CN104744566A - Antibacterial peptide separated from skin secretions of wood frog and preparation method of antibacterial peptide - Google Patents
Antibacterial peptide separated from skin secretions of wood frog and preparation method of antibacterial peptide Download PDFInfo
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Abstract
The invention discloses an antibacterial peptide separated from skin secretions of a wood frog and a preparation method of the antibacterial peptide, and belongs to the technical fields of biomedicine and biology. The antibacterial peptide is characterized in that an amino acid sequence of the antibacterial peptide is SEQ ID No.1 (Sequence Identifier Number 1): Phe-Leu-Pro-Phe-Phe-Ala-Ala-Cys-Ala-Ile-Thr-Lys-Lys-Cys, and the molecular weight of the antibacterial peptide is 1556.8 Dalton. The preparation method of the antibacterial peptide comprises the steps of 1) extracting the skin secretions and 2) separating and purifying, wherein in the step 1), an alive wood frog is adopted, an ear-side gland and a back of the wood frog are stimulated with 10V stabilized voltage discontinuously, and the secretions are collected for later use; in the step 2), ultracentrifugation filtration is performed; a filtrate is collected; liquid chromatography separation is performed; a solution A is trifluoroacetic acid; a solution B is acetonitrile solution containing trifluoroacetic acid; a chromatographic column is balanced with the solution A before being used; after the column is balanced, a filtrate is added to the column; the loading quantity of a sample is 2mL/time; then the solution B is added; a peak with the best antibacterial activity is collected; and the filtrate is further separated by utilizing a reversed phase high performance liquid chromatography. The antibacterial peptide has the beneficial effects that the antibacterial peptide has better resistance to escherichia coli and staphylococcus, and also has an inhibiting effect on clinically separated MRSA (Methicillin-Resistant Staphylococcus Aureus). The antibacterial peptide has the inhibiting effect on MRSA, due to the particularity of an antibacterial mechanism of the antibacterial peptide, no drug resistance can be generated, and the antibacterial peptide can become a novel antibacterial agent resisting to clinical drug-resistant strain or even superbacteria.
Description
Technical field
The invention belongs to biomedicine and biological technical field.
Background technology
Antibacterial peptide, also known as peptide antibiotic or antimicrobial peptide, is distributed widely in virus, bacterium, insect and various vegeto-animal body, is to have the small molecule polypeptide eliminating vivo mutations cell, the infringement of opposing external microbe.Antibacterial peptide not only has the anti-microbial activity of wide spectrum, and has the advantages such as very high antimicrobial efficiency, and its maximum advantage not easily causes bacterial drug resistance, is the focus in current anti-pathogenic microorganism medicine development research.
Based on the vital role of antibacterial peptide, many scholars carry out searching and the preparation research of novel antimicrobial peptide.Current searching and prepare antibacterial peptide and have three kinds of methods: chemosynthesis, genetically engineered and screen natural antibacterial peptide class from biology.The antibacterial peptide of chemosynthesis often easily changes space conformation, thus loses activity.Although the antibacterial peptide output that genetically engineered obtains is high, also there is expression product less stable, and posttranslational modification may change the problems such as active.From abundance, cheap biology, screening is prepared antibacterial peptide and is had very high application prospect, therefore the favor of extremely investigator.
Amphibian animal build is various, of a great variety, widely distributed, has scientific research value and economic worth two major features concurrently.Owing to there being several functions biologically active substance that is complicated, that can resist the invasion and attack of extraneous injurious factor to be present in the skin of amphibian animal, therefore, the skin of Amphibians is important resource treasure-house, has very large potentiality to be exploited in natural antibacterial peptide screening.
Summary of the invention
The object of the invention is: provide a kind of antibacterial peptide be separated from wood frog skin secretion and preparation method thereof, it can isolate the strong antibacterial peptide of bacteriostatic activity from wood frog skin secretion.
Antibacterial peptide of the present invention and preparation method thereof is:
Wood frog skin antibacterial peptide, its aminoacid sequence is SEQ ID No.1:Phe-Leu-Pro-Phe-Phe-Ala-Ala-Cys-Ala-Ile-Thr-Lys-Lys-Cys, and molecular weight is 1556.8 dalton.
A disulfide linkage is formed between described antibacterial peptide aminoacid sequence the 8th and 14.
Its preparation method is
1) extract skin secretion: the live body wood frog adopting 8-10 cm long, cleans up with ultrapure water, be placed on after alcohol swab wiping on clean glass dish.Stimulate ear rear gland and the back of the frog with the multiple spot of 10 volts of voltage stabilizing discontinuity, to be seenly occur a large amount of juice to skin, with ultrapure water, juice is rinsed, these juices are collected, be placed on-70 DEG C for subsequent use, be wood frog skin secretion crude extract;
2) separation and purification: get skin secretion crude extract ultracentrifugation filtering and collecting filter liquid.Filtrate carries out liquid chromatography separation by C18 semipreparative column.Determined wavelength 217nm, solution A is the aqueous solution containing 0.025% trifluoroacetic acid, and solution B is the acetonitrile solution containing 0.025% trifluoroacetic acid; By solution A balance before chromatographic column uses, be added on pillar by filtrate after pillar balance, applied sample amount 2mL/ time, flow velocity 2mL/min, then adds solution B with gradient 10%-60%, continues 56min, collects the peak with best antimicrobial activity.RPLC is utilized to be separated further it.Adopt C18 analytical column, solution A 1 component is the aqueous solution containing 0.1% trifluoroacetic acid, and solution B 1 component is the acetonitrile solution containing 0.1% trifluoroacetic acid.With front, pillar solution A 1 balances, then with the flow velocity of 1mL/min, sample is added on pillar, solution B 1 is added with gradient 10%-40%, time length 25min, then add solution B 1 with gradient 40%-60%, time length 45min, collect the elutriant at the 4th peak, lyophilize is described antibacterial peptide.
The invention has the beneficial effects as follows: this Wood frog antibiotic peptides not only has stronger resistance to conditionality pathogenic bacterium intestinal bacteria and streptococcus aureus, and also inhibited for the methicillin-resistant staphylococcus aureus (MRSA) of clinical separation.Streptococcus aureus is one of clinical modal pathogenic bacterium, wherein particularly important with MRSA, cause the report of human infection and death more and more frequent by MRSA in recent years, virulence is more and more stronger, MRSA multidrug resistant sex chromosome mosaicism is also increasingly severe, illustrates MRSA resistance mechanism and development of new microbiotic is study hotspot always.Antibacterial peptide of the present invention is inhibited to MRSA, due to the singularity of its antibacterial mechanisms, can not produce resistance clinically, is expected to the novel antibacterial medicine that exploitation becomes anti-clinical drug-resistant bacterial strain and even " superbacteria ".
In addition, this Wood frog antibiotic peptides is inhibited to human breast cancer cell line Bcap-37 growth, and polypeptide of the present invention belongs to small molecular protein, compared with conventional anticancer drugs, it is easier to body and absorbs, and toxic side effect is little, can be used as and is preparing the application in antitumor drug.
Accompanying drawing explanation
Fig. 1: utilize preparative liquid chromatography to be separated the schematic diagram of wood frog skin secretion;
Fig. 2: the schematic diagram utilizing analysis mode liquid chromatography isolating active peak;
Fig. 3: antibacterial peptide is to the effect of intestinal bacteria ATCC25922;
Fig. 4: antibacterial peptide is to the effect of streptococcus aureus ATCC25923;
Fig. 5: antibacterial peptide is to the effect of resistant Staphylococcus aureus MRSA;
Fig. 6: antibacterial peptide is to the growth-inhibiting effect schematic diagram of human breast cancer cell line Bcap-37, and wherein ordinate zou is human breast cancer cell line Bcap-37 inhibiting rate, and X-coordinate is antibacterial peptide concentration.
Fig. 7: Wood frog antibiotic peptides minimal inhibitory concentration.
Embodiment
Embodiment 1:
The preparation of wood frog skin antibacterial peptide
1 extracts Rana temporaria chensinensis David Skin exudate: 8-10 centimetre wood frog 10 only cleans up with ultrapure water, ear rear gland and the back of the frog is stimulated with the multiple spot of 10 volts of voltage stabilizing discontinuity, juice is collected after a large amount of juice to be generated with ultrapure water, centrifugal 15 minutes of 12000 rpm, after getting supernatant cryoconcentration, with 10KDa super filter tube in 4 DEG C of ultracentrifugations, collect filtrate.
2 preparative liquid chromatographies: with the aqueous equilibrium containing 0.025% trifluoroacetic acid before C18 semipreparative column uses, get above-mentioned filtrate upper prop, applied sample amount 2mL/ time, flow velocity 2mL/min, gradient: 0 ~ 25min, 0 ~ 10%(v/v) containing the acetonitrile solution of 0.025% trifluoroacetic acid; 25 ~ 81min, 10% ~ 60%(v/v) containing the acetonitrile solution of 0.025% trifluoroacetic acid.Determined wavelength 217nm, elution curve as shown in Figure 1.Collect 36 elution peaks altogether, after the elutriant of each elution peak is concentrated, carry out anti-microbial activity detection.
3 high performance liquid chromatography: with the aqueous equilibrium containing 0.1% trifluoroacetic acid before C18 analytical column uses, get the above-mentioned elutriant upper prop with anti-microbial activity, applied sample amount 25 μ L/ time, flow velocity 1mL/min, gradient: 0 ~ 25min, 0 ~ 10%(v/v) containing the acetonitrile solution of 0.1% trifluoroacetic acid; 25 ~ 50min, 10% ~ 40% containing the acetonitrile solution of 0.1% trifluoroacetic acid; 50 ~ 95min, 40% ~ 60% containing the acetonitrile solution of 0.1% trifluoroacetic acid.Determined wavelength 217nm, elution curve as shown in Figure 2.Collect 5 elution peaks, only have peak 4 to have anti-microbial activity, after lyophilize, be antibacterial peptide of the present invention.
The order-checking of Wood frog antibiotic peptides
Wood frog skin antibacterial peptide Edman edman degradation Edman provided by the present invention measures-terminal amino acid order, use ABI company Procise 491 type protein sequence analyzer, automatic edman degradation method is adopted to check order, amino acid derivative adopts capillary liquid chromatography to detect, compare with the retention time of standard substance benzene thiohydantoin derivative (PTH-AA), obtain the primary structure of polypeptide.
Adopt MALDI-TOF mass-spectrometric technique qualification aminoacid sequence simultaneously.According to the antibacterial peptide molecular weight measured, determine the parent ion of second order ms, utilize second order ms measuring method to obtain second order ms figure, on second order ms figure, utilize Y ion and B ion to splice the contrary two sequences of sequence respectively simultaneously.
Polypeptide provided by the present invention is through above-mentioned two kinds of methods order-checking, and sequence is Phe-Leu-Pro-Phe-Phe-Ala-Ala-Cys-Ala-Ile-Thr-Lys-Lys-Cys, and forms disulfide linkage between the 8th and 14.By the aminoacid sequence of this antibacterial peptide by NCBI(http: //www. ncbi. nlm.nih.gov/) and Antimicrobial Peptide Database (http://aps.unmc.edu/AP/ main.htmL) two large database concepts carry out search comparison, do not find identical sequence.
Agar coating method detects antibacterial peptide bacteriostatic activity of the present invention
This example antibacterial peptide used is synthesized according to sequence SEQ ID No.1 by the biochemical company limited of Shanghai gill, and purity is 95%.Bacterial classification used is the methicillin-resistant staphylococcus aureus (MRSA) of reference culture intestinal bacteria (Escherichia coli ATCC25922), reference culture streptococcus aureus (Staphylococcus aureus ATCC25923) and clinical separation.Concrete operations are as follows:
Choose single colony inoculation in nutrient broth medium, (37 DEG C, 180rpm) are cultivated in jolting of spending the night, and be inoculated in 5mL broth culture by 50 μ L incubated overnight bacterium liquid, jolting cultivates 4 hours.Cultured bacteria suspension dilutes 1000 times, gets 100 μ L and adds MH slat chain conveyor primary surface, and coating evenly.Stick at the plate culture medium surface uniform of coated bacterium liquid the circular filter paper sheet that diameter is 5mm, filter paper adds 10 μ L testing samples.The flat-plate inverted adding sample is placed in 37 DEG C of constant incubators to cultivate 24 hours, use kind of calliper antibacterial circle diameter, unit is mm.Experimental result as shown in Figure 4.As shown in Figure 4, occur without inhibition zone around the filter paper that A group is added with antibacterial peptide sample, illustrate that antibacterial peptide of the present invention is to intestinal bacteria unrestraint effect.There is obvious inhibition zone around the filter paper that B group and C group are added with antibacterial peptide sample, show that antibacterial peptide of the present invention has obvious restraining effect to streptococcus aureus and resistant Staphylococcus aureus.
Liquid growth is adopted to suppress method to detect the suppression vigor of antibacterial peptide of the present invention to bacterium
This example antibacterial peptide used is synthesized according to sequence SEQ ID No.1 by the biochemical company limited of Shanghai gill, and purity is 95%.
Above-mentioned bacterial strains is chosen single colony inoculation in LB substratum, 37 DEG C of overnight incubation, dilution bacterium liquid makes bacteria concentration reach 2 × 10
5cFU/mL.Polypeptide 50 μ L after having diluted the bacterium liquid 50 μ L of concentration and dilution is by a certain percentage added in 96 well culture plates, mixes.After 96 orifice plates being placed in 37 DEG C of cultivation 18 h, microplate reader detects the light absorption value of 600 nm wavelength.The results are shown in Figure 7.
The minimum peptide concentration that can't detect bacterial growth is minimum inhibition concentration.Calculating principle: get the hole that just can't detect bacterial growth and be adjacent and have the hole of bacterial growth, the mean value of the two concentration is minimal inhibitory concentration (MIC is defined as the minimum concentration of remarkable bacteria growing inhibiting).
From detected result, the minimal inhibitory concentration of this antibacterial peptide to streptococcus aureus (ATCC25923) and methicillin-resistant staphylococcus aureus (MRSA) all reaches Gamma Magnitude, has extremely strong bacteriostatic activity.
Mtt assay detects the restraining effect that antibacterial peptide of the present invention grows breast cancer cell MCF-7.
The present embodiment antibacterial peptide used is synthesized according to sequence SEQ ID No.1 by the biochemical company limited of Shanghai gill, and purity is 95%.Concrete operations are as follows:
Breast cancer cell MCF-7 is incubated at containing 10% heat-inactivated fetal bovine serum, in the MEM substratum of Streptomycin sulphate and each 100U/mL of penicillin.
Collecting logarithmic phase cell, is 5 × 10 by cell concn dilution
4individual/mL.Plating cells, every hole 100 μ L, cell count is 5000/hole.Be placed in 37 DEG C of CO2gas incubator and cultivate 12h.
After continuing culturing cell 24 h with the 100 μ L substratum containing different concns antibacterial peptide, then under the condition of lucifuge, every hole adds 10 μ L MTT(5mg/mL), hatch 4 h.
Discard substratum, 100 μ L DMSO are added in 96 orifice plates, dissolving crystallized.37 DEG C, 80 rpm, shaking table hatches 20 min, fully dissolves to crystallization.
Microplate reader detects 535nm light absorption value.Inhibiting rate is calculated as follows:
Inhibiting rate %=(1-(sample light absorption value-blank absorbency)/(negative control light absorption value-blank absorbency)) × 100%
Result show, when antibacterial peptide concentration is 5 μ g/mL, be 6.3% to the inhibiting rate of MCF-7, during other concentration to the inhibiting rate of cancer cells be respectively 7.6%, 9.6%, 13.8%, 20.6%, 32.5%, 50.7%, 73.4% and 75.8%(as shown in Figure 5).
As can be seen from the above results, antibacterial peptide of the present invention has growth-inhibiting effect to human breast cancer cell line Bcap-37, and has dose-effect relationship.
Sequence table
< 110 > MILITARY VETERINARY INST ACADE
< 120 > antibacterial peptide be separated from wood frog skin secretion and preparation method thereof
〈160〉 1
〈170〉 PatentIn Version 2.1
〈210〉 1
〈211〉 14
〈212〉 PRT
< 213 > Rana temporaria chensinensis David (Rana chensinensis)
〈220〉
〈221〉 PEPTIDE，DISULFID
〈221〉 （1）…（14），（8）…（14）
〈223〉
〈400〉 Phe Leu Pro Phe Phe Ala Ala Cys Ala Ile Thr Lys Lys Cys
1 5 10
< 110 > MILITARY VETERINARY INST ACADE
< 120 > antibacterial peptide be separated from wood frog skin secretion and preparation method thereof
〈160〉 1
〈170〉 PatentIn Version 2.1
〈210〉 1
〈211〉 14
〈212〉 PRT
< 213 > Rana temporaria chensinensis David (Rana chensinensis)
〈220〉
〈221〉 PEPTIDE，DISULFID
〈221〉 （1）…（14），（8）…（14）
〈223〉
〈400〉 Phe Leu Pro Phe Phe Ala Ala Cys Ala Ile Thr Lys Lys Cys
1 5 10
Claims (3)
1. the antibacterial peptide be separated from wood frog skin secretion, is characterized in that: its aminoacid sequence is SEQ ID No.1:Phe-Leu-Pro-Phe-Phe-Ala-Ala-Cys-Ala-Ile-Thr-Lys-Lys-Cys, and molecular weight is 1556.8 dalton.
2. a preparation method for the antibacterial peptide be separated from wood frog skin secretion, its preparation method is:
1) extract skin secretion: the live body wood frog adopting 8-10 cm long, cleans up with ultrapure water, be placed on clean glass dish after alcohol swab wiping; Stimulate ear rear gland and the back of the frog with the multiple spot of 10 volts of voltage stabilizing discontinuity, to be seenly occur a large amount of juice to skin, with ultrapure water, juice is rinsed, these juices are collected, be placed on-70 DEG C for subsequent use, be wood frog skin secretion crude extract;
2) separation and purification: get skin secretion crude extract ultracentrifugation filtering and collecting filter liquid; Filtrate carries out liquid chromatography separation by C18 semipreparative column; Determined wavelength 217nm, solution A is the aqueous solution containing 0.025% trifluoroacetic acid, and solution B is the acetonitrile solution containing 0.025% trifluoroacetic acid; By solution A balance before chromatographic column uses, be added on pillar by filtrate after pillar balance, applied sample amount 2mL/ time, flow velocity 2mL/min, then adds solution B with gradient 10%-60%, continues 56min, collects the peak with best antimicrobial activity; RPLC is utilized to be separated further it; Adopt C18 analytical column, solution A 1 component is the aqueous solution containing 0.1% trifluoroacetic acid, and solution B 1 component is the acetonitrile solution containing 0.1% trifluoroacetic acid; With front, pillar solution A 1 balances, then with the flow velocity of 1mL/min, sample is added on pillar, solution B 1 is added with gradient 10%-40%, time length 25min, then add solution B 1 with gradient 40%-60%, time length 45min, collect the elutriant at the 4th peak, lyophilize is described antibacterial peptide.
3. a kind of antibacterial peptide be separated from wood frog skin secretion as claimed in claim, is characterized in that: form a disulfide linkage between antibacterial peptide aminoacid sequence the 8th and 14.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998489A (en) * | 2018-08-10 | 2018-12-14 | 通化县金汇生态农业发展有限公司 | A kind of preparation method of Wood frog antibiotic peptides |
| CN110183528A (en) * | 2019-06-05 | 2019-08-30 | 苏州立豪生物科技有限公司 | Antibacterial peptide and its purposes for pharmacy and beauty |
Citations (2)
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| CN101333247A (en) * | 2008-07-29 | 2008-12-31 | 辽宁大学 | Antimicrobial peptide separated from skin of Northeast China brown frog and applications in antibacterials |
| CN102786583A (en) * | 2012-08-13 | 2012-11-21 | 中国科学院烟台海岸带研究所 | Rana kunyuensis kunyuenin kunyuenin-3 as well as preparation method and application of rana kunyuensis kunyuenin kunyuenin-3 |
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2015
- 2015-04-08 CN CN201510162430.8A patent/CN104744566A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333247A (en) * | 2008-07-29 | 2008-12-31 | 辽宁大学 | Antimicrobial peptide separated from skin of Northeast China brown frog and applications in antibacterials |
| CN102786583A (en) * | 2012-08-13 | 2012-11-21 | 中国科学院烟台海岸带研究所 | Rana kunyuensis kunyuenin kunyuenin-3 as well as preparation method and application of rana kunyuensis kunyuenin kunyuenin-3 |
Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998489A (en) * | 2018-08-10 | 2018-12-14 | 通化县金汇生态农业发展有限公司 | A kind of preparation method of Wood frog antibiotic peptides |
| CN110183528A (en) * | 2019-06-05 | 2019-08-30 | 苏州立豪生物科技有限公司 | Antibacterial peptide and its purposes for pharmacy and beauty |
| CN110183528B (en) * | 2019-06-05 | 2020-06-16 | 广州市涵美化妆品有限公司 | Antibacterial peptide and its use in pharmacy and cosmetology |
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