CN100365018C - Antibiotic peptides and their prepn process and application - Google Patents
Antibiotic peptides and their prepn process and application Download PDFInfo
- Publication number
- CN100365018C CN100365018C CNB200310108033XA CN200310108033A CN100365018C CN 100365018 C CN100365018 C CN 100365018C CN B200310108033X A CNB200310108033X A CN B200310108033XA CN 200310108033 A CN200310108033 A CN 200310108033A CN 100365018 C CN100365018 C CN 100365018C
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- Prior art keywords
- antibacterial peptide
- lys
- preparation
- cell
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Abstract
The present invention provides a group of antibacterial peptides which have stronger bactericidal activity than natural antibacterial peptides and particularly have good killing effect on gram-negative bacteria. The present invention also discloses a method for preparing the antibacterial peptides. The antibacterial peptides can be synthesized by using a solid phase chemical method or obtained by gene engineering expression. The synthesized antibacterial peptides of the present invention can be used for preparing medicines for treating diseases caused by the infection of gram-positive bacteria, gram-negative bacteria, fungi or viruses.
Description
Technical field
The present invention relates to one group of antibacterial peptide, its preparation method and the application in preparation treatment bacterium, fungi, medicine for treating viral infections.
Background technology
Antibacterial peptide is the micromolecule polypeptide of a kind of biologically active of organism through inducing generation, generally is made up of 20-60 amino acid, and molecular weight is about 2000-7000D.Along with Medical Immunology and molecular biological developing rapidly, the research of antibacterial peptide more and more becomes the heat subject in biotechnology and the biomedicine field.Up to now, found to surpass more than 200 kind of antibacterial peptide on many animals (especially insect), plant, microorganism and human body, this class small peptide not only has the germicidal action of wide spectrum to bacterium, fungi, and virus, protozoon and cancer cells are also had attack function.Clinical trial also shows, maybe may cause under the situation of courses of infection the organism infection germ, and antibiotic Toplink is killed the germ of having invaded fast, and can stop germ continue infect.
Along with to the deepening continuously of antibacterial peptide primary structure and higher structure research, existing many investigators study the 26S Proteasome Structure and Function of these antibacterial peptides, find that alpha-helix degree and its fungicidal activity in the molecule are closely related under the hydrophobic environment of analogue membrane.Result of study shows that antibacterial peptide is to make bacterial cell membrane seepage and killing bacteria (Nakajima Y.etal., J.Biol.Chem, 262:1665-1669 by the integrity of destroying film in addition; Zasloff M.Nature, 2002,415:389-395).Therefore there is the people to attempt by α-Luo Xuanjiegou in the increase molecule or improve in the polypeptide to contain polypeptide (the Broth W.B.etal. that the amino acid whose ratio of positive charge is sought stronger anti-microbial activity, Antimicrobial Agents Chemotherapy, 2001,45:1894-1895; HongS.Y.etal., Peptides, 2001,22:1669-1674).
Summary of the invention
One of technical issues that need to address of the present invention provide one group of antibacterial peptide.
Two of the technical issues that need to address of the present invention provide the preparation method of one group of antibacterial peptide.
Three of the technical issues that need to address of the present invention are the application that disclose described antibacterial peptide.
Antibacterial peptide provided by the invention is to design synthetic on to the sequence of natural antibacterial peptide, basis of structural analysis, and their sequence is as follows:
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu Arg
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Leu Leu Lys Leu Leu Lys Leu Leu Arg
Lys Trp Lys Leu Phe Lys Lys Ile Pro Lys Val Arg Ala Lys Ala Lys Thr Arg
Lys Trp Lys Leu Phe Lys Lys Ile Pro Val Gly Arg Val His Arg Leu Leu Arg Lys
Lys Trp Lys Leu Phe Lys Lys Ile Pro Lys Gly Arg Gly Lys Gln Gly Gly Lys
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Val Arg Ala Lys Ala Lys Thr Arg
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg Gly Lys Gln Gly Gly Lys Val Arg
Lys Trp Lys Leu Phe Lys Lys Ile Pro Gly Arg Gly Lys Gln Gly Gly Lys Val Arg
One group of antibacterial peptide provided by the invention, its preparation method can be a mechanochemical method, also the encoding gene of antibacterial peptide can be cloned on the carrier, expresses the back then and obtain in host cell.Wherein expression vector can be a kind of in plasmid or the virus; host cell can be a prokaryotic cell prokaryocyte; comprise intestinal bacteria, subtilis etc., host cell can be eukaryotic cell also, comprise yeast cell, vegetable cell, insect cell and mammalian cell etc.The antibacterial peptide of preparation can be identified by mass spectrum.
In order to further investigate the structure-function relationship of this quasi-biology active antibacterial peptide of the present invention, the Pioneer Peptide synthesizer that utilizes U.S. application system biotech firm to produce prepares one group of polypeptide, to study.Narrate as an example with the GKIII-1 with following amino acid sequences and the GKIII-2 antibacterial peptide of preparation below:
GKIII-1：
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Leu Leu Lys Lys Leu Leu Lys
Lys Leu Leu Arg
GKIII-2：
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Leu Leu Lys Leu Leu Lys Leu
Leu Arg
Utilize 96 well plate method to detect fungicidal activity (the In Yup Park etc of polypeptide; FEBS Letters; 437 (1998) 258-262) with synthetic natural antibacterial peptide GramicidinS in advance, nisin, magaininII is contrast, carries out fungicidal activity and detects.The result shows that the fungicidal activity of antibacterial peptide of the present invention is better than the fungicidal activity of described three kinds of natural antibacterial peptides.
Antibacterial peptide also might act on high organism and comprise human body cell in efficient sterilizing, because the mode of action of antibacterial peptide all is that perforation makes the death of cell generation seepage on cytolemma.So can make red corpuscle generation seepage as its virose standard whether antibacterial peptide, if antibiotic Toplink makes the oxyphorase generation seepage in the red corpuscle, just can be by detecting OD
490Value is determined toxic size.Therefore the present invention has also detected the hemolytic activity of antibacterial peptide to human erythrocyte, and experiment shows that antibacterial peptide hemolysis rate value is very low, confirms that the hemolytic toxicity of antibacterial peptide of the present invention is minimum.
In addition, again antibacterial peptide of the present invention is carried out acute toxicity test in the animal body, prove the antibacterial peptide free of toxic effects.Carry out the test of antibacterial peptide again, show that antibacterial peptide has significant inhibitory effect to infection of staphylococcus aureus small white mouse acute infection aureus with inhibition.
The invention provides one group of new artificial design synthetic antibacterial peptide.Can adopt mechanochemical method preparation easily or with the gene clone of encoding antimicrobial peptide to carrier, enter then and express the back in the host cell and obtain.This antibacterial peptide has the wide spectrum killing activity to Gram-negative bacteria, gram-positive microorganism, fungi, virus, and than natural antibacterial peptide stronger fungicidal activity is arranged, but the animal and plant cell is not had any toxic action.And the mouse test of streptococcus aureus acute infection fungicidal activity is shown that antibacterial peptide gives 0.25mg/kg dosage by antibacterial peptide, just can reach 100% sterilization inhibiting rate, just can reach 100% sterilization inhibiting rate and will give 4.0mg/kg dosage as the vancomycin of killing the streptococcus aureus specifics, show that antibacterial peptide of the present invention has very significant sterilization effect to the streptococcus aureus acute infection, so antibacterial peptide of the present invention can be applicable to prepare the medicine of the disease that treatment gram-positive microorganism, Gram-negative bacteria or fungi infestation causes.
Description of drawings
Fig. 1 is the mass spectrum of antibacterial peptide GKIII-1.
Embodiment
The preparation of antibacterial peptide and separation and purification
Prepare GKIII-1 and GKIII-2 by above-mentioned sequence, prepare GramicidinS simultaneously, nisin and magaininII are in contrast.
The sequence of GramicidinS:
Val Orn Leu Phe Pro Val Orn Leu Phe Pro
The sequence of nisin:
Ile Thr Ser Ile Ser Leu Cys Thr Pro Gly Cys Lys Thr Gly Ala Leu Met Gly
Cys Asn Met Lys Thr Ala Thr Cys His Cys Ser Ile His Val Ser Lys
The sequence of magaininII:
Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe Val Gly Glu
Ile Met Asn Ser
Present embodiment adopts mechanochemical method synthetic, the Pioneer Peptide synthesizer that used instrument is produced for u.s.a. applied biosystem company.The synthetic polypeptide is used reverse column purification after the TFA of excessive concentrations shears, the polypeptide behind the purifying is identified by mass spectrum.Concrete testing sequence is as follows:
1, the preparation of antibacterial peptide (GKIII-1 with preparation 0.1mmol amount is an example)
Below all the preparation antibacterial peptides reagent all purchase in u.s.a. applied biosystem company.
The GKIII-1 peptide sequence of preparation is
N-Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu
Leu Arg-C
Preparation holds the N end to carry out one by one from C, is controlled automatically by synthesizer.At first weighing 0.1mmol's combines the resin that first amino acid is Arg (purchasing in u.s.a. applied biosystem company); the dress post; use 20% piperidines dimethyl formamide solution deprotection again; dimethyl formamide cleans; the total free aminoacids of 9-tablet held before the breast by officials methoxycarbonyl (Fmoc) protection is dissolved in carbodiimide (DCC); hydroxybenzotriazole (HOBt)/diisopropyl ethyl amine (DIPEA); solution after the dissolving is post cocycle coupled reaction 30 minutes, dimethyl formamide clean repeat above deprotection to the coupled reaction step up to preparation end (the concrete operations step is seen pioneer Peptide synthesizer operational guidance).
Polypeptide after the preparation is sheared through following steps:
Take off reacted resin, add Type B and shear liquid (88% trifluoroacetic acid, 5% phenol, 5% water, 2% tri isopropyl silane), room temperature reaction 2 hours filters, the precooling anhydrous diethyl ether that adds 10 times of volumes in the filtrate, 4000 rev/mins centrifugal 10 minutes, collecting precipitation and drying at room temperature.
2, antibacterial peptide purifying
The a certain amount of dried polypeptide of weighing is dissolved in 0.1% trifluoroacetic acid, and sample preparation is after elution peak is collected in oppositely post separation (elutriant is 0.1% trifluoroacetic acid that contains 80% second cyanogen).
3, the evaluation of antibacterial peptide
As shown in Figure 1, the antibacterial peptide GKIII-1 of preparation is through mass spectroscopy, and the molecular weight that GKIII-1 shows in mass spectrum is calculated as follows: the molecular weight that 932.2 * 3=2796.6,2796.6-3=2793.6 calculate GKIII-1 is 2793.6.The theoretical value that is calculated by peptide sequence is 2793.7.The polypeptide of proof preparation is the GKIII-1 antibacterial peptide of design.The antibacterial peptide product of accreditation is standby.
Antibacterial peptide GKIII-2, natural antibacterial peptide GramicidinS, nisin and magaininII can adopt the preparation method's preparation that is similar to the GKIII-1 antibacterial peptide.
The fungicidal activity of antibacterial peptide detects
Employed various bacterial strains are purchased in Chinese biological goods calibrating institute in following examples.
Adopt 96 well plate method that the fungicidal activity of antibacterial peptide is detected, and with mechanochemical method synthetic antibacterial peptide GramicidinS, nisin and magaininII in contrast, with estimate among the present invention as an example three antibacterial peptide GKIII-1 and the fungicidal activity of GKIII-2.
Measure the fungicidal activity of antibacterial peptide according to the following steps:
The bacterial classification recovery, the 37 ℃ of overnight incubation in inoculation inclined-plane are chosen bacterium in common LB substratum, 37 ℃ of overnight incubation, it is 10 that dilution bacterium liquid makes bacteria concentration
4-10
5CFU/ml is inoculated in 96 orifice plates by every hole 100ul bacterium liquid, after polypeptide is diluted with certain proportion, adds 10ul in every hole, and 96 orifice plates are placed 37 ℃ of overnight incubation, and microplate reader detects OD620 value (In Yup Park etc; FEBS Letters; 437 (1998) 258-262).Detected result sees Table 1.
Growth concentration (the OD that contains the bacterium of antibacterial peptide
620) be minimal inhibitory concentration (minimal inhibitory concentration (MIC) is defined as the minimum concentration of remarkable bacteria growing inhibiting) with the ratio of the growth concentration of the bacterium that does not add antibacterial peptide greater than 90% o'clock antibacterial peptide concentration.
Five kinds of antibacterial peptides of table 1 are to the comparison of the anti-microbial activity minimal inhibitory concentration (MIC) of different bacterium
| Strain name | The minimal inhibitory concentration of several antibacterial peptides (ug/ml) | |||||
| GramicidinS | nisin | magaininII | GKIII-1 | GKIII-2 | ||
| Gram-positive microorganism | Streptococcus aureus CMCC26003 Bacillus subtilus DB430 bacillus pumilus CMCC63202 micrococcus lysodeikticus S1.634 micrococcus luteus CMCC28001 | 8 24 24 12 24 | 4 6 6 8 8 | 12 ＞100 48 24 48 | 4 4 8 2 4 | 8 4 4 2 4 |
| Gram-negative bacteria | Intestinal bacteria ATCC8099 Klebsiella pneumonia CMCC46117 moscow' paratyphi B CMCC50094 Pseudomonas aeruginosa CMCC10104 | ＞100 ＞100 48 ＞100 | ＞100 ＞100 ＞100 ＞100 | 48 24 48 ＞100 | 0.5 0.5 1 8 | 2 2 8 8 |
| Fungi | Candida albicans ATCC10231 cereuisiae fermentum ATCC9736 | ＞100 ＞100 | ＞100 ＞100 | ＞100 ＞100 | 2 4 | 4 8 |
In the last table the minimal inhibitory concentration value more little, then represent antibacterial ability strong more, as can be seen from the above table, the MIC of antibacterial peptide of the present invention compares GramicidinS, nisin and magaininII are all little, particularly to the effect of Gram-negative bacteria, the antibacterial ability of antibacterial peptide of the present invention is better than correlated two antibacterial peptides greatly.
Embodiment 3
External hemolytic activity detects
Present embodiment is used to detect antibacterial peptide whether human erythrocyte is had hemolytic activity, and with mechanochemical method synthetic antibacterial peptide GramicidinS, nisin and magaininII are in contrast.Use blood sample be taken at normal human blood.
The detection step of antibacterial peptide hemolytic activity is:
The release of fresh red blood cell suspension oxyphorase under 414nm of 4% detects.HRBC is through PBS (PBS:35mM phosphoric acid buffer/0.15MNaCl, PH7.0) washing, the 8% HRBC suspension of getting 100ul is in 96 orifice plates, add 100ul antibacterial peptide solution in every hole, 37 ℃ after one hour, centrifugal 5 minutes of 1500rpm shifts the 100ul supernatant in 96 new orifice plates, by the absorption under the microplate reader detection 414nm.Negative control PBS, positive control 0.1%TritonX-100.Detected result sees Table 2
Five kinds of antibacterial peptide hemolytic activities of table 2 detected result
| Antibacterial peptide concentration (ug/ml) | Hemolysis rate (%) | ||||
| GramicidinS | nisin | magaininII | GKIII-1 | GKIII-2 | |
| 12.5 25 50 100 200 | 0.6 3.2 25.5 100 100 | 0.3 1.5 10.5 54.3 80.5 | 0 0 0.8 3.5 11.2 | 0 0 0.5 2.3 9.8 | 0 0 0.9 2.3 9.5 |
| 500 | 100 | 100 | 40.0 | 31.2 | 20.0 |
The hemolysis rate value of antibacterial peptide is more little in the table 2, then represents the hemolytic toxicity of antibacterial peptide more little.
Embodiment 4
Acute toxicity test in the animal body
Present embodiment is in order to detecting the toxicity of antibacterial peptide to animal, and with mechanochemical method synthetic antibacterial peptide GramicidinS, nisin and magaininII measure the toxicity of antibacterial peptide GKIII-1 provided by the invention and GKIII-2 in contrast.
Adopt 60 of Kunming small white mouses, male and female half and half, body weight 33.5 ± 0.25g, antibacterial peptide press 1mg/kg dosage, observe animal toxic reaction under maximal dose through the intramuscular injection small white mouse in continuous once a day 7 days.Experimental result shows that animal via intramuscular injection antibacterial peptide is after 7 days, and no abnormal reaction, activity are normally.Observed through 7 days, 60 small white mouses all survive.Proof antibacterial peptide free of toxic effects.
Antibacterial peptide and vancomycin suppress the comparison of effect to mouse infection of staphylococcus aureus model
Use the streptococcus aureus acute infection model of Kunming small white mouse, experimental procedure is as follows:
Place the veal that contains 5% pig gastric mucoitin to inculcate in the meat soup streptococcus aureus CMCC26003, shaking culture is spent the night.Injection 10 in the mouse peritoneal of the about 32.6 ± 0.25g of body weight
6-10
7Survivaling cell has 3 mouse in each treatment group.Intravenous injection antibacterial peptide GKIII-1 (being dissolved in 0.1 milliliter of 5% dextrose) for injection, injection in 10 minutes.The subcutaneous injection vancomycin (but vancomycin is complete biological utilisation to the mouse subcutaneous administration, no matter subcutaneous give or vein has similar activity).
Table 3 antibacterial peptide GKIII-1 and vancomycin are to the restraining effect of streptococcus aureus acute infection small white mouse model
| Dosage (mg/kg) | Inhibiting rate (%) | |
| 0 0.125 0.25 0.5 1.0 2.0 4.0 8.0 | Antibacterial peptide GKIII-1 intravenous injection 0 40 100 100 100 | Vancomycin subcutaneous injection 0 40 80 100 100 |
As shown in table 3, intravenous injection gives 0.25mg/kg, antibiotic Toplink 100% protection infecting mouse.Vancomycin is just 100% effective under 4.0 milligrams/kg dosage.All untreated mice are all dead in less than 24 hours.
This experimental example shows that antibacterial peptide is highly effective to killing streptococcus aureus with the chmice acute infection model of the fatal bacterium dosage of height.
Sequence table
＜110〉Shanghai Hi-Tech United Biotechnology R ﹠ D Ltd
＜120〉one group of antibacterial peptide and its production and application
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<160>8
<170>Patent In Ver s ion2.1
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Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Leu Leu Lys
5 10 15
Lys Leu Leu Lys Lys Leu Leu Arg
20
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5 10 15
Leu Leu Lys Leu Leu Arg
20
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＜213〉artificial sequence
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Lys Trp Lys Leu Phe Lys Lys Ile Pro Lys Val Arg Ala Lys Ala
5 10 15
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Lys Trp Lys Leu Phe Lys Lys Ile Pro Val Gly Arg Val His Arg
5 10 15
Leu Leu Arg Lys
<210>5
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＜213〉artificial sequence
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Lys Trp Lys Leu Phe Lys Lys Ile Pro Lys Gly Arg Gly Lys Gln
5 10 15
Gly Gly Lys
<210>6
<211>20
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＜213〉artificial sequence
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Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Lys Val Arg Ala
5 10 15
Lys Ala Lys Thr Arg
20
<210>7
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＜213〉artificial sequence
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Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg Gly Lys Gln
5 10 15
Gly Gly Lys Val Arg
20
<210>8
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＜213〉artificial sequence
<220>
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<400>8
Lys Trp Lys Leu Phe Lys Lys Ile Pro Gly Arg Gly Lys Gln Gly
5 10 15
Gly Lys Val Arg
Claims (10)
1. an antibacterial peptide is characterized in that, its aminoacid sequence is shown in SEQ ID NO:1 in the sequence table.
2. the preparation method of antibacterial peptide according to claim 1 is characterized in that, described preparation method is synthetic for mechanochemical method.
3. the preparation method of antibacterial peptide according to claim 1 is characterized in that, described preparation method will encode the gene clone of described antibacterial peptide to carrier, enters then to express the back in the host cell and obtain described antibacterial peptide.
4. according to the preparation method of the described antibacterial peptide of claim 3, it is characterized in that the carrier of being addressed is a kind of in plasmid or the virus.
5. according to the preparation method of the described antibacterial peptide of claim 3, it is characterized in that the host cell of being addressed is a prokaryotic cell prokaryocyte.
6. according to the preparation method of the described antibacterial peptide of claim 5, it is characterized in that the prokaryotic cell prokaryocyte of being addressed comprises intestinal bacteria, subtilis.
7. according to the preparation method of the described antibacterial peptide of claim 3, it is characterized in that the host cell of being addressed is an eukaryotic cell.
8. according to the preparation method of the described antibacterial peptide of claim 7, it is characterized in that the eukaryotic cell of being addressed is yeast cell, vegetable cell, insect cell or mammalian cell.
9. application of antibacterial peptide according to claim 1 is characterized in that the application of described antibacterial peptide in the medicine of preparation treatment gram-positive microorganism, Gram-negative bacteria or fungi infestation.
10. application according to claim 9 is characterized in that described medicine comprises the antibacterial peptide in the claim 1, and is mixed with one or more pharmaceutically acceptable carrier and additives.
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| CN106519000B (en) * | 2016-11-25 | 2019-06-18 | 青岛农业大学 | A kind of antibacterial peptide TO2-24 and its application |
| CN107383175B (en) * | 2017-09-01 | 2019-07-05 | 遵义医学院 | A kind of antibacterial peptide VK-21 and its application |
| CN108003223B (en) * | 2017-12-04 | 2019-08-09 | 遵义医科大学 | A kind of antibacterial peptide FR-31 and its application |
| CN112724202B (en) * | 2021-02-08 | 2022-07-26 | 中国海洋大学 | Antibacterial peptide and application thereof |
| CN113527458B (en) * | 2021-08-31 | 2022-05-27 | 中国科学院南海海洋研究所 | Antibacterial peptide HeHamp II-2 (63-86) and application thereof |
| CN114195869B (en) * | 2021-12-22 | 2022-11-25 | 杭州吉越生物科技有限公司 | Peptide and preparation method thereof |
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| CN1365981A (en) * | 2001-01-19 | 2002-08-28 | 山东大学 | Antibacterial peptide gene of fly and its cloning process |
| CN1398897A (en) * | 2002-09-02 | 2003-02-26 | 上海高科联合生物技术研发有限公司 | Serial synthetic antibacterial peptide |
| CN1415203A (en) * | 2002-10-11 | 2003-05-07 | 上海高科联合生物技术研发有限公司 | A kind of disinfection preparation and disinfecting wet towel its method of preparation and application |
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| CN1365981A (en) * | 2001-01-19 | 2002-08-28 | 山东大学 | Antibacterial peptide gene of fly and its cloning process |
| CN1398897A (en) * | 2002-09-02 | 2003-02-26 | 上海高科联合生物技术研发有限公司 | Serial synthetic antibacterial peptide |
| CN1415203A (en) * | 2002-10-11 | 2003-05-07 | 上海高科联合生物技术研发有限公司 | A kind of disinfection preparation and disinfecting wet towel its method of preparation and application |
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