CN102924574A - 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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- CN102924574A CN102924574A CN2012104227513A CN201210422751A CN102924574A CN 102924574 A CN102924574 A CN 102924574A CN 2012104227513 A CN2012104227513 A CN 2012104227513A CN 201210422751 A CN201210422751 A CN 201210422751A CN 102924574 A CN102924574 A CN 102924574A
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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 the 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 were found, the treatment of directed toward bacteria infectious diseases had had basic improvement, and countless patients' life has been saved in antibiotic use, had prolonged human mean lifetime.But along with antibiotic extensive use or abuse, especially in developing country, generation and the diffusion of some drug-fast bacteriums have been caused, comprising the very strong pathogenic bacterium of some virulence, as staphylococcus and streptococcus pneumoniae etc.Therefore, the antibacterials of the safe and effective and difficult generation resistance character of searching become the direction of whole world scientist competition and effort.
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 be comprised of 12 to 100 amino acid, size does not wait to tens kDa from several kDa.Antibacterial peptide is extensively to exist as the effective defense molecule of body, identifies thousands of kinds of antibacterial peptides in microorganism, plant, insect, arthropods, Amphibians, Mammals even human body at present.Antibacterial peptide is different from the microbiotic that traditional microorganism (comprising bacterium, fungi and streptomycete etc.) produces at aspects such as synthesis mechanism, amino acid composition and mechanism of action.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.The antibacterial peptide Antibacterial Mechanism is complicated, but most theories thinks that all 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 at bacterial cell membrane, cause at last the bacterium content to leak and dead.Therefore, antibacterial peptide will be higher than traditional microbiotic far away on the speed of kill bacteria, and unlike the growth of microbiotic anti-bacteria under lower concentration, antibacterial peptide nearly all is 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, all have wide practical use in fields such as medical and health, agriculture production, foodstuffs industry.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 the organism, also can utilize the genetic engineering means restructuring to obtain antibacterial peptide, can directly utilize again at short notice synthetic a large amount of small molecules antibacterial peptides of chemosynthesis means.
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 there is cytotoxicity, can cause that maybe the aspects such as haemolysis have limited antibacterial peptide applying as antibacterials, therefore, seek molecular weight less, anti-microbial activity is stronger, especially can not exist the antibacterial peptide of haemolysis or cytotoxicity to be only the solution antibacterial peptide as the factor of the most critical of antibacterials popularization.In recent years, scientists also begins to be devoted to original natural antibacterial peptide is carried out structure of modification or redesign when seeking novel antimicrobial peptide, for example change some amino-acid residue or directly design as required the amino acid whose primary structure of antibacterial peptide, active higher, more targeted simultaneously to the antibacterial peptide of host cell toxicological harmless effect to obtaining.
Transformation to antibacterial peptide mainly concentrates on Cecropins, Magainins, Defensins and cathelicidin family, especially maximum with what the transformation of Cecropin class antibacterial peptide was studied, the novel antimicrobial peptide molecular designing is the basis mainly with such antibacterial peptide greatly also, 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 among the present invention in the preparation antimicrobial drug.
Beneficial effect of the present invention is:
Antibacterial peptide LZ1 among 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.
Description of drawings:
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, and above-mentioned experimental result is the mean value of 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.Above-mentioned experimental result is the mean value of 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; Above-mentioned experimental result is the mean value of 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 the 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 with 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, measures the aminoacid sequence structure with automatic Protein Sequencer.
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 two: 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, such as Fig. 1, concrete grammar is as follows:
Microbionation is in Luria-Bertani(LB) on the solid medium, be inverted in 37 ℃ of incubators and cultivate.After bacterium colony grows, be transferred in the LB liquid nutrient medium with transfering loop picking list bacterium colony, 37 ℃ of incubator concussions are cultured to logarithmic phase.Detect bacterium liquid OD600 at ultraviolet spectrophotometer, according to 1OD600=1 * 10
9CFU/ml is diluted to 2 * 10 with bacterium liquid with liquid LB substratum
5CFU/ml.In each hole of aseptic 96 orifice plates, add in advance 100 μ l LB liquid nutrient mediums, then in the first hole, add 100 μ l and be diluted to certain density antibacterial peptide sample through 0.22 μ m filtering with microporous membrane degerming, get 100 μ l behind the mixing and add the 2nd hole, doubling dilution successively, 100 μ l discard from the 12nd hole sucking-off, and so far two times of concentration gradient samples namely prepare.
Add in each hole and diluted good bacterium liquid 100 μ l, behind the mixing in 37 ℃ slowly concussion cultivate 16h, measure the absorbance value at 600nm place.The result calculates: 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, cultivates the substratum that uses and is brain heart infusion agar (brain heart infusion BHI), and other conditions are similar.
The 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 being comprised 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 just illustrates 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 that separates clinically also has good sterilization effect, and minimum concentration can reach the concentration of 1.17 μ g/ml.In addition, antibacterial peptide LZ1 is to the minimal inhibitory concentration of two strain propionibacterium acneses even reached 0.6 μ g/ml, illustrates that antibacterial peptide LZ1 is especially remarkable to gram-positive bacillus effect.
Embodiment three: the hemolytic activity experiment of antibacterial peptide LZ1:
Rabbit heart blood sampling or people's venous blood collection, institute is 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, transfer pH to 6.1,4 ℃ of preservations behind the autoclaving) place centrifuge tube in the mixing of 1:1 ratio, till the centrifugal 5min of 1000rpm, physiological saline wash and no longer take on a red color to supernatant liquor.The red corpuscle that above-mentioned washing is good adds normal saline dilution and becomes 10
7-10
8The suspension of concentration.37 ℃ of insulations of sample 30min of the good red blood cell suspension of above-mentioned dilution and the different concns that is dissolved in physiological saline, in the centrifugal 5min of 1000rpm, supernatant liquor is surveyed absorption value in 540nm again.Negative control uses physiological saline, and positive control uses Triton X-100.Hemolytic activity is directly proportional with the 540nm absorption value.
The result as shown in Figure 3, Figure 4.
By Fig. 3, Fig. 4 as can be known, even antibacterial peptide LZ1 can not cause human blood and rabbit blood generation haemolysis yet under the high density of 320 μ g/ml.
Embodiment four: 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, transfer pH value to 7.2) wash three times after, the tryptic digestion with 0.25% gets 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, wait cell attachment after, the sample that adds different concns, at 37 ℃, cultivated altogether under 5% the carbon dioxide conditions 24 hours, after cultivation finishes, the every hole of 96 porocyte culture plates adds 20 μ l 5mg/ml MTT solution (with the preparation of cell cultures PBS damping fluid), continue to cultivate 4h, with liquid in the liquid-transfering gun sucking-off hole, every hole adds 100 μ l DMSO, jog is 10 minutes under the room temperature, detects the photoabsorption of 490nm wavelength with microplate reader.
The 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. the purposes of each antibacterial peptide in the preparation antimicrobial drug according to claim 1-2.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104974228A (en) * | 2015-06-15 | 2015-10-14 | 四川合泰新光生物科技有限公司 | Small molecule polypeptide ZY4 and application thereof |
| CN107759664A (en) * | 2017-11-08 | 2018-03-06 | 中国科学院新疆理化技术研究所 | A kind of micromolecule polypeptide AKK10 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 |
| CN111184855A (en) * | 2020-02-19 | 2020-05-22 | 厦门大学 | Composition for treating acne |
| CN112724198A (en) * | 2019-10-28 | 2021-04-30 | 于荣敏 | Methicillin-resistant staphylococcus aureus-resistant antibacterial peptide and preparation method and application thereof |
| CN113896768A (en) * | 2021-11-01 | 2022-01-07 | 深圳市维琪医药研发有限公司 | Antibacterial peptides and their cosmetic or pharmaceutical compositions and uses |
| CN114681588A (en) * | 2022-02-17 | 2022-07-01 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
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| CN1683399A (en) * | 2005-02-23 | 2005-10-19 | 中国科学院昆明动物研究所 | Wasp antibacterial peptide and preparation method and application thereof |
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| CN1683399A (en) * | 2005-02-23 | 2005-10-19 | 中国科学院昆明动物研究所 | Wasp antibacterial peptide and preparation method and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104974228A (en) * | 2015-06-15 | 2015-10-14 | 四川合泰新光生物科技有限公司 | Small molecule polypeptide ZY4 and application thereof |
| WO2016201972A1 (en) * | 2015-06-15 | 2016-12-22 | 四川合泰新光生物科技有限公司 | Small-molecule polypeptide zy4 and application thereof |
| JP2018521041A (en) * | 2015-06-15 | 2018-08-02 | 四川合泰新光生物科技有限公司Ｓｉｃｈｕａｎ Ｈｅｔａｉ Ｓｙｎｌｉｇｈｔ Ｂｉｏｔｅｃｈ Ｌｔｄ | Low molecular polypeptide ZY4 and use thereof |
| US10208088B1 (en) | 2015-06-15 | 2019-02-19 | Sichuan Synlight Biotech Ltd. | Low molecular polypeptide ZY4 and applications thereof |
| 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 |
| CN107759664A (en) * | 2017-11-08 | 2018-03-06 | 中国科学院新疆理化技术研究所 | A kind of micromolecule polypeptide AKK10 and its application |
| CN112724198A (en) * | 2019-10-28 | 2021-04-30 | 于荣敏 | Methicillin-resistant staphylococcus aureus-resistant antibacterial peptide and preparation method and application thereof |
| CN111184855A (en) * | 2020-02-19 | 2020-05-22 | 厦门大学 | Composition for treating acne |
| CN113896768A (en) * | 2021-11-01 | 2022-01-07 | 深圳市维琪医药研发有限公司 | Antibacterial peptides and their cosmetic or pharmaceutical compositions and uses |
| CN114681588A (en) * | 2022-02-17 | 2022-07-01 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
| 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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