CN102382176A - Oral cavity cariogenic bacterium-resisting polypeptide Pm11 and preparation method thereof - Google Patents
Oral cavity cariogenic bacterium-resisting polypeptide Pm11 and preparation method thereof Download PDFInfo
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- CN102382176A CN102382176A CN2011103812341A CN201110381234A CN102382176A CN 102382176 A CN102382176 A CN 102382176A CN 2011103812341 A CN2011103812341 A CN 2011103812341A CN 201110381234 A CN201110381234 A CN 201110381234A CN 102382176 A CN102382176 A CN 102382176A
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Abstract
The invention belongs to the technical field of medication biology, and relates to an Fmoc (Fluorenylmethyloxycarbonyl chloride) chemical synthetic polypeptide, in particular to an oral cavity cariogenic bacterium-resisting polypeptide Pm11 and a preparation method thereof. The preparation method comprises the following steps of: searching for the active center of a natural polypeptide pleurocidin which has certain antimicrobial and bactericidal capabilities for common oral cavity cariogenic bacteria; and on the basis of determining the active center, performing deletion and replacement of amino acids on the natural polypeptide pleurocidin to form a novel antibacterial polypeptide having the bactericidal capability, wherein the sequence of the novel antibacterial polypeptide Pm11 is WFKFFKKFFKKWK; and the common oral cavity cariogenic bacteria mentioned in the preparation method refer to streptococcus mutans, streptococcus sobrinus and streptococcus sanguis. By adopting the polypeptide Pm11, the problems of over large length and relatively high synthesis cost of a novel antibacterial peptide are solved.
Description
Technical field
The invention belongs to the medical biotechnology field, relate to the Fmoc chemically synthesized polypeptide, i.e. a kind of polypeptide Pm11 and preparation method of anti-cariogenic bacteria, this polypeptide has the bacterium of anti-cariogenic bacteria ability, especially streptococcus.
Background technology
Though dropped into great amount of manpower and material resources, dental caries sick (dental caries) remain one of worldwide noninfectious three big diseases.The dental caries disease is in the internal and external environment of oral cavity under the multiple factor affecting, a kind of chronic progressive external destructive bacterial infection disease that hard tooth tissue takes place.Streptococcus mutans and streptococcus sobrinus are main cariogenic bacterias.Nineteen forty-six, McClure and Hewitt confirm that penicillium mould can suppress the sick generation of rat dental caries, dominant mechanism be suppress that bacteria cell wall is synthetic, the nucleic acid of inhibition or interfere with bacterial and proteinic metabolism and route of synthesis reach the antimicrobial purpose.Yet these traditional microbiotic lure that easily bacterium undergos mutation and produce the resistance of microbiotic into.Therefore, Chinese scholars is devoted to the microbiotic of development of new always, and polypeptide is one section amino acid with weak point of anti-microbial activity.
Fish have strong natural immune system, thereby can tackle a large amount of pathogenic micro-organisms.We before the linear cationic polypeptide Pleurocidin of verified α spiral (GWGSFFKKAAHVGKHVGKAALTHYL-NH
2) have the ability of anti-cariogenic bacteria, fungi, and the Streptococcus mutans microbial film is had certain restraining effect.But the length of pleurocidin is 25 amino acid, and long amino acid has improved synthetic cost and to the penetrating power of bacterial biof iotalm, and therefore the small peptide that makes new advances of exploitation is just more necessary.
Summary of the invention
The object of the present invention is to provide a kind of polypeptide Pm11 and preparation method of anti-cariogenic bacteria, long to overcome natural antibacterial peptide length, the synthetic relative problem of higher of cost.
The technical scheme that the present invention adopts is: a kind of polypeptide Pm11 of anti-cariogenic bacteria; Its preparation method is: through searching the active site of natural polypeptides pleurocidin; The common cariogenic bacteria in oral cavity there are certain antibiotic, sterilizing ability; On the basis of confirming the active site, it is carried out amino acid whose deletion and the novel antimicrobial polypeptide with sterilizing ability of replacement formation; Said novel antibacterial polypeptide Pm11, sequence is WFKFFKKFFKKWK.
The common cariogenic bacteria in oral cavity described in its preparation method is a Streptococcus mutans.
The common cariogenic bacteria in oral cavity described in its preparation method is a streptococcus sobrinus.
The common cariogenic bacteria in oral cavity described in its preparation method is a Streptococcus sanguis.
Polypeptide makes the bacterial cell membrane surface lose original proterties, becomes coarse even the hole of appearance is arranged, and through forming ionic channel at bacterium surface, causes the outflow of bacterium content, thereby has caused the death of bacterium.
The polypeptide Pm1 preparation method of anti-cariogenic bacteria is; It is characterized in that: through keeping, improve the α spinning behaviour; Natural polypeptides pleurocidin active site amino acid is deleted and replaced,, be built with the polypeptide of antibacterial ability through direct chemical Fmoc synthetic method.
Characteristics of the present invention are: find and synthesized novel antibiotic small peptide.This polypeptide causes disintegration, the death of bacterium through the principle that forms ionic channel at bacterium surface.Improve synthetic cost and utilising efficiency, thereby better be used for dental caries sick prevention and treatment.The present invention has constituted new antimicrobial polypeptide through deletion and replacement to natural polypeptides pleurocidin active site.This polypeptide all has certain antibacterial ability to the Streptococcus mutans in the oral cavity, Streptococcus sanguis, streptococcus sobrinus.Polypeptide is 8-32 μ g/ml to the minimal inhibitory concentration scope of bacterium, and the MBC scope is between the 8-64 μ g/ml.Polypeptide is littler than the length of natural polypeptides, and antibacterial, antibacterial ability is suitable.The antibacterial mechanisms of polypeptide of the present invention is: polypeptide makes bacterium surface lose original proterties, becomes coarse even the hole of appearance is arranged; Form ionic channel at bacterium surface, cause the outflow of bacterium content, thereby caused disintegration, the death of bacterium; Thereby ability with anti-cariogenic bacteria.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed description:
Fig. 1 is polypeptide Pm11 and the α screw structure software supposition figure of Pm14 among the present invention;
Fig. 2 is the electromicroscopic photograph that polypeptide is handled bacterium;
Fig. 3 is the result that polypeptide was handled 2 hours;
Fig. 4 is the result that polypeptide was handled 2 hours.
Embodiment
A kind of polypeptide Pm11 of anti-cariogenic bacteria; Its preparation method is: through searching the active site of natural polypeptides pleurocidin; The common cariogenic bacteria in oral cavity there are certain antibiotic, sterilizing ability; On the basis of confirming the active site, it is carried out amino acid whose deletion and the novel antimicrobial polypeptide with sterilizing ability of replacement formation; Said novel antibacterial polypeptide Pm11, sequence is WFKFFKKFFKKWK.
The common cariogenic bacteria in oral cavity described in its preparation method is a streptococcus sobrinus.
The common cariogenic bacteria in oral cavity described in its preparation method is a Streptococcus sanguis.
Polypeptide makes the bacterial cell membrane surface lose original proterties, becomes coarse even the hole of appearance is arranged, and through forming ionic channel at bacterium surface, causes the outflow of bacterium content, thereby has caused the death of bacterium.
The polypeptide preparation method of anti-cariogenic bacteria is; It is characterized in that: through keeping, improve the α spinning behaviour; Natural polypeptides pleurocidin active site amino acid is deleted and replaced,, be built with the polypeptide of antibacterial ability through direct chemical Fmoc synthetic method.
Embodiment:
Fig. 1 is the α screw structure software supposition figure of polypeptide Pm11 and Pm14.
Fig. 2 is the electromicroscopic photograph that polypeptide is handled bacterium.A, the normal variant suis; B, Pm11 handles Streptococcus mutans later; C, Pm14 handles Streptococcus mutans later; D, normal Streptococcus sanguis; E, Pm11 handles Streptococcus mutans later; F, Pm14 handles Streptococcus mutans later.Can see that the bacterium surface after polypeptide is handled changes, and vesicular or bacterium disintegration occur, the phenomenon of shrinkage.
1. the design of polypeptide is synthetic
1.1 the design of polypeptide
Polypeptide adopts the method in intercepting pleurocidin active site, deletion, the amino acid whose method design of replacement.The physical properties such as the table 1 of design back polypeptide.The parameter of α screw structure is seen table 1, and synoptic diagram is seen Fig. 2.
1.2 the Fmoc method is synthesized polypeptide.
The synthetic polypeptide of conventional Fmoc solid phase synthesis process.This compound method is comparatively ripe, reliable product quality, and synthetic product is through the HPLC purifying.Polypeptide purity all need greater than 90%.
2. the main cariogenic bacteria active testing in the external anti-oral cavity of polypeptide
2.1 bacterial isolates and culture condition
Bacterial strain: Streptococcus mutans (
Streptococcus mutans)UA 159; Streptococcus sanguis (
Streptococcus sanguinis) ATCC 10556; Streptococcus sobrinus (
Streptococcus sobrinus) ACTT 6715 (available from West China stomatological hospital centralab of Sichuan University).
Culture condition: 37 degree constant temperature, anaerobism is cultivated in the BHI substratum.
2.2 the mensuration of minimal inhibitory concentration (MIC) and MBC (MBC)
According to U.S. CLSI standard, use double dilution method to carry out the detection of MIC and MBC.Bacterium after the incubated overnight, is diluted to 10 in the BHI substratum
6CFU/ml is subsequent use.After using double dilution method to dilute, puts into polypeptide 96 orifice plates in 200 μ L/ holes.Add subsequent use bacterium liquid, challenge polypeptide ultimate density is that 0.125 μ g/ml is to 256 μ g/ml.Tri-distilled water adds in other holes as negative control.Whole 96 orifice plates are put into 37 ° of static cultivations of C anaerobism thermostat container 16 to 24 hours.The opacity of direct viewing substratum and confirm the MIC value with spectrophotometer (600nm).In clarifying hole, take out 100 μ l inoculums, bed board, 37 ° of C incubated overnight.The peptide concentration that can not observe colony growth is a MBC.Each experiment repetition 3 times.
2.3 electron microscopic observation behind the polypeptide processing cariogenic bacteria
Use ESEM to observe the influence of polypeptide to the bacterial film surface.
S. mutansUA 159,
S. sanguinis10556 grow into logarithmic phase under the anaerobic condition in fresh culture.At room temperature, polypeptide handle bacterial suspension (100 μ l) after 24 hours centrifugal (4500 rpm) collect the bacterium liquid precipitate.Bacterial precipitation uses PBS to clean 2 times, and acetonitrile gradient dehydration (50%, 70%, 80% and 90% handled 20 minutes, and 100% handles 20 minutes 2 times) is fixed and used to the LUTARALDEHYDE of use 2.5%.Use drying machine (ES-2030, Hitachi, Japan) lyophilize then, metal spraying (Ion Sputter E-1045, Hitachi, Japan).Untreated bacterium is as negative control, and other steps are similar.Sample behind the metal spraying uses awkward silence at a meeting emission electron microscopic observation (S-4800, Hitachi, Japan).
2.4 the mensuration of killing curve
In order to detect the short period of time sterilizing ability of polypeptide, we kill the experiment of curve the time of having carried out.Streptococcus mutans, streptococcus sobrinus and Streptococcus sanguis grow into logarithmic phase in the BHI substratum, be diluted to 10 then
5CFU/ml.Under the anaerobic culture condition, use the method for killing curve detection time that the sterilizing ability of this polypeptide is estimated.The concentration of using is the MIC value of 8 times of each polypeptide.Concrete experimental procedure is following: after using polypeptide processing Streptococcus mutans; Putting into 490 μ l substratum the bacterial suspension of each time point (0,2,5,10,20,30,60,120 minute) sucking-off 10 μ l dilutes; The concentration of polypeptide is reduced under the bacteriocidal concentration, and remain on it is stopped growing.According to different bacterial concentrations, 20-500 μ l dilutes back bacterium liquid bed board after the concussion, and 37 degree anaerobism are counted after cultivating.
3. experimental result proves, its beneficial effect is following:
3.1 MIC and MBC detected value show that these several peptide species all have certain antibacterial ability to cariogenic bacteria, see table 2.
3.2 Electronic Speculum result shows: the surface of normal bacteria is smooth relatively, structural integrity, bacterium size, shape consistent (A, Streptococcus mutans; B, Streptococcus sanguis).After polypeptide Pm11, Pm14 processing, the cytolemma of Streptococcus mutans and Streptococcus sanguis receives the influence of polypeptide.Antimicrobial polypeptide makes bacterium surface lose original proterties, becomes coarse even the hole of appearance (among B, the E shown in the white arrow) is arranged.Explain that this polypeptide is through forming ionic channel at bacterium surface, cause the outflow of bacterium content, thereby caused the death of bacterium, seeing Fig. 2.
3.3 the result of killing curve shows: polypeptide is handled 2 hours result like Fig. 3, shown in 4.Pm1, Pm4, Pm8, Pm11 and Pm14 have well fragmentation effect fast for Streptococcus mutans.Wherein Pm1 and Pm4 just can kill Streptococcus mutans fully in 60 minutes.Pm14 can kill Streptococcus mutans fully in 2 hours.Pm8 and Pm11 even can in 20-30 minute, kill fully.
Sequence table
SEQUENCE LISTING
< 110>Ni, dragon is emerging
<120> PM11
<130> PEPTIDES
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 13
<212> PRT
<213> Pleuronectes americanus
<400> 1
Trp Phe Lys Phe Phe Lys Lys Phe Phe Lys Lys Trp Lys
1 5 10
Claims (6)
1. the polypeptide Pm11 of an anti-cariogenic bacteria; Its preparation method is: through searching the active site of natural polypeptides pleurocidin; The common cariogenic bacteria in oral cavity there are certain antibiotic, sterilizing ability; On the basis of confirming the active site, it is carried out amino acid whose deletion and the novel antimicrobial polypeptide with sterilizing ability of replacement formation; Said novel antibacterial polypeptide Pm11, sequence is WFKFFKKFFKKWK.
2. the polypeptide Pm11 of anti-cariogenic bacteria according to claim 1, the common cariogenic bacteria in oral cavity described in its preparation method is a Streptococcus mutans.
3. the polypeptide Pm11 of anti-cariogenic bacteria according to claim 1, the common cariogenic bacteria in oral cavity described in its preparation method is a streptococcus sobrinus.
4. the polypeptide Pm11 of anti-cariogenic bacteria according to claim 1, the common cariogenic bacteria in oral cavity described in its preparation method is a Streptococcus sanguis.
5. the polypeptide Pm11 of anti-cariogenic bacteria according to claim 1; It is characterized in that: polypeptide makes the bacterial cell membrane surface lose original proterties; Become coarse even the hole of appearance is arranged; Through forming ionic channel, cause the outflow of bacterium content, thereby caused the death of bacterium at bacterium surface.
6. the polypeptide Pm11 preparation method of anti-cariogenic bacteria according to claim 1 is; It is characterized in that: through keeping, improve the α spinning behaviour; Natural polypeptides pleurocidin active site amino acid is deleted and replaced; Through direct chemical Fmoc synthetic method, be built with the polypeptide of antibacterial ability.
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| CN 201110381234 CN102382176B (en) | 2011-11-26 | 2011-11-26 | Oral cavity cariogenic bacterium-resisting polypeptide Pm11 and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961216A (en) * | 2021-03-01 | 2021-06-15 | 温州医科大学附属口腔医院 | Streptococcus mutans specific targeting antibacterial peptide and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1678632A (en) * | 2002-08-22 | 2005-10-05 | 加拿大国家研究所 | A genomic approach to identification of novel broad-spectrum antimicrobial peptides from bony fish |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1678632A (en) * | 2002-08-22 | 2005-10-05 | 加拿大国家研究所 | A genomic approach to identification of novel broad-spectrum antimicrobial peptides from bony fish |
Non-Patent Citations (2)
| Title |
|---|
| RUI TAO等: "Antimicrobial and antibiofilm activity of pleurocidin against cariogenic microorganisms", 《PEPTIDES》 * |
| YUMIKA FUKUOKA等: "Structure-activity relationship of model peptides based on Pleurocidin, an antibacterial peptide", 《BULL.CHEM.SOC.JPN》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961216A (en) * | 2021-03-01 | 2021-06-15 | 温州医科大学附属口腔医院 | Streptococcus mutans specific targeting antibacterial peptide and application thereof |
| CN112961216B (en) * | 2021-03-01 | 2022-06-14 | 温州医科大学附属口腔医院 | Streptococcus mutans specific targeting antibacterial peptide and application thereof |
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