CN102924574B - Antibacterial peptide LZ1 and application of antibacterial peptide in preparation of antibacterial medicament - Google Patents
Antibacterial peptide LZ1 and application of antibacterial peptide in preparation of antibacterial medicament Download PDFInfo
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- CN102924574B CN102924574B CN2012104227513A CN201210422751A CN102924574B CN 102924574 B CN102924574 B CN 102924574B CN 2012104227513 A CN2012104227513 A CN 2012104227513A CN 201210422751 A CN201210422751 A CN 201210422751A CN 102924574 B CN102924574 B CN 102924574B
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Abstract
An antibacterial peptide LZ1 is an artificially designed and synthesized active polypeptide and contains 15 amino acid residues, the molecular weight is 2,228.77Da, and the isoelectric point is 12.05; and the full sequence of the antibacterial peptide is valine-lysine-arginine-tryptophan-lysine-lysine-tryptophan-tryptophan-arginine-lysine-tryptophan-lysine-lysine-tryptophan-valine-NH2. The antibacterial peptide LZ1 is small in molecular weight, strong in bactericidal effect and wide in antibacterial spectrum, almost does not have hemolytic activity or eukaryocyte toxicity, is a small molecular antibacterial peptide with application value, is convenient to artificially synthesize, and can be used for preparing an antibacterial medicament.
Description
Technical field:
The invention belongs to the polypeptide drugs technical field in biological chemistry, be specifically related to antibacterial peptide LZ1 and this antibacterial peptide purposes in the preparation antibacterials.
Background technology:
Since the microbiotic such as penicillin are found, the treatment of directed toward bacteria infectious diseases has had basic improvement, and countless patients' life has been saved in antibiotic use, has extended the mankind's mean lifetime.But along with antibiotic extensive use or abuse, especially in developing country, caused generation and the diffusion of some drug-fast bacteriums, the very strong pathogenic bacterium comprising some virulence, as staphylococcus and streptococcus pneumoniae etc.Therefore, the direction that the antibacterials of finding safe and effective and difficult generation resistance character become whole world scientist competition and make great efforts.
Antibacterial peptide be when biological be subject to microorganism and invade after body produce rapidly efficiently, the antibacterial peptide quasi-molecule of wide spectrum, participate in the immune response of body.Generally 12 to 100 amino acid, consist of, size does not wait to tens kDa from several kDa.Antibacterial peptide is extensively to exist as the effective defense molecule of body, from microorganism, plant, insect, arthropods, Amphibians, Mammals, even in human body, identifies thousands of kinds of antibacterial peptides at present.Antibacterial peptide is different from aspects such as synthesis mechanism, amino acid composition and mechanism of action the microbiotic that traditional microorganism (comprising bacterium, fungi and streptomycete etc.) produces.Antibacterial peptide not only has the anti-microbial activity of wide spectrum, and it also shows higher biological activity at antiviral, antimycotic, parasiticide and the aspect such as antitumor.Antibacterial peptide Antibacterial Mechanism complexity, but most theories all thinks that its mechanism relates to the effect of the cationic of antibacterial peptide and hydrophobicity and electronegative microorganism after birth, after the cytolemma of antibacterial peptide and bacterium contacts, cause that membrane permeability changes, or form the hole of cross-film on bacterial cell membrane, finally cause the bacterium content to leak and dead.Therefore, antibacterial peptide will be far away higher than traditional microbiotic on the speed of kill bacteria, and, unlike the growth of microbiotic anti-bacteria under lower concentration, antibacterial peptide is nearly all lethality to the effect of bacterium.
The discovery of antibacterial peptide and fast development provide huge resources bank for development of new antibacterial peptide class antibacterials, also, for the problem that solves the clinical drug-resistant bacterial strain provides great possibility, in fields such as medical and health, agriculture production, foodstuffs industry, all have wide practical use.Along with the discovery of people to the further understanding of antibacterial peptide Antibacterial Mechanism and new antibacterial peptide, people not only can obtain antibacterial peptide from direct separation and purification in organism, also can utilize the genetic engineering means restructuring to obtain antibacterial peptide, can directly utilize again chemosynthesis means synthetic a large amount of small molecules antibacterial peptides at short notice.
The antibacterial peptide of natural origin partly can be because of the larger immunogenicity that exists of molecular weight, anti-microbial activity is low, host cell is had to cytotoxicity, maybe can cause that the aspects such as haemolysis have limited antibacterial peptide applying as antibacterials, therefore, find molecular weight less, anti-microbial activity is stronger, especially can not exist the antibacterial peptide of haemolysis or cytotoxicity to be only the factor of antibacterial peptide as the most critical of antibacterials popularization that solve.In recent years, scientists also starts to be devoted to original natural antibacterial peptide is carried out to structure of modification or redesign when finding novel antimicrobial peptide, for example change some amino-acid residue or directly design as required the amino acid whose primary structure of antibacterial peptide, to obtain active higher, more targetedly simultaneously to the antibacterial peptide of host cell toxicological harmless effect.
Transformation to antibacterial peptide mainly concentrates on Cecropins, Magainins, Defensins and cathelicidin family, that especially with the transformation to Cecropin class antibacterial peptide, studies is maximum, the novel antimicrobial peptide molecular designing is also basis mainly with such antibacterial peptide greatly, the structure of such antibacterial peptide is very clear and definite, its length is 33～39 amino-acid residues, molecular weight is about 4kDa, peptide chain N end is rich in hydrophilic amino acid, particularly basic aminoacids Lys, Arg etc., the C end is rich in hydrophobic amino acid Leu, Ile, Val etc., and C-terminal is the phthalein amination.
Summary of the invention:
The object of the present invention is to provide a kind of new antibacterial peptide LZ1 with pharmaceutical use.
Another object of the present invention is to provide the purposes of above-mentioned antibacterial peptide aspect the preparation antibacterials.
Antibacterial peptide LZ1, described antibacterial peptide LZ1 is the synthetic active polypeptide of artificial design, comprise 15 amino-acid residues, molecular weight is 2228.77Da, iso-electric point is 12.05, and its total order is classified as: α-amino-isovaleric acid-Methionin-arginine-tryptophane-Methionin-Methionin-Trp-Trp-arginine-Methionin-tryptophane-Methionin-Methionin-tryptophane-α-amino-isovaleric acid-NH2.
Preferably, the C-of described complete sequence end amidation.
The purposes of antibacterial peptide in the present invention in preparing antimicrobial drug.
Beneficial effect of the present invention is:
Antibacterial peptide LZ1 in the present invention is synthetic, has that molecular weight is little, synthetic is convenient, the advantages such as germicidal action is strong, has a broad antifungal spectrum, and antibacterial peptide LZ1 also has the characteristics of extremely low hemolytic activity and eukaryotic cell toxicity in addition.
The accompanying drawing explanation:
Fig. 1 is the doubling dilution schematic diagram.
The anti-microbial activity of Fig. 2 antibacterial peptide LZ1, wherein LZ1 MIC: i.e. the minimal inhibitory concentration of antibacterial peptide LZ1, above result is three independent repeated experiments mean values.
Fig. 3 antibacterial peptide LZ1 hemolytic activity is analyzed (human blood), wherein LZ1: antibacterial peptide LZ1; NC: physiological saline, its application of sample volume is identical with sample sets; PC:Triton X-100, its application of sample volume is identical with sample sets, the mean value that above-mentioned experimental result is three independent experiments.
Fig. 4 antibacterial peptide LZ1 hemolytic activity is analyzed (rabbit blood), wherein LZ1: antibacterial peptide LZ1; NC: physiological saline, its application of sample volume is identical with sample sets; PC:Triton X-100, its application of sample volume is identical with sample sets.The mean value that above-mentioned experimental result is three independent experiments.
Fig. 5 antibacterial peptide LZ1 cytotoxicity analysis (embryonic kidney cell HEK), LZ1: antibacterial peptide LZ1; Contrast: physiological saline, its application of sample volume is identical with sample sets; The mean value that above-mentioned experimental result is three independent experiments.,
Embodiment:
The following examples can make the present invention of professional and technical personnel's comprehend, but do not limit the present invention in any way.
Embodiment 1: the preparation of antibacterial peptide LZ1
The chemical synthesis process of I, antibacterial peptide LZ1: according to the aminoacid sequence described in summary of the invention, with synthetic its complete sequence of automatic Peptide synthesizer (433A, Applied Biosystems), by HPLC reversed phase column chromatography desalting and purifying.
II, molecular weight determination adopt ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF).
The antibacterial peptide LZ1 of III, purifying identifies its purity by the high-efficient liquid phase chromatogram HPLC method, molecular weight determination adopts ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF), isoelectric focusing electrophoresis is measured iso-electric point, with automatic Protein Sequencer, measures the aminoacid sequence structure.
Antibacterial peptide LZ1 comprises altogether 15 amino-acid residues, and molecular weight is 2228.77Da, and iso-electric point is 12.05.The complete sequence of LZ1 antibacterial peptide is as follows: α-amino-isovaleric acid-Methionin-arginine-tryptophane-Methionin-Methionin-Trp-Trp-arginine-Methionin-tryptophane-Methionin-Methionin-tryptophane-α-amino-isovaleric acid-NH2 (C-holds amidation).
Embodiment bis-: the antibacterial experiment of antibacterial peptide LZ1:
Minimal inhibitory concentration (minimal inhibitory concentration, MIC): for can't detect the minimum sample concentration of bacterial growth.Adopt doubling dilution, as Fig. 1, concrete grammar is as follows:
Microbionation is in Luria-Bertani(LB) on solid medium, in 37 ℃ of incubators, be inverted and cultivate.After bacterium colony grows, with transfering loop picking list bacterium colony, be transferred in the LB liquid nutrient medium, 37 ℃ of incubator concussions are cultured to logarithmic phase.Detect bacterium liquid OD600 on ultraviolet spectrophotometer, according to 1OD600=1 * 10
9CFU/ml is diluted to 2 * 10 by bacterium liquid with liquid LB substratum
5CFU/ml.Add in advance 100 μ l LB liquid nutrient mediums in each hole of aseptic 96 orifice plates, then in the first hole, add 100 μ l to be diluted to the certain density antibacterial peptide sample through 0.22 μ m filtering with microporous membrane degerming, get 100 μ l after mixing and add the 2nd hole, doubling dilution successively, from the 12nd hole sucking-off 100 μ l, discard, so far two times of concentration gradient samples prepare.
Add the bacterium liquid 100 μ l that diluted in each hole, after mixing, in 37 ℃, slowly shake and cultivate 16h, measure the absorbance value at 600nm place.Result is calculated: get the hole that can't detect bacterial growth and with it the mean value of the adjacent hole sample concentration sum that bacterial growth is arranged as the sample minimal inhibitory concentration.
In addition, propionibacterium acnes is anerobe, and cultivating the substratum used is brain heart infusion agar (brain heart infusion BHI), and other conditions are similar.
Result as shown in Figure 2.
As shown in Figure 2, antibacterial peptide LZ1 to tested bacterial classification very strong lethal effect is arranged, as staphylococcus comprised to the minimum 1.17 μ g/ml that reach of MIC value of the Resistant strain of separating clinically, the highest also lower concentration of 4.7 μ g/ml, illustrate that antibacterial peptide LZ1 just can suppress staphylococcic growth under extremely low concentration.Simultaneously, antibacterial peptide LZ1 comprises that to several strains the Candida albicans separated clinically also has good sterilization effect, and minimum concentration can reach the concentration of 1.17 μ g/ml.In addition, antibacterial peptide LZ1 has even reached 0.6 μ g/ml to the minimal inhibitory concentration of two strain propionibacterium acneses, illustrates that antibacterial peptide LZ1 is especially remarkable to gram-positive bacillus effect.
Embodiment tri-: the hemolytic activity experiment of antibacterial peptide LZ1:
Rabbit heart blood sampling or people's venous blood collection, by gathered blood and A Shi liquid (Alsever Solution, 8.0g Trisodium Citrate, 0.55g citric acid, 20.5g glucose, 4.2g NaCl, add deionized water to 1L, adjust pH to 6.1,4 ℃ of preservations after autoclaving) mix and be placed in centrifuge tube in the 1:1 ratio, the centrifugal 5min of 1000rpm, till physiological saline washs and no longer takes on a red color to supernatant liquor.By above-mentioned washing, good red corpuscle adds normal saline dilution and becomes 10
7-10
8The suspension of concentration.37 ℃ of insulation 30min of sample of the good red blood cell suspension of above-mentioned dilution and the different concns that is dissolved in physiological saline, then, in the centrifugal 5min of 1000rpm, supernatant liquor is surveyed absorption value in 540nm.Negative control is used physiological saline, and positive control is used Triton X-100.Hemolytic activity is directly proportional to the 540nm absorption value.
Result as shown in Figure 3, Figure 4.
From Fig. 3, Fig. 4, even antibacterial peptide LZ1 can not cause human blood and rabbit blood generation haemolysis under the high density of 320 μ g/ml yet.
Embodiment tetra-: the cellulotoxic experiment of antibacterial peptide LZ1:
Human normal cell line embryonic kidney cell HEK 293 is in containing 10% foetal calf serum and two DMEM culture medium culturing that resists (each 100U/ml of penicillin and Streptomycin sulphate) to logarithmic phase, cell PBS(NaCl 8.0g, KCl 0.2g, Na2HPO
4H
2O 1.56g, KH
2PO
40.20g, add deionized water to 1000ml, adjust pH value to 7.2) wash three times after, with 0.25% tryptic digestion, get off, with fresh DMEM substratum suspension cell, the adjustment cell density is to 1*10
6Individual/ml, spread 96 orifice plates with the volume of every hole 200 μ l, after waiting cell attachment, the sample that adds different concns, at 37 ℃, under 5% carbon dioxide conditions, cultivate altogether 24 hours, after cultivating end, the 96 every holes of porocyte culture plate add 20 μ l 5mg/ml MTT solution (with the preparation of cell cultures PBS damping fluid), continue to cultivate 4h, with liquid in liquid-transfering gun sucking-off hole, every hole adds 100 μ l DMSO, under room temperature, jog is 10 minutes, detects the photoabsorption of 490nm wavelength by microplate reader.
Result as shown in Figure 5.
As shown in Figure 5, even there is very low approximately 10% cytotoxicity in antibacterial peptide LZ1 to human normal cell line embryonic kidney cell HEK 293 under the concentration of 200 μ g/ml.
Claims (3)
1. antibacterial peptide LZ1, it is characterized in that: described antibacterial peptide LZ1 is the synthetic active polypeptide of artificial design, comprise 15 amino-acid residues, molecular weight is 2228.77Da, iso-electric point is 12.05, and its total order is classified as: α-amino-isovaleric acid-Methionin-arginine-tryptophane-Methionin-Methionin-Trp-Trp-arginine-Methionin-tryptophane-Methionin-Methionin-tryptophane-α-amino-isovaleric acid-NH2.
2. a kind of antibacterial peptide LZ1 according to claim 1, is characterized in that: the C-end amidation of described complete sequence.
3. according to the purposes in any antimicrobial drug in preparation treatment intestinal bacteria, staphylococcus, Candida albicans, propionibacterium acnes, subtilis of any one antibacterial peptide in claim 1-2.
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| CN104623628A (en) * | 2015-01-13 | 2015-05-20 | 中国科学院昆明动物研究所 | Application of micro-molecule polypeptide LZ1 |
| CN104974228B (en) * | 2015-06-15 | 2017-10-17 | 四川合泰新光生物科技有限公司 | A kind of micromolecule polypeptide ZY4 and its application |
| CN105294838B (en) * | 2015-09-22 | 2018-07-03 | 徐州市玛泰生物科技有限公司 | A kind of antibacterial peptide and its application |
| CN107759664B (en) * | 2017-11-08 | 2021-02-12 | 中国科学院新疆理化技术研究所 | Small molecular polypeptide AKK10 and application thereof |
| CN112724198A (en) * | 2019-10-28 | 2021-04-30 | 于荣敏 | Methicillin-resistant staphylococcus aureus-resistant antibacterial peptide and preparation method and application thereof |
| CN111184855B (en) * | 2020-02-19 | 2021-08-31 | 厦门大学 | Composition for treating acne |
| CN113896768B (en) * | 2021-11-01 | 2022-10-14 | 深圳市维琪医药研发有限公司 | Antibacterial peptides and their cosmetic or pharmaceutical compositions and uses |
| CN114681588B (en) * | 2022-02-17 | 2023-06-06 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
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| CN102294019A (en) * | 2011-09-07 | 2011-12-28 | 中国科学院昆明动物研究所 | Application of cathelicidin-BF |
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