CN104292307B - A kind of peptide molecule - Google Patents
A kind of peptide molecule Download PDFInfo
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- CN104292307B CN104292307B CN201410512200.5A CN201410512200A CN104292307B CN 104292307 B CN104292307 B CN 104292307B CN 201410512200 A CN201410512200 A CN 201410512200A CN 104292307 B CN104292307 B CN 104292307B
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Abstract
The invention discloses a kind of peptide molecule, the peptide molecule is included：(a) peptide molecule amino acid sequence：Amino acid sequence shown in SEQ ID No.1；(b) (a) limit peptide molecule in by missing, insert or replace one or several amino acid and with peptide molecule (a) described have identical biological function as peptide molecule derived from (a)；The peptide molecule molecular weight is 2138.68g/mol, and isoelectric point is 10.6；It can be applied in the material of preparation antibacterial and promotion Oesteoblast growth.The present invention is a kind of peptide molecule, with good antibacterial ability, can also promote Oesteoblast growth, its stability is strong, it is easy to combined with other compounds, with apparent application potential.
Description
Technical field
The present invention relates to biomedicine field, more particularly to a kind of peptide molecule.
Background technology
Over nearly 40 years, the development of canine implant technology is more and more faster, since the eighties, also carries out rapidly in China
Come, effect is notable, but also many problem urgent need to resolve.Tooth implant is to be implanted into human body by way of surgical operation to lack
In the upper mandibular at tooth position, after after the healing of its wound, the device for repairing artificial tooth is installed at an upper portion thereof, for planting body material
The requirement of material is especially high, need to be without any side effects to human body, therefore, and it is necessary to find a kind of suitable planting body material
's.
Pure titanium or titanium alloy micro-implant allows bone tissue to be deposited on its surface, is formed relatively in micro-implant-bone interface
Relatively firm Integrated implant, but easily come off and fracture when bearing larger orthodontic force, and bacterial plaque implant surface assemble with
Osseointegrated implants interface resistivity is low, and peptide molecule structure is made up of amino acid, the bio-compatible generally in human body
Property it is good, develop a kind of peptide molecule significant as the material of planting body.
The content of the invention
One of the object of the invention is to provide a kind of peptide molecule with antibacterial and promotion Oesteoblast growth；The present invention
The second purpose is to provide application of the peptide molecule in preparing antibacterial and promoting the material of Oesteoblast growth.
The technical scheme that the present invention is provided is：
A kind of peptide molecule, the peptide molecule is included：
(a) peptide molecule amino acid sequence：Amino acid sequence shown in SEQ ID No.1；
(b) (a) limit peptide molecule in by missing, insert or replace one or several amino acid and with (a) institute
State peptide molecule have identical biological function as peptide molecule derived from (a).
Preferably, the peptide molecule molecular weight is 2138.68g/mol.
Preferably, the peptide molecule isoelectric point is 10.6.
Preferably, it is preparing antibacterial and the application in the material for promoting Oesteoblast growth.The titanium surface of planting body
It can be pre-processed with connexon, and the amino acid containing-SH groups, such as cysteine can be introduced peptide molecule of the present invention
In N-terminal or C-terminal, with the functional group with reference to the connexon.Also can be with connexon to connecting peptides after titanium surface preparation
- NH in molecule2Group, titanium surface is connected to by peptide molecule, for example, with reference to connexon, peptide molecule of the present invention is in N-terminal
Containing cysteine to be easily introduced into by means of the interaction between connexon and cysteine in planting body.Separately
Outside, in order to which peptide molecule of the present invention is stably introduced into the titanium of planting body (Ti) surface, preferably by silane-connexon-peptide
Annexation these peptide molecules are introduced into implant surface.So the obtained titanium of processing can as tooth implant and
Bone planting body is used, due to its excellent anti-microbial property and promotion Oesteoblast growth function, for avoiding planting body bacterium sense
Dye and redemption patients ' lives are of great importance.
In addition, the present invention peptide molecule also can simple application in other material surfaces used in everyday, such as plastics, glass
With other metal surfaces, it can effectively prevent bacterium infection caused by everyday exposure.
The beneficial effects of the invention are as follows a kind of peptide molecule that the present invention is provided, peptide molecule of the invention, which has, to be promoted into
The ability of bone cell differentiation, while having good antibacterial ability, can effectively be sterilized for a variety of strains, and bactericidal effect
99% can be reached, wherein 99.97% can be reached for the sterilizing ability of candida albicans；It is thin that peptide molecule also possesses raising skeletonization
The ability of cytoactive；Pass through cytotoxicity analysis, it is known that the material non-toxic made using the present invention, available for manufacture planting body material
Material；The invention provides a kind of peptide molecule, its structure is more stablized, it is easy to combined with other compounds, can be with simple application
In various forms of materials.
The term definition that the present invention relates to
Unless otherwise defined, otherwise all technologies used herein and scientific terminology all have with it is of the art
Those of ordinary skill generally understands identical implication.Although can be used and described herein in the practice or test of the present invention
Similar or equivalent any method, device and material, but method for optimizing, device and material will now be described.
Term " amino acid " means to constitute the base unit of protein, assigns the specific molecular morphosis of protein, make
Its molecule has biochemical activity.Protein is bioactive molecule important in organism, includes the enzyme of catalysis metabolism, two
Or more than two chemistry of amino acids aggregate into peptide, the original segments of a protein are proteinogenous precursor, amino acid
Broadly refer to not only containing a basic amine group but also containing the organic compound of an acidic carboxypolymer, as described in its name
Like that.But general amino acid, then refer to the structural units for constituting protein.In living nature, the ammonia of native protein is constituted
There is base acid its specific design feature, i.e. its amino to be connected directly between on alpha -carbon atom, and this amino acid is referred to as alpha-amido
Acid.300 several amino acids, wherein 21 kinds of a-amino acid are had in nature.A-amino acid is the component point of peptide and protein
Son, is also to constitute one of basic masonry of life mansion.
Term " polypeptide " mean a-amino acid with peptide bond link together formed by compound, it be also protein hydrolysis
Intermediate product.Dipeptides is called by the compound of two amino acid molecular dehydrating condensations, also tripeptides, four are similarly analogized
Peptide, pentapeptide etc., generally by the compound of 10~100 amino acid molecular dehydrating condensations polypeptide, their molecular weight is less than
10000 dalton, can pass through pellicle, not precipitated by trichloroacetic acid and ammonium sulfate, also have document by 2~10 amino acid
The peptide of composition is referred to as oligopeptides (small-molecular peptides)；The peptide of 10~50 amino acid compositions is referred to as polypeptide；Amino acid more than 50
The peptide of composition is known as protein.
Term " missing " means that a normal chromosomal lost the gene in a fragment, this fragment also therewith after being broken
Lose, mean peptide molecule in the present invention in addition to for the amino acid sequence shown in SEQ ID No.1, in addition to polypeptide point
The peptide molecule of the new amino acid sequence for not influenceing its function of one or several amino acid is lost in sub- amino acid sequence.
Term " insertion " means to access in another segment DNA or in cloning vector section of DNA using gene manipulation techniques,
Mean peptide molecule in the present invention in addition to for the amino acid sequence shown in SEQ ID No.1, in addition to peptide molecule ammonia
The peptide molecule of the new amino acid sequence for not influenceing its function of one or several amino acid is inserted in base acid sequence.
Term " displacement " means a kind of gene recombined vector, general to be replaced certain section of gene in genome using homologous recombination
Change, and reach the purpose of gene knockout, mean peptide molecule except for the amino acid sequence shown in SEQ ID No.1 in the present invention
Outside row, in addition in peptide molecule amino acid sequence one or several amino acid by after other kinds of amino acid replacement not
Influence the peptide molecule of the new amino acid sequence of its function.
Term " isoelectric point " means in a certain pH solution, and amino acid or protein dissociation are into cation and anion
Trend or degree are equal, as hybrid ion, in electroneutral, and what now the pH of solution turned into the amino acid or protein waits electric
Point, biological amphiphatic molecule is the functional group also containing alkalescence containing acidity such as protein.The amino acid of constitutive protein matter may
Positively charged, negatively charged, neutral or this life is both sexes, their electric charge be added together be protein electricity
Lotus, the total electrical charge of protein is positive when pH value is less than isoelectric point, is negative during more than isoelectric point.
Brief description of the drawings
Fig. 1 is influence of the peptide molecule of the present invention to activity of osteoblast proliferation and ordinary culture medium to Gegenbaur's cell
The comparative result compares figure of proliferation activity influence；
Fig. 2 is for peptide molecule of the present invention to initial cell differentiation capability and ordinary culture medium to initial cell differentiation potency
The comparative result compares figure of power.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
Embodiment 1：
A kind of peptide molecule, amino acid sequence is as shown in SEQ ID No.1；
SEQ ID No.1：TYKKPVPIIY CNKKRGKC.
Embodiment 2：
The preparation of peptide molecule：
From amino acid-Wang resin as carrier (resin), resin is fully swelled with dichloromethane, dimethyl formyl is used
Amine is cleaned several times, and with the DBLK of debita spissitudo, Fmoc- blocking groups are deviate from, is cleaned several times, washed with dimethylformamide afterwards
DBLK is removed, suitable condensing agent BTA-N, N, N ', N '-tetramethylurea hexafluorophosphate and activator methyl is weighed
Second Fmoc- protected amino acid (Fmoc-Pro-OH) of quinoline and C-terminal is coupled, and is detected by ninhydrin detection method
Ensure that connection is more complete, cleaned with dimethylformamide several times, wash away the various residues of residual, activator and condensing agent, according to
Amino acid sequence is coupled, and after all amino acid connections are terminated, is sloughed last Fmoc- blocking groups, is used cutting liquid
Cracking, removes resin and amino acid protective group, obtains the crude product of peptide molecule, send mass spectrum to confirm molecular weight product
2138.68g/mol meet theoretical value.
Embodiment 3：
Antibacterial ability is tested：
First, experimental method
It is 1 × 10 that 1mL concentration is added in sterilizing test tubes6Individual/mL bacterium solution, adds 1mg polypeptides provided by the present invention
Molecule, after 37 DEG C are cultivated 24 hours, culture medium, which is collected, uses doubling dilution, and extension rate is that 10 times and spread plate method are detected
Viable count.
2nd, experimental result：
The sterilizing rate (%) of the peptide molecule of the present invention of table 1
| Bacterium | ATCC6538 | ATCC25922 | ATCC10231 | ATCC9372 |
| Sterilizing rate | 100% | 99.98% | 99.96% | 99.97% |
As shown in Table 1, peptide molecule provided by the present invention, is wished to staphylococcus aureus (ATCC6538), large intestine angstrom
Salmonella (ATCC25922), candida albicans (ATCC10231), Bacillus subtilis endophyticus (ATCC9372) all have very
Strong bactericidal properties, its sterilizing rate is up to more than 99%.
Embodiment 4：
Activity of osteoblast proliferation is tested：
First, experimental method
1mg peptide molecules of the present invention are placed in 24 orifice plates, 1mL density is 2 × 104Individual/mL mouse bone-forming cell hangs
Liquid is inoculated in 24 orifice plates, then cultivates 1,4 and 7 days.To after predetermined point of time, the 3- that 200uL concentration is 5mg/mL is added per hole
(4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides (MTT) and 800uL serum-frees are without phenol red improvement Du Shi Igers
Culture medium (DMEM), 37 DEG C be incubated 4 hours after inhale and abandon supernatant, add the crystallization of 1ml dimethyl sulfoxide (DMSO)s (DMSO) dissolving generation
Thing, takes three parts of 200uL lysates to go to 96 well culture plates, surveys its OD value (OD at 490nm with spectrophotometer per hole
Value).
2nd, experimental result
As shown in figure 1, mouse bone-forming cell is inoculated in ordinary culture medium and carried added with the present invention as seen from Figure 1
In the culture medium of the peptide molecule of confession, its OD value is in increased trend, as number of days increases, added with provided by the present invention
The mouse bone-forming cell OD values increase of peptide molecule is more notable, and OD values can reflect the activity of mouse bone-forming cell, so from
Fig. 1 is it is recognised that peptide molecule provided by the present invention can significantly improve mouse bone-forming cell proliferation activity.
Embodiment 5：
Initial cell differentiation capability is analyzed：
By the alkaline phosphatase for comparing the peptide molecule provided by the present invention as the initial differentiation marker of Gegenbaur's cell
Enzyme (ALP) activity, checks the influence that peptide molecule provided by the present invention breaks up to mouse bone-forming cell.
First, experimental method
It is 1 × 10 by 1mL concentration4The mouse bone-forming cell of individual/mL cells is inoculated into 24 orifice plates, and is cultivated 1 day.With
Afterwards, cell is handled with the culture medium without the present invention and the differential medium containing peptide molecule provided by the present invention respectively,
And in CO2Content is cultivates in 5% 37 DEG C of insulating boxs, the 7th day and the 14th day measurement ALP is active after incubation.
2nd, experimental result
As shown in Fig. 2 experiment is found, the differential medium containing peptide molecule provided by the present invention was in display in the 7th day
ALP activity is higher by 93% than the culture medium without peptide molecule provided by the present invention, and this shows to provide peptide molecule in invention
The lower osteoblast differentiation ability of effect is obviously improved.
Embodiment 6：
Cytotoxicity analysis：
Peptide molecule provided by the present invention is assessed using the activity of lactic dehydrogenase (LDH) as cytotoxicity index
Cytotoxicity size.LDH is a kind of protein of stabilization, is present in the kytoplasm of normal cell, once damaged membrane, LDH
It is released to extracellular.By detecting the activity of LDH in cells and supernatant, the degree of cell damage, LDH activity can determine whether
It is higher to show that cell damage degree is higher.
First, experimental method
It is 1 × 10 by 1mL concentration4The mouse bone-forming cell of individual/mL cells is inoculated into 24 orifice plates, and is cultivated 1 day.With
Afterwards, cell is handled with the culture medium without peptide molecule of the present invention and the differential medium containing peptide molecule of the present invention respectively,
And in CO2Content takes supernatant to be detected for LDH activity to be cultivated 24 hours in 5% 37 DEG C of insulating boxs after centrifugation, LDH lives
Property built up with Nanjing Bioengineering Research Institute production LDH kits detection.
2nd, experimental result
The LDH activity (U/L) of the peptide molecule of the present invention of table 2
| Culture medium | Peptide molecule of the present invention | |
| LDH activity | 308 | 286 |
As shown in table 2, experiment is found, the LDH activity of the differential medium containing peptide molecule provided by the present invention is lower slightly
In the culture medium without peptide molecule provided by the present invention, show peptide molecule no cytotoxicity provided by the present invention.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (4)
1. a kind of peptide molecule, it is characterised in that the amino acid sequence of the peptide molecule such as sequence table SEQ ID No.1 institutes
Show.
2. peptide molecule according to claim 1, it is characterised in that the peptide molecule molecular weight is 2138.68g/
mol。
3. peptide molecule according to claim 1, it is characterised in that the peptide molecule isoelectric point is 10.6.
4. the peptide molecule described in claim 1, it is characterised in that it is preparing antibacterial and is promoting the material of Oesteoblast growth
In application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410512200.5A CN104292307B (en) | 2014-09-29 | 2014-09-29 | A kind of peptide molecule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410512200.5A CN104292307B (en) | 2014-09-29 | 2014-09-29 | A kind of peptide molecule |
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| Publication Number | Publication Date |
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| CN104292307A CN104292307A (en) | 2015-01-21 |
| CN104292307B true CN104292307B (en) | 2017-07-28 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108059656B (en) * | 2017-12-29 | 2020-07-17 | 广西中医药大学 | Small molecule polypeptide and application thereof |
| CN107936093B (en) * | 2017-12-29 | 2020-06-30 | 广西中医药大学 | Small molecule polypeptide and application thereof |
| CN107987133B (en) * | 2017-12-29 | 2020-07-17 | 广西中医药大学 | Small molecule polypeptide and application thereof |
| CN107892712B (en) * | 2017-12-29 | 2020-06-30 | 广西中医药大学 | Small molecule polypeptide and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173004A (en) * | 2007-10-30 | 2008-05-07 | 沈子龙 | Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof |
| WO2013183048A1 (en) * | 2012-06-03 | 2013-12-12 | Ben-Gurion University Of The Negev Research And Development Authority | Functionalized titanium binding peptides and implants coated with same |
-
2014
- 2014-09-29 CN CN201410512200.5A patent/CN104292307B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173004A (en) * | 2007-10-30 | 2008-05-07 | 沈子龙 | Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof |
| WO2013183048A1 (en) * | 2012-06-03 | 2013-12-12 | Ben-Gurion University Of The Negev Research And Development Authority | Functionalized titanium binding peptides and implants coated with same |
Non-Patent Citations (2)
| Title |
|---|
| The emerging physiological roles of the glycerophosphodiesterase family;Corda D.等;《The FEBS Journal》;20140228;第281卷(第4期);1050-1058 * |
| 大肠杆菌ER2566表达的重组Thanatin的敏感抗菌活性;王沛珍等;《南方医科大学学报》;20101231;第30卷(第5期);998-1016 * |
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| CN104292307A (en) | 2015-01-21 |
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Effective date of registration: 20191225 Address after: No. 659, lushong Road, Changsha hi tech Development Zone, Changsha, Hunan 410000 Patentee after: Hunan Tianjin Pharmaceutical Co., Ltd. Address before: 13 No. 530213 the Guangxi Zhuang Autonomous Region Nanning Qingxiu District five Avenue Patentee before: Guangxi University of Chinese Medicine |