance increase does not occur or reduces, therefore in the concentration range of experiment, the splenocyte of Cbf-14 to mouse does not have toxicity.
Sequence table
Ying Haiyue bio tech ltd, <110> Nanjing
<120> anti-drug resistance bacteriological infection peptide C bf-14 and uses thereof
<130> 2013
<160> 1
<170> PatentIn version3.3
<210> 1
<211> 14
<212> PRT
<213> artificial sequence
<400> 1
Arg Leu Leu Arg Lys Phe Phe Arg Lys Leu Lys Lys Ser Val
1 5 10
Claims (3)
1. an antimicrobial polypeptide, its aminoacid sequence is as shown in SEQ ID:NO:1.
2. a pharmaceutical composition, the antimicrobial polypeptide wherein containing claim 1 and pharmaceutically acceptable carrier.
3. the polypeptide of claim 1 is for the preparation of the purposes of anti-drug resistance bacteriological infection medicine, and described drug tolerant bacteria is streptococcus aureus, staphylococcus epidermidis, escherichia coli, the single bacterium of the false born of the same parents of verdigris or streptococcus pneumoniae.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103920137B (en) * | 2014-04-28 | 2016-03-30 | 中国药科大学 | A kind of pharmaceutical composition with the effect of anti-drug resistance gram-positive bacteria |
| CN104971342A (en) * | 2015-07-13 | 2015-10-14 | 中国药科大学 | Pharmaceutical composition for resisting methicillin-resistant staphylococcus aureus (mrsa) |
| CN105031609B (en) * | 2015-07-22 | 2018-10-12 | 中国药科大学 | The disinfectant and its preparation and use of the Cbf-14 containing antibacterial peptide |
| CN105497872A (en) * | 2015-12-23 | 2016-04-20 | 中国药科大学 | Medical application of polypeptide Cbf-14 in resistance to fungal infection |
| CN106543271B (en) * | 2016-12-08 | 2019-07-30 | 中国药科大学 | Anti-drug resistance infects peptide C bf-14-2 and application thereof |
| CN113480627B (en) * | 2021-06-25 | 2022-06-14 | 华中农业大学 | Antibacterial peptide and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102702333A (en) * | 2012-05-29 | 2012-10-03 | 中国药科大学 | Drug-resistant pathogen infection resistant polypeptide and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102702333A (en) * | 2012-05-29 | 2012-10-03 | 中国药科大学 | Drug-resistant pathogen infection resistant polypeptide and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| Snake Cathelicidin from Bungarus fasciatus Is a Potent Peptide Antibiotics;Yipeng Wang et al;《PLoS ONE》;20080916;第13卷(第9期);参见全文，尤其是第3页右栏第一段Antimicrobial testing部分第1-2行；第6页左栏第1段倒数第1-3行 * |
| 金环蛇抗菌肽Cathelicidin-BF 的体外抗菌活性研究;周慧敏等;《药物生物技术》;20111231;第18卷(第4期);全文，尤其是是第317页左栏第2段第1-2行、最后一段第1-4行；第318-319页表1、2 * |
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Denomination of invention: Antibiotic resistant bacterial infection polypeptide cbf-14 and its application Effective date of registration: 20220719 Granted publication date: 20150923 Pledgee: China Construction Bank Corporation Nanjing Gulou sub branch Pledgor: NANJING YINGHAIYUE BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd. Registration number: Y2022980010791 |