CN104817629A - Sequence of anti-bacterial peptide capable of being specially combined with hydroxylapatite crystal on tooth surface and application thereof - Google Patents
Sequence of anti-bacterial peptide capable of being specially combined with hydroxylapatite crystal on tooth surface and application thereof Download PDFInfo
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- CN104817629A CN104817629A CN201510225140.3A CN201510225140A CN104817629A CN 104817629 A CN104817629 A CN 104817629A CN 201510225140 A CN201510225140 A CN 201510225140A CN 104817629 A CN104817629 A CN 104817629A
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Abstract
The invention discloses a sequence of an anti-bacterial peptide capable of being specially combined with hydroxylapatite crystals on tooth surface and an application thereof. In the invention, a peptide fragment GGG is used as a connecting peptide for combining an broad-spectrum antibacterial peptide fragment (KSLW): KKVVFWVKFK (Seq ID No:2) with a hydroxylapatite specially combining peptide fragment (HBP7): NNHYLP (Seq ID No:3) to obtain a peptide sequence (HBAMP): NNHYLPGGGKKVVFWVKFK (Seq ID No:1), which has both an anti-bacterial effect and a teeth surface adhesion effect. The peptide not only maintains inhibiting and killing effects on various oral pathogenic bacteria, but also has a specific adhesion effect on the hydroxylapatite crystal which is the main component of enamel and dentin. The peptide can form an anti-bacterial surface layer on teeth surface to inhibit generation of dental plaque thereon, and has huge clinical application prospect in the fields of preventing and treating oral dental plaque related diseases, such as caries, periodontal diseases and the like.
Description
Technical field
The present invention relates to field of molecular microbiology, refer to particularly a kind of can with the antibacterial peptide of tooth surface hydroxyapatite crystal specific binding and application thereof.
Background technology
Many oral diseases, such as dental caries disease, periodontal disease, peri-implantitis etc., be by bacterial chronic infectious disease.Plaque is by some bacterium, as suis, Bacterium lacticum, actinomycetes etc., is formed, be considered to the main pathogenic causing multiple oral disease in dental surface field planting propagation.Effectively suppressing the propagation of pathogenic bacteria in oral cavity and plaque in the formation of dental surface, is prevention and the key of these oral diseases for the treatment of.
Many antiseptic-germicides, such as microbiotic, Tubulicid, phenolic compound etc., can anti-bacteria and plaque very effectively.But these antiseptic-germicides often bring obvious side effect, such as resistance, gastrointestinal reaction, dental stain, calculus etc., and due to the dilution of saliva and Degradation, these antiseptic-germicides often can only provide the antibacterial effect of transient short-term in the oral cavity.Therefore find non-evident effect, and the antiseptic-germicide that can retain for a long time in oral environment is popular with the research become in recent years.
Antibacterial peptide (antimicrobial peptides) is considered to antibiotic ideal and substitutes articles for use, no matter is natural or synthetic, can shows good antibacterial effect and not have obvious side effect.However, because it is of short duration in intraoral action time, its clinical application is still restricted.In recent years along with deepening continuously to polypeptide structure and functional study, invented and manyly there is multi-functional polypeptide, such as STAMPs, and a kind ofly specificity can kill the antibacterial peptide of streptococcus mutans; The antibacterial peptide be also such as derived by group ammonia element, a kind of polypeptide that can be adsorbed onto titanium metal and show to suppress biofilm formation.There are now many synthetic peptide classes to attach to biomaterial surface, formed and there is bioactive top layer, and antibacterial peptide is applied to dental surface, form long acting antibiotic top layer, then have no report.
Summary of the invention
Technical problem to be solved by this invention is just to provide a kind of novel multifunctional antibacterial peptide, make it possess feature that is antibacterial, stable in saliva, specific adhesion simultaneously, namely the antibacterial peptide designed is while having the germ resistance of wide spectrum, can retain for a long time in saliva and not easily be degraded, and specific sticking can be produced to the main moiety of dental surface-hydroxyapatite, thus form anti-microbial activity top layer at dental surface, overcome the defect of conventional antimicrobial peptide in clinical oral application.
For solving the problems of the technologies described above, provided by the invention a kind of can with the antibacterial peptide of tooth surface hydroxyapatite crystal specific binding, it comprises broad spectrum antimicrobial peptide section (KSLW): KKVVFWVKFK (SEQ ID NO:2), hydroxyapatite specific binding peptides section (HBP7): NNHYLP (SEQ ID NO:3) and connection peptides: GGG.The primary structure of this antibacterial peptide is (HBAMP): NNHYLPGGGKKVVFWVKFK (SEQ IDNO:1).
1. the antibacterial peptide prepared by the present invention has good suppression and the effect of kill bacteria, can be used for the preparation of antiseptic-germicide.
2. the antibacterial peptide prepared by the present invention can with tooth surface (hydroxyapatite crystal) specific binding, thus formed at dental surface there is the top layer of anti-microbial activity.
3. the antibacterial peptide prepared by the present invention is not easily degraded in saliva, can in saliva long-acting performance antibacterial efficacy.
Beneficial effect of the present invention is:
Compared with the embodiment of existing oral antimicrobial agents, antibacterial peptide prepared by the present invention is stable in saliva, can with tooth surface (hydroxyapatite crystal) specific binding, anti-microbial activity top layer can be formed at dental surface, thus add antibacterial peptide in intraoral action time, restrained effectively the formation of dental surface bacterial plaque, at prevention and therapy Dental Plaque relative disease, as dental caries disease, periodontopathy etc., there is very high potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 is that fluorescently-labeled polypeptide HBAMP and KSLW is to the comparison diagram of the hydroxyapatite surface amount of sticking;
Fig. 2 is the antibacterial effect figure after hydroxyapatite surface adhesion polypeptide: the process of (Control) surface free; (TK) surface is through KSLW process; (TH) surface is through HBAMP process.(TOP) microbial film upper epidermis; (Bottom) microbial film layer.
Fig. 3 is the sterilization dynamic curve of 0.25mg/mL and 0.5mg/mL HBAMP;
Fig. 4 is the sterilization effect figure of HBAMP to bacterium in microbial film of different concns.(A) untreated microbial film; (B) with the HBAMP process microbial film of 10 minutes of 0.25mg/mL; (C) with the HBAMP process microbial film of 10 minutes of 0.5mg/mL; (D) microbial film of 10 minutes is processed with the CHX (Tubulicid) of 0.12%.
Fig. 5 is the mass spectrum of human saliva and polypeptide: the mass spectrum of (a) human saliva; B () polypeptide adds people's saliva after, the mass spectrum of 0 minute; C () polypeptide adds people's saliva after, the mass spectrum of 60 minutes; The degradation curve of (d) polypeptide in people's saliva.
Fig. 6 behaves the HBAMP of Gingival Fibroblasts through 0.25mg/mL and 0.5mg/mL, processes the propagation comparison diagram after 5,60,240 minutes.
Embodiment
In order to explain the present invention better, illustrate main contents of the present invention further below in conjunction with specific embodiment, but content of the present invention is not only confined to following examples.
The antibacterial peptide trust Shanghai Sheng Gong biotechnology company limited synthesis that following embodiment is used.
Because enamel exists autofluorescence effect, affect the microscopical imaging of confocal laser, and enamel forms primarily of hydroxyapatite, so we use hydroxyapatite flag to carry out experiment test to replace enamel sheet in the following embodiments, the hydroxyapatite flag (diameter 8mm, thick 2mm) used entrusts Sichuan University's National Biomedical material engineering research centre to prepare.
Embodiment 1 polypeptide stick experiment
1, the process of hydroxyapatite flag
Polish with 400,800, No. 1200 surfaces of sand paper to hydroxyapatite flag successively, then use ethanolic soln ultrasonic cleaning.
2, the fluorescent mark of polypeptide
The polypeptide of synthesis FITC mark: FITC-NNHYLPGGGKKVVFWVKFK (SEQ ID NO:1) and FITC-KKVVFWVKFK (SEQ ID NO:2).
3, to the detection that hydroxyapatite sticks
Fluorescently-labeled polypeptide ultrapure water is diluted to identical volumetric molar concentration: 92.60 μm of ol/L (for broad spectrum antimicrobial peptide KSLW is for 2 times of streptococcus mutans minimum inhibitory concentration), respectively get 100uL solution to drip on hydroxyapatite flag, make dissolution homogeneity covering surfaces.After processing 2,5,10,20 minutes respectively, clean 3 times with ultrapure water, allow rear laser confocal microscope observe.
4, result: as shown in Figure 1, under same molar ratio, HBAMP sooner, more can attach to hydroxyapatite flag surface than KSLW.At 5 minutes, HBAMP can cover the surface of 67.3%, and KSLW cover only 23.3%.
Antibacterial experiment after embodiment 2 surface polypeptide sticks
1, the surface treatment of hydroxyapatite flag
Polish with 400,800, No. 1200 surfaces of sand paper to hydroxyapatite flag successively, then use ethanolic soln ultrasonic cleaning.Polypeptide HBAMP and polypeptide KSLW ultrapure water are diluted to identical volumetric molar concentration: 92.60 μm of ol/L, respectively get 100ul solution and drip on hydroxyapatite flag, make dissolution homogeneity covering surfaces, process and be placed in 24 orifice plates respectively after 5 minutes.The hydroxyapatite flag processed is not had to be placed in 24 orifice plates as a control group yet.
2, antibacterial surface experiment
2mL is contained 10
6the BHIS (2% sucrose brain heart infusion agar) of CFU/mL streptococcus mutans is added in 24 orifice plates respectively.24 hollow plates are placed in 37 DEG C of incubator Anaerobic culturel after 12 hours, with ultrapure water rinsing hydroxyapatite flag 2 times, to wash away the flcating germ for sticking.Then use
bacLight
tMbacterial Viability Kit dyes about 20 minutes to the microbial film on hydroxyapatite flag surface under lucifuge condition.Laser confocal microscope is used successively to scan stained biological film.
3, result:
As shown in Figure 2: the shown in green fluorescence of the viable bacteria in microbial film, dead bacterium then shown in red fluorescence.Compared with control group, almost do not have (top layer viable bacteria average out to 97.4%) microbial film top layer by the hydroxyapatite surface of HBAMP and KSLW process; But with the surface of HBAMP process, relative to control group and KSLW treatment group (bottom viable bacteria average out to 58.3%), obvious lethal effect (bottom viable bacteria average out to 27.1%) is then created to the bacterium of microbial film bottom.Lethal effect can be played to the bacterium initially attaching to surface after surface adhesion HBAMP is described, thus affect biomembranous formation to a certain extent.
Embodiment 3 flcating germ sensitivity experiments
1, the cultivation of bacterium
Streptococcus mutans (Streptococcus mutans, ATCC 25175), Streptococcus sanguis (Streptococcus sanguinis, ATCC 10556), with actinomyces viscosus (Actinomycesviscosus, ATCC 19246) use BHI (brain heart infusion agar) to cultivate, Lactobacterium acidophilum (Lactobacillus acidophilus, ATCC 4356) uses MRS substratum (lactic-acid-bacterium substratum) to cultivate.All bacteriums are all at anaerobic environment (80%N
2, 10%H
2and 10%CO
2), cultivate at 37 DEG C.10 are diluted at the exponential phase substratum of bacterial growth
6cFU/mL is for subsequent use.
2, the gradient dilution of polypeptide
In 96 holes, use substratum to carry out gradient dilution to HBAMP (SEQ ID NO:1), make every hole have the solution of 200 μ L, the concentration range of polypeptide is from 2mg/mL to 0.03125mg/mL.And the CHX of 0.12% is as positive controls, substratum is as orifice plate blank group.
3, flcating germ sensitivity experiments
20 μ L are contained 10
6the bacterium liquid of CFU/mL is added in each hole, and mixing is placed in anaerobism thermostat container and cultivates 24 hours.With visual inspection, in hole, solution is clarified, without the polypeptide minimum concentration corresponding to obvious bacterial growth, is the MIC (minimum inhibitory concentration) of tested bacterium.Settled solution is got and is applied in right amount on solid medium, do not have bacterium colony to form corresponding polypeptide minimum concentration, be the MBC (minimum bactericidal concentration) of tested bacterium.
4, result is as shown in table 1: (unit of MIC and MBC is mg/mL)
The sterilization kinetics inspection of embodiment 4 polypeptide
1, sterilization kinetic measurement
The streptococcus mutans substratum being in exponential phase of growth is diluted to 10
5cFU/mL. be then divided into 4 groups, first group adds HBAMP, and final concentration is 0.25mg/mL (MBC); Second group adds HBAMP, and final concentration is 0.5mg/mL; 3rd group adds CHX, and final concentration is 0.12%, is positive controls; 4th group adds substratum, as blank group.0,10,20,30,40,60,90 minute time, the bacterium liquid PBS buffer memory liquid taking out 10 μ l carries out serial dilution, and the diluent then getting 50 μ l is applied to BHI agar plate.Agar plate is placed in 37 DEG C of incubator Anaerobic culturel after 24 hours to enumeration.And when drawing corresponding m-viable bacteria curve to react sterilization speed.
2, result
As shown in Figure 3: the HBAMP of 0.25mg/mL can kill all bacteriums at about 40 minutes, the HBAMP of 0.5mg/mL then can kill all bacteriums at about 20 minutes, illustrated that HBAMP has stable and sterilization effect fast.
Embodiment 5 microbial film sensitivity experiments
1, the biomembranous formation of streptococcus mutans
Polish with 400,800, No. 1200 surfaces of sand paper to hydroxyapatite flag successively, ethanolic soln ultrasonic cleaning, as in 24 orifice plates after drying.The BHIS (2% sucrose brain heart infusion agar) 2mL being contained 106CFU/mL streptococcus mutans is added in 24 orifice plates respectively.24 hollow plates are placed in 37 DEG C of incubator Anaerobic culturel after 12 hours, with ultrapure water rinsing hydroxyapatite flag 2 times, to wash away the flcating germ for sticking.
2, microbial film sensitivity experiments
The preparation HBAMP of 0.25mg/mL and 0.5mg/mL and the CHX of 0.12%, gets 100uL respectively and drips in biofilm surface, process 10 minutes.Allow rear ultrapure water rinsing hydroxyapatite flag 1 time, then use
bacLight
tMbacterialViability Kit dyes about 20 minutes to the microbial film on hydroxyapatite flag surface under lucifuge condition.Laser confocal microscope is used successively to scan stained biological film.
3, result:
As shown in Figure 4, undressed microbial film viable bacteria has accounted for 93.4%, and only remains 3.8% through the 0.12%CHX process microbial film viable bacteria of 10 minutes.HBAMP is relevant to concentration on biomembranous impact, and 0.25mg/mL process is after 10 minutes, and viable bacteria account for 55.3%; 0.5mg/mL process is after 10 minutes, and viable bacteria only remains 35.0%.Although HBAMP is to the bacterial killer effect in microbial film not as good as CHX (may be perviousness not as good as CHX) in microbial film, it is at short notice on biomembranous impact or gratifying.
The stability test of embodiment 6 polypeptide in people's saliva
1, the collection of people's saliva
The collection of people's saliva is from 4 volunteers (2 male sex and 2 women).In the morning, under the prerequisite of oral cavity cleaning of not taking food, do not do, saliva collection is carried out.By the saliva of collecting with whizzer 2,000rpm centrifugal 20 minutes immediately, then use the membrane filtration supernatant liquor of 0.45mm, acquisition people saliva sample.
2, the test of polypeptide stability
Polypeptide HBAMP is added in people's saliva, makes the ultimate density of polypeptide be 0.5mg/mL.By the saliva containing polypeptide as in the thermostat container of 37 DEG C, 0,5,20,60 minute time, take out sample segment liquid chromatography (HPLC) and quantitative analysis is done to the HBAMP in saliva, measure its degradation rate in saliva.Do not add the saliva of polypeptide as blank group.
3, result:
As shown in Figure 5: the HBAMP of 0.5mg/mL adds saliva still has 80.5% maintenance complete after 60 minutes, illustrate that HBAMP has satisfactory stability in people's saliva.
The cytotoxicity test of embodiment 7 polypeptide
1, the cultivation of people's Gingival Fibroblasts (HGFs)
Gather healthy gingiva tissue, by the gingiva tissue block of collection with containing 15% foetal calf serum DMEM nutrient solution 37 DEG C, carry out original cuiture under 5%CO2 saturated humidity condition, adopt enzyme digestion and adherent method purifying HGFs repeatedly, get the 6th generation HGFs and test.
2, cytotoxicity test
Say that HGFs is inoculated in 96 orifice plates, in each hole, have cell 2 × 10
3.Cultivate after 48 hours, abandon original nutrient solution, and 96 holes are divided into 3 groups at random, first group of new substratum added containing 0.25mg/mL HBAMP, second group of new substratum added containing 0.5mg/mLHBAMP, the 3rd group is control group, only adds substratum and cultivates.Through the cultivation of 10,60,240 minutes, nutrient solution is abandoned, and add new nutrient solution, then cultivate 24 hours.The proliferative conditions of HGFs uses CCK-8 test kit, and is set as that the microplate reader of 450nm measures with wavelength.
3, result:
As shown in Figure 6, with the HBAMP of 0.25mg/mL and 0.5mg/mL, HGFs process is not almost affected its propagation for 10,60,240 minutes, show that HBAMP has good biocompatibility.
Other unspecified part is prior art.Although above-described embodiment is to invention has been detailed description; but it is only the present invention's part embodiment; instead of whole embodiment, people can also obtain other embodiments according to the present embodiment under without creative prerequisite, and these embodiments all belong to scope.
Claims (3)
1. one kind can with the antibacterial peptide of tooth surface hydroxyapatite crystal specific binding, its aminoacid sequence is as shown in SEQ ID NO:1: NNHYLPGGGKKVVFWVKFK, shown antibacterial peptide comprises peptide section GGG and broad spectrum antimicrobial peptide section KKVVFWVKFK, shown peptide section GGG is as connection peptides, and described broad spectrum antimicrobial peptide section KKVVFWVKFK will combine with hydroxyapatite specific binding peptides section: NNHYLP.
2. according to claim 1 can with an application for the antibacterial peptide of tooth surface hydroxyapatite crystal specific binding, described antibacterial peptide is for the preparation of antiseptic-germicide.
3. according to claim 1 can with an application for the antibacterial peptide of tooth surface hydroxyapatite crystal specific binding, it is characterized in that: described antibacterial peptide treatment and prevention oral disease in application.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114832155A (en) * | 2022-04-29 | 2022-08-02 | 大连理工大学 | Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity |
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| CN102286073A (en) * | 2011-06-27 | 2011-12-21 | 华中科技大学同济医学院附属同济医院 | Heptapeptide capable of axially and specifically bonding with surface of tooth enamel and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102286073A (en) * | 2011-06-27 | 2011-12-21 | 华中科技大学同济医学院附属同济医院 | Heptapeptide capable of axially and specifically bonding with surface of tooth enamel and use thereof |
Non-Patent Citations (2)
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| 李冀寅等: "抗菌肽对种植体周围炎主要致病菌生长抑制作用的研究", 《中国微生态学杂志》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114832155A (en) * | 2022-04-29 | 2022-08-02 | 大连理工大学 | Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity |
| CN114832155B (en) * | 2022-04-29 | 2022-11-15 | 大连理工大学 | Preparation and application of biomineralization-based hydroxyapatite with controllable hydrophilicity and hydrophobicity |
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