CN104327164B - A kind of synthesis polypeptide and application thereof - Google Patents
A kind of synthesis polypeptide and application thereof Download PDFInfo
- Publication number
- CN104327164B CN104327164B CN201410512199.6A CN201410512199A CN104327164B CN 104327164 B CN104327164 B CN 104327164B CN 201410512199 A CN201410512199 A CN 201410512199A CN 104327164 B CN104327164 B CN 104327164B
- Authority
- CN
- China
- Prior art keywords
- synthesis polypeptide
- amino acid
- polypeptide
- present
- synthesis
- 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.)
- Active
Links
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a kind of synthesis polypeptide, the synthesis polypeptide is included：(a) synthesis polypeptide amino acid sequence：Amino acid sequence shown in SEQ ID No.1；(b) in the synthesis polypeptide that (a) is limited by missing, insert or replace one or several amino acid and with polypeptide (a) described have identical biological function as polypeptide derived from (a)；The synthesis polypeptide molecular weight is 1691.05g/mol, and isoelectric point is 9.7；During it can be applied to prepare antibacterial and promote the material of Oesteoblast growth.The present invention is a kind of synthesis polypeptide, with antibacterial ability, can also promote the growth of Gegenbaur's cell, and stability is strong, it is easy to combined with other compounds, can be with simple application in various forms of materials, and the present invention is nontoxic, with certain application potential.
Description
Technical field
The present invention relates to biomedicine field, more particularly to a kind of synthesis polypeptide and application thereof.
Background technology
Planting body is implanted into by human body agomphosis position by surgical operation, the plantation that reparation artificial tooth is installed at an upper portion thereof is referred to as tooth
Plantation, planting body counts for much and the biocompatibility and Integrated implant of human body between, and it can perch for pathogenic microorganism is provided
Place, once infecting, can all cause plantation to fail.Tooth implant is different by its material, is divided into Metal and Alloy material class,
Ceramic material class, carbon materials class, macromolecular material class, composite class.
Protein, enzyme, polypeptide with specific function etc. can promote the increasing of bone pluripotent cell and Gegenbaur's cell in bone matrix
Grow, break up, can be by being fixed in implant surface, the structure and composition for improveing implant surface promote early stage Integrated implant.
The bioactivators such as the protein with specific function, enzyme, polypeptide are fixed on implant surface, bone pluripotent cell can be promoted
And propagation, the differentiation of Gegenbaur's cell, good osteogenic action is reached, therefore, finding a kind of compound is used to improve planting body
It is necessary for and preventing planting body infections relating.
The content of the invention
One of the object of the invention is to provide a kind of synthesis polypeptide with antibacterial and promotion Oesteoblast growth；The present invention
The second purpose is the application for providing the synthesis polypeptide in the material for preparing antibacterial and promoting Oesteoblast growth.
The present invention provide technical scheme be：
A kind of synthesis polypeptide, the synthesis polypeptide is included：
(a) synthesis polypeptide amino acid sequence：Amino acid sequence shown in SEQ ID No.1；
(b) in the synthesis polypeptide that (a) is limited by missing, insert or replace one or several amino acid and with (a) institute
State polypeptide have identical biological function as polypeptide derived from (a).
Preferably, the synthesis polypeptide molecular weight is 1691.05g/mol.
Preferably, the synthesis polypeptide isoelectric point is 9.7.
Preferably, its application in the material for preparing antibacterial and promoting Oesteoblast growth.The titanium surface of planting body
Can be pre-processed with connexon, and the amino acid containing-SH groups, such as cysteine can be introduced synthesis polypeptide of the present invention
In N-terminal or C-terminal, to combine the functional group of the connexon.Also can be with connexon to connecting peptides after titanium surface preparation
In-NH2Group, titanium surface is connected to by polypeptide.For example, with reference to connexon, synthesis polypeptide of the present invention contains in N-terminal
Cysteine by means of the interaction between connexon and cysteine to be easily introduced into planting body.In addition, being
Synthesis polypeptide of the present invention is stably introduced into the titanium of planting body (Ti) surface, preferably by the connection of silane-connexon-peptide
Be introduced into these synthesis polypeptides in implant surface by relation.The titanium that so treatment is obtained can be planted as tooth implant and bone
Body is used, due to its excellent anti-microbial property and promotion Oesteoblast growth function, for avoiding planting body bacterium infection and drawing
Patients ' lives are rescued to be of great importance.
Additionally, synthesis polypeptide of the invention also can simple application in other material surfaces used in everyday, such as plastics, glass
With other metal surfaces, the bacterium infection that can effectively prevent everyday exposure to cause.
It is of the invention it is beneficial effect be：
Firstth, synthesis polypeptide of the present invention is nontoxic, it is adaptable to planting body material, understands that the present invention is closed by cytotoxicity analysis
The culture medium of synthesis polypeptide of the present invention, lactic acid dehydrogenase activity table higher are not added slightly below into polypeptide lactic acid dehydrogenase activity
The clear-cells extent of damage is higher, it can thus be appreciated that synthesis polypeptide of the present invention is nontoxic；
Secondth, synthesis polypeptide of the present invention has good bactericidal effect, to staphylococcus aureus (ATCC 6538), greatly
Intestines Escherichia (ATCC 25922), candida albicans (ATCC 10231), Bacillus subtilis endophyticus (ATCC 9372)
All there are very strong bactericidal properties, its sterilizing rate is up to more than 99%；
3rd, synthesis polypeptide of the present invention has raising osteoblast activity, promotes the ability of osteoblast differentiation；
4th, synthesis polypeptide cost of manufacture of the present invention is lower compared to other materials cost, it is easy to mass produce；
5th, the synthesis polypeptide stability that the present invention is provided is strong, and structure is easy to regulation and control, it is easy to combined with other compounds,
Can with simple application in various forms of materials, with apparent application potential, also can simple application in other routine uses
Material surface, such as plastics, glass and other metal surfaces, the bacterium infection that can effectively prevent everyday exposure to cause.
The term definition that the present invention relates to
Unless otherwise defined, all technologies otherwise used herein and scientific terminology all have with it is of the art
Those of ordinary skill generally understands identical implication.Although be can be used and described herein in practice of the invention or test
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, makes
Its molecule has biochemical activity.Protein is bioactive molecule important in organism, including the metabolic enzyme of catalysis, two
Or more than two chemistry of amino acids aggregate into peptide, an original segments for protein are proteinogenous precursor, amino acid
Broadly refer to not only containing a basic amine group but also containing an organic compound for 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 its specific design feature, i.e. its amino to be connected directly between on alpha -carbon atom for base acid, 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 one of basic masonry of composition life mansion.
Term " polypeptide " means the compound that a-amino acid is formed so that peptide bond links together, and it is 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；By more than 50 amino acid
The peptide of composition is known as protein.
Term " missing " means to lost a fragment after the fracture of normal chromosomal, and the gene in this fragment is also therewith
Lose, synthesis polypeptide is meant in the present invention in addition to the amino acid sequence shown in SEQ ID No.1, it is also many including synthesizing
The synthesis polypeptide of the new amino acid sequence for not influenceing its function of one or several amino acid is lost in peptide amino acid sequence.
Term " insertion " means to access in another segment DNA or in cloning vector section of DNA using gene manipulation techniques,
Mean synthesis polypeptide in the present invention in addition to the amino acid sequence shown in SEQ ID No.1, also including synthesis polypeptide ammonia
The synthesis polypeptide 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 synthesis polypeptide except the amino acid sequence shown in SEQ ID No.1 in the present invention
Outside row, also including one or several amino acid in synthesis polypeptide amino acid sequence by after other kinds of amino acid replacement not
Influence the synthesis polypeptide of the new amino acid sequence of its function.
Term " isoelectric point " means in the solution of a certain pH, and amino acid or protein dissociation are into cation and anion
Trend or degree are equal, and as hybrid ion, in electroneutral, what now the pH of solution turned into the amino acid or protein waits electric
Point, biological amphiphatic molecule such as protein is, containing acid, also to contain the functional group of alkalescence.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 synthesis polypeptide 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 synthesis polypeptide 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.
Specific 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 synthesis polypeptide, amino acid sequence is as shown in SEQ ID No.1；
SEQ ID No.1：QKKPVPIIYC NGTCQ.
Embodiment 2：
The preparation of synthesis polypeptide：
It is with dichloromethane that resin is fully swelling from amino acid-Wang resin as carrier (resin), use dimethyl formyl
Amine is cleaned several times, with the DBLK of debita spissitudo, the abjection of Fmoc- blocking groups is cleaned several times with dimethylformamide afterwards, is washed
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 relatively completely, is cleaned several times with dimethylformamide, wash away the various residues of residual, activator and condensing agent, according to
Amino acid sequence is coupled, and after the connection of all of amino acid is terminated, sloughs last Fmoc- blocking groups, uses cutting liquid
Cracking, removes resin and amino acid protective group, obtains the crude product of synthesis polypeptide, send mass spectrum to confirm molecular weight product
1691.05g/mol meets theoretical value.
Embodiment 3：
Antibacterial ability is tested：
First, experimental technique
It is 1 × 10 that 1mL concentration is added in sterilizing test tubes6The bacterium solution of individual/mL, adds 1mg synthesis provided by the present invention
Polypeptide, after 37 DEG C are cultivated 24 hours, culture medium 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 synthesis polypeptide of the present invention of table 1
| Bacterium | ATCC 6538 | ATCC 25922 | ATCC 10231 | ATCC 9372 |
| Sterilizing rate | 99.99% | 99.99% | 99.96% | 99.98% |
As shown in Table 1, synthesis polypeptide provided by the present invention, wishes to staphylococcus aureus (ATCC 6538), large intestine angstrom
Salmonella (ATCC 25922), candida albicans (ATCC 10231), Bacillus subtilis endophyticus (ATCC 9372) 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 technique
1mg synthesis polypeptides of the present invention are placed in 24 orifice plates, 1mL density is 2 × 104The mouse bone-forming cell of individual/mL hangs
Liquid is inoculated in 24 orifice plates, then cultivates 1,4 and 7 days.To after predetermined point of time, it is the 3- of 5mg/mL that 200uL concentration 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 generations
Thing, takes three parts of 200uL lysates and goes to 96 well culture plates per hole, surveys its OD value (OD at 490nm with spectrophotometer
Value).
2nd, experimental result
As shown in figure 1, mouse bone-forming cell is inoculated in ordinary culture medium and is added with of the invention being carried as seen from Figure 1
In the culture medium of the synthesis polypeptide of confession, its OD value is in increased trend, as number of days increases, is added with provided by the present invention
The mouse bone-forming cell OD values of synthesis polypeptide increase more significantly, and OD values can reflect the activity of mouse bone-forming cell, so from
Fig. 1 is it is recognised that synthesis polypeptide 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 synthesis polypeptide provided by the present invention as the initial differentiation marker of Gegenbaur's cell
Enzyme (ALP) activity, checks the influence that synthesis polypeptide provided by the present invention is broken up to mouse bone-forming cell.
First, experimental technique
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 processed with the differential medium without culture medium of the invention and containing synthesis polypeptide provided by the present invention respectively,
And in CO2Content be 5% 37 DEG C of insulating boxs in cultivate, the 7th day and the 14th day measurement ALP is active after incubation.
2nd, experimental result
As shown in Fig. 2 experiment finds, the differential medium containing synthesis polypeptide provided by the present invention was in display in the 7th day
ALP activity is higher by 93% than the culture medium without synthesis polypeptide provided by the present invention, and this shows to provide synthesis polypeptide in invention
The lower osteoblast differentiation ability of effect is obviously improved.
Embodiment 6：
Cytotoxicity analysis：
Synthesis polypeptide 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 are can determine whether
It is higher to show that cell damage degree is higher.
First, experimental technique
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 processed with the culture medium without synthesis polypeptide of the present invention and the differential medium containing synthesis polypeptide of the present invention respectively,
And in CO2Content be 5% 37 DEG C of insulating boxs in cultivate 24 hours, taken after centrifugation supernatant for LDH activity detect, LDH live
Property Nanjing build up Bioengineering Research Institute production LDH kits detection.
2nd, experimental result
The LDH activity (U/L) of the synthesis polypeptide of the present invention of table 2
| Culture medium | Synthesis polypeptide of the present invention | |
| LDH activity | 308 | 298 |
As shown in table 2, experiment finds that the LDH activity of the differential medium containing synthesis polypeptide provided by the present invention is lower slightly
In the culture medium without synthesis polypeptide provided by the present invention, show synthesis polypeptide no cytotoxicity provided by the present invention.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in specification and implementation method
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
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 synthesis polypeptide, it is characterised in that the amino acid sequence of the synthesis polypeptide such as sequence table SEQ ID No.1 institutes
Show.
2. synthesis polypeptide according to claim 1, it is characterised in that the synthesis polypeptide molecular weight is 1691.05g/
mol。
3. synthesis polypeptide according to claim 1, it is characterised in that the synthesis polypeptide isoelectric point is 9.7.
4. the synthesis polypeptide 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 |
|---|---|---|---|
| CN201410512199.6A CN104327164B (en) | 2014-09-29 | 2014-09-29 | A kind of synthesis polypeptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410512199.6A CN104327164B (en) | 2014-09-29 | 2014-09-29 | A kind of synthesis polypeptide and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104327164A CN104327164A (en) | 2015-02-04 |
| CN104327164B true CN104327164B (en) | 2017-06-13 |
Family
ID=52401980
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410512199.6A Active CN104327164B (en) | 2014-09-29 | 2014-09-29 | A kind of synthesis polypeptide and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104327164B (en) |
Families Citing this family (3)
| 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 |
| CN107892712B (en) * | 2017-12-29 | 2020-06-30 | 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1234042A1 (en) * | 1999-10-25 | 2002-08-28 | Millennium Pharmaceuticals, Inc. | Protein 27875, a human adam-ts homolog |
| CN104005016A (en) * | 2014-06-06 | 2014-08-27 | 重庆大学 | Medical titanium alloy with antibacterial and osteocyte-facilitating functions and preparation method thereof |
-
2014
- 2014-09-29 CN CN201410512199.6A patent/CN104327164B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104327164A (en) | 2015-02-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104292309B (en) | Micro-molecule polypeptide | |
| CN103554225B (en) | Synthetic antibacterial peptides and application thereof | |
| CN104292307B (en) | A kind of peptide molecule | |
| KR101655781B1 (en) | Bacillus tequilensis HD15 with antibacterial activity and purification method of bacteriocin using the same | |
| CN104327164B (en) | A kind of synthesis polypeptide and application thereof | |
| CN104327168B (en) | A kind of micromolecule polypeptide and its application | |
| KR20160018549A (en) | Antimicrobial Peptide | |
| EP3138572A1 (en) | Antimicrobial peptides derived from phage of acinetobacter baumannii and use thereof | |
| CN104327165B (en) | A kind of peptide molecule and its application | |
| ES2382466T3 (en) | Bacteriocin inducing peptides | |
| CN104017085A (en) | PAMP37-PR39 fused antibacterial peptide and preparation method thereof | |
| JP2006160640A (en) | Antibacterial peptide and its utilization | |
| CN102140130B (en) | Antibacterial peptide and use thereof | |
| WO2013180011A1 (en) | Anti-bacterial peptide and use thereof | |
| JP2011072294A (en) | New antibacterial peptide | |
| TWI403330B (en) | Low hemolysis antibacterial peptide pharmaceutical compositions and use thereof | |
| CN103214583A (en) | Secretory expression method of bacteriocin lacticin Q | |
| Chi et al. | Biosurfactins production by Bacillus amyloliquefaciens R3 and their antibacterial activity against multi-drug resistant pathogenic E. coli | |
| CN102391362B (en) | Group of animal-derived cationic antibacterial peptides and its application | |
| CN103103196A (en) | Modified goat defensin gene and preparation method and application thereof | |
| CN105175525A (en) | Hylarana guentheri antibacterial peptide and application thereof | |
| JP4524671B2 (en) | Antibacterial peptides and their use | |
| CN109867710B (en) | Novel broad-spectrum antibacterial peptide SAMP1-A3 and preparation method thereof | |
| KR20150012505A (en) | A method for designing antimicrobial peptides for reducing the hemolysis thereof | |
| CN110074179B (en) | Application of antibacterial peptide Jelleine-I in preparation of medicine for preventing and treating postharvest green mold of citrus fruits |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210125 Address after: 710000 Shaanxi Xi'an economic and Technological Development Zone, Fengcheng four road, Ming Guang road, southeast corner of the new business building 1808 Patentee after: SHAANXI ZHUANYI INTELLECTUAL PROPERTY OPERATION Co.,Ltd. Address before: 530213 No. 13 Wuhe Avenue, Qingxiu District, Nanning City, Guangxi Zhuang Autonomous Region Patentee before: Guangxi University of Chinese Medicine |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210902 Address after: 710000 room 407, floor 4, building B1, standard workshop community, textile industrial park, Baqiao District, Xi'an City, Shaanxi Province Patentee after: Shaanxi future polypeptide Biotechnology Co.,Ltd. Address before: 710000 Shaanxi Xi'an economic and Technological Development Zone, Fengcheng four road, Ming Guang road, southeast corner of the new business building 1808 Patentee before: SHAANXI ZHUANYI INTELLECTUAL PROPERTY OPERATION Co.,Ltd. |