CN104327165A - Polypeptide molecule and application thereof - Google Patents
Polypeptide molecule and application thereof Download PDFInfo
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- CN104327165A CN104327165A CN201410512789.9A CN201410512789A CN104327165A CN 104327165 A CN104327165 A CN 104327165A CN 201410512789 A CN201410512789 A CN 201410512789A CN 104327165 A CN104327165 A CN 104327165A
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Abstract
The invention discloses a polypeptide molecule including: (a) an amino sequence of the polypeptide molecule, wherein the amino sequence is represented as the SEQ ID No.1; and (b) a polypeptide molecule which is derived from (a) and has same biological functions of the polypeptide molecule (a), wherein the polypeptide molecule (b) is obtained since one or more amino acids are deleted, inserted or replaced in the polypeptide molecule limited in (a). A molecular weight of the polypeptide molecule is 2289.91 g/mol and an isoelectric point of the polypeptide molecule is 11.1. The polypeptide molecule can be applied for preparing materials which is anti-bacterial and can promote growth of osteoblast. The invention provides the polypeptide molecule which can be compounded with other compounds simply, can be applied in materials in various types and has effects of promoting growth of the osteoblast and being anti-bacterial. The polypeptide molecule is free of toxic and harmful effect on human body. The polypeptide molecule is simpler in synthetic method compared with a large-molecular polypeptide and can be industrially produced.
Description
Technical field
The present invention relates to biomedicine field, particularly a kind of peptide molecule and application thereof.
Background technology
Tooth implant is the agomphosis position of implant into body, different by its material, is divided into Metal and Alloy material type, stupalith class, carbon materials class, macromolecular material class, matrix material class.Planting body material is mainly used in the treatment of defect of dentition and bone loss, and this treatment infection rate is very high, may cause a large amount of antibiotic therapy, remove the serious consequences such as planting body is even dead.Planting body infections relating major cause occurred frequently has 2 points: bacterial plaque implant surface assemble and Osseointegrated implants interface resistivity low.In order to avoid planting body infections relating, graft should have the premium properties of two aspects: excellent anti-microbial property and promote Oesteoblast growth function, and the material that exploitation has these two kinds of effects has great importance for the treatment of relative disease.
Summary of the invention
One of the object of the invention is that providing a kind of has peptide molecule that is antibacterial and promotion Oesteoblast growth; Two of the object of the invention is to provide the antibacterial application with promoting in the material of Oesteoblast growth in preparation of this peptide molecule.
Technical scheme provided by the invention is:
A kind of peptide molecule, described peptide molecule comprises:
(a) peptide molecule aminoacid sequence: the aminoacid sequence shown in SEQ ID No.1;
In b peptide molecule that () limits at (a) through disappearance, insert or replace one or several amino acid and with peptide molecule (a) described, there is the peptide molecule derivative by (a) of identical biological function.
Preferably, described peptide molecule molecular weight is 2289.91g/mol.
Preferably, described peptide molecule iso-electric point is 11.1.
Preferably, its antibacterial application with promoting in the material of Oesteoblast growth in preparation.The titanium available connexon pre-treatment in surface of planting body, and can by the N-terminal that introduce peptide molecule of the present invention containing the amino acid (such as halfcystine) of-SH group or C-terminal, with the functional group in conjunction with described connexon.Also can with connexon to the-NH in connecting peptides molecule after titanium surface preparation
2group, is connected to titanium surface by peptide molecule.Such as, in conjunction with connexon, peptide molecule of the present invention contains halfcystine to be easily introduced in planting body by means of the interaction between connexon and halfcystine at N-terminal.In addition, in order to stably be introduced in titanium (Ti) surface of planting body by peptide molecule of the present invention, the annexation of silane-connexon-peptide is preferably utilized to be introduced in implant surface by these peptide molecules.The titanium metal that such process obtains can be used as tooth implant and bone planting body uses, and due to anti-microbial property and the promotion Oesteoblast growth function of its excellence, is of great importance for avoiding planting body bacteriological infection and saving patients ' lives.
In addition, peptide molecule of the present invention also can simple application in other material surfaces used in everyday, as plastics, glass and other metallic surfaces, the bacteriological infection that can effectively prevent everyday exposure to cause.
The invention has the beneficial effects as follows a kind of peptide molecule provided by the invention, there is good antibacterial ability, can kill except streptococcus aureus reaches 99.99%, all can reach 99% for other bacterial classification sterilizing rate; Peptide molecule of the present invention possesses the ability improving osteoblast activity simultaneously, known by optical density value, the bone cell activity adding peptide molecule of the present invention will improve greatly than the activity not adding peptide molecule of the present invention, promotes the effect of osteoblast differentiation ability; By cytotoxicity analysis, the material non-toxic that the known the present invention of utilization makes, acts on human body and can not cause Health hazard; Peptide molecule provided by the invention, stability is strong, and structure is easy to regulation and control, is easy to be combined with other compound, can simple application in various forms of material, there is application potential clearly, be easy to scale operation.
The term definition arrived involved in the present invention
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.Although any method, device and the material similar or equivalent with person described herein can be used in practice of the present invention or test, preferred method, device and material are described now.
Term " amino acid " means the fundamental unit forming protein, gives protein specific molecular morphosis, makes its molecule have biochemical activity.Protein is bioactive molecule important in organism, comprise the metabolic enzyme of catalysis, two or more chemistry of amino acids aggregates into peptide, the original segments of a protein, it is proteinogenous precursor, amino acid broadly refers to the organic compound not only containing a basic amine group but also containing an acidic carboxypolymer, and the name as it is said.But general amino acid, then refer to the structure unit forming protein.In organic sphere, the amino acid forming natural protein has its specific constructional feature, and namely its amino is connected directly between on alpha-carbon atom, and this seed amino acid is called as a-amino acid.Have 300 multiple amino acids at occurring in nature, wherein a-amino acid 21 kinds, a-amino acid is the building block molecule of peptides and proteins, is also one of basic masonry forming life mansion.
Term " polypeptide " means a-amino acid and links together with peptide bond and the compound formed, and it is also the intermediate product of proteolysis.Dipeptides is called by the compound of two amino acid molecular dehydrating condensations, in like manner analogize and also have tripeptides, tetrapeptide, pentapeptide etc., usually by the compound of 10 ~ 100 amino acid molecular dehydrating condensations polypeptide, their molecular weight is lower than 10000 dalton, can through semi-permeable membranes, not by trichoroacetic acid(TCA) and ammonium sulfate precipitate, also by document, the peptide be made up of 2 ~ 10 amino acid is called oligopeptides (small-molecular peptides); The peptide of 10 ~ 50 amino acid compositions is called polypeptide; The peptide be made up of the amino acid of more than 50 is just called protein.
Term " disappearance " lost a fragment after meaning a normal chromosomal fracture, gene in this fragment also loses thereupon, mean peptide molecule in the present invention except for except the aminoacid sequence shown in SEQ ID No.1, also comprise in peptide molecule aminoacid sequence and lose one or several amino acid whose peptide molecule not affecting the new aminoacid sequence of its function.
Term " insertion " means to utilize gene manipulation techniques that section of DNA is accessed in another segment DNA or in cloning vector and goes, mean peptide molecule in the present invention except for except the aminoacid sequence shown in SEQ ID No.1, also comprise in peptide molecule aminoacid sequence and insert one or several amino acid whose peptide molecule not affecting the new aminoacid sequence of its function.
Term " displacement " means a kind of gene recombined vector, certain fragment gene in genome is replaced by general homologous recombination, and reach the object of gene knockout, mean peptide molecule in the present invention except for except the aminoacid sequence shown in SEQ ID No.1, also to comprise in peptide molecule aminoacid sequence one or several amino acid by the peptide molecule not affecting the new aminoacid sequence of its function after the amino-acid substitution of other kinds.
Term " iso-electric point " means in the solution of a certain pH, amino acid or protein dissociation become the trend of positively charged ion and negatively charged ion or degree equal, become zwitterion, in electric neutrality, now the pH of solution becomes this amino acid or isoelectric point of protein, biological amphipathic molecule, if protein is namely containing acid, also contains the functional group of alkalescence.The amino acid of constitutive protein matter may be positively charged, electronegative, neutrality or this life is both sexes, it is the electric charge of protein that their electric charge is added together, when pH value is less than iso-electric point, the total charge of protein is positive, is negative when being greater than iso-electric point.
Accompanying drawing explanation
Fig. 1 is that peptide molecule of the present invention contrasts figure with ordinary culture medium to the comparative result that activity of osteoblast proliferation affects on the impact of activity of osteoblast proliferation;
Fig. 2 is that peptide molecule of the present invention contrasts figure to initial cell differentiation capability with the comparative result of ordinary culture medium to initial cell differentiation capability.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
Embodiment 1:
A kind of peptide molecule, aminoacid sequence is as shown in SEQ ID No.1;
SEQ ID No.1：KKPVPIIYCN KKRGKCQRM。
Embodiment 2:
The preparation of peptide molecule:
Select amino acid-king's resin as carrier (resin), with methylene dichloride, resin is fully swelling, several times are cleaned with dimethyl formamide, with the DBLK of proper concn, Fmoc-blocking group is deviate from, several times are cleaned afterwards with dimethyl formamide, wash away DBLK, take applicable condensing agent benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate and activator methylmorpholine and C hold second Fmoc-protected amino acid (Fmoc-Pro-OH) to carry out coupling, carry out detection by triketohydrindene hydrate detection method and guarantee that connection is more complete, several times are cleaned with dimethyl formamide, wash away residual various residues, activator and condensing agent, coupling is carried out according to aminoacid sequence, all amino acid is connected after terminating, slough last Fmoc-blocking group, use cutting liquid cracking, remove resin and amino acid protective group, obtain the crude product of peptide molecule, mass spectrum is sent to confirm molecular weight product 2289.91g/mol coincidence theory value.
Embodiment 3:
Antibacterial ability is tested:
One, experimental technique
In sterilizing test tubes, add 1mL concentration is 1 × 10
6the bacterium liquid of individual/mL, adds 1mg peptide molecule provided by the present invention, and cultivate after 24 hours for 37 DEG C, substratum collects with doubling dilution, and extension rate is 10 times and spread plate method detection viable count.
Two, experimental result:
The sterilizing rate (%) of table 1 peptide molecule of the present invention
| Bacterium | ATCC 6538 | ATCC 25922 | ATCC 10231 | ATCC 9372 |
| Sterilizing rate | 99.99％ | 99.92％ | 99.91％ | 99.93％ |
As shown in Table 1, peptide molecule provided by the present invention, all have very strong bactericidal properties to streptococcus aureus (ATCC 6538), colon bacillus (ATCC 25922), Candida albicans (ATCC 10231), Bacillus subtilis endophyticus (ATCC 9372), its sterilizing rate reaches more than 99%.
Embodiment 4:
Activity of osteoblast proliferation is tested:
One, experimental technique
Peptide molecule provided by the present invention for 1mg is placed in 24 orifice plates, and 1mL density is 2 × 10
4the mouse bone-forming cell suspension inoculation of individual/mL, in 24 orifice plates, then cultivates 1,4 and 7 day.After predetermined point of time, every hole adds the 3-(4 that 200uL concentration is 5mg/mL, 5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT) and 800uL serum-free are without phenol red improvement Du Shi Eagle's medium (DMEM), 37 DEG C hatch 4 hours after inhale abandon supernatant liquor, add 1ml dimethyl sulfoxide (DMSO) (DMSO) and dissolve the crystallisate generated, every hole is got three parts of 200uL lysates and is forwarded 96 well culture plates to, surveys its optical density value (OD value) with spectrophotometer in 490nm place.
Two, experimental result
As shown in Figure 1, mouse bone-forming cell is inoculated in ordinary culture medium and is added with in the substratum of peptide molecule provided by the present invention as seen from Figure 1, its OD value is all in the trend increased, along with number of days increases, the mouse bone-forming cell OD value being added with peptide molecule provided by the present invention increases more remarkable, OD value can reflect the activity of mouse bone-forming cell, so we can know that peptide molecule provided by the present invention can significantly improve mouse bone-forming cell proliferation activity from Fig. 1.
Embodiment 5:
Initial cell differentiation capability is analyzed:
Active by the alkaline phosphatase (ALP) compared as the peptide molecule provided by the present invention of scleroblast initial differentiation marker thing, check the impact that peptide molecule provided by the present invention breaks up mouse bone-forming cell.
One, experimental technique
Be 1 × 10 by 1mL concentration
4the mouse bone-forming cell of individual/mL cell is inoculated in 24 orifice plates, and cultivates 1 day.Subsequently, respectively with not containing substratum of the present invention and the division culture medium process cell containing peptide molecule provided by the present invention, and at CO
2content is cultivate in 37 DEG C of thermostat containers of 5%, within the 7th day and the 14th day, measures ALP after incubation active.
Two, experimental result
As shown in Figure 2, experiment finds, division culture medium containing peptide molecule provided by the present invention is high by 93% containing the substratum of peptide molecule provided by the present invention in the ALP specific activity of display in the 7th day, this show invention the effect of peptide molecule is provided under osteoblast differentiation ability obviously improve.
Embodiment 6:
Cytotoxicity analysis:
The cytotoxicity size of peptide molecule provided by the present invention is assessed using the activity of serum lactic dehydrogenase (LDH) as cytotoxicity index.LDH is a kind of stable protein, is present in Normocellular kytoplasm, once damaged membrane, namely LDH is released to extracellular.By detecting the activity of LDH in cells and supernatant, can judge the degree of cell damage, LDH activity is higher shows that cell damage degree is higher.
One, experimental technique
Be 1 × 10 by 1mL concentration
4the mouse bone-forming cell of individual/mL cell is inoculated in 24 orifice plates, and cultivates 1 day.Subsequently, respectively with not containing the substratum of peptide molecule of the present invention and the division culture medium process cell containing peptide molecule of the present invention, and at CO
2content is cultivate 24 hours in 37 DEG C of thermostat containers of 5%, gets supernatant liquor and detect for LDH activity after centrifugal, and the LDH test kit detection that Bioengineering Research Institute produces is built up in LDH activity Nanjing.
Two, experimental result
The LDH activity (U/L) of table 2 peptide molecule of the present invention
| Substratum | Peptide molecule of the present invention | |
| LDH activity | 308 | 295 |
As shown in table 2, experiment finds, the LDH activity of the division culture medium containing peptide molecule provided by the present invention, a little less than the substratum not containing peptide molecule provided by the present invention, shows peptide molecule no cytotoxicity provided by the present invention, can be used on planting body.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (4)
1. a peptide molecule, is characterized in that, described peptide molecule comprises:
(a) peptide molecule aminoacid sequence: the aminoacid sequence shown in SEQ ID No.1;
In b peptide molecule that () limits at (a) through disappearance, insert or replace one or several amino acid and with peptide molecule (a) described, there is the peptide molecule derivative by (a) of identical biological function.
2. peptide molecule according to claim 1, is characterized in that, described peptide molecule molecular weight is 2289.91g/mol.
3. peptide molecule according to claim 1, is characterized in that, described peptide molecule iso-electric point is 11.1.
4. peptide molecule according to claim 1, is characterized in that, it is the antibacterial application with promoting in the material of Oesteoblast growth in preparation.
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| CN201410512789.9A CN104327165B (en) | 2014-09-29 | 2014-09-29 | A kind of peptide molecule and its application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107892712A (en) * | 2017-12-29 | 2018-04-10 | 广西中医药大学 | Micromolecule polypeptide and application thereof |
| CN107936093A (en) * | 2017-12-29 | 2018-04-20 | 广西中医药大学 | Micromolecule polypeptide and application thereof |
| CN108059656A (en) * | 2017-12-29 | 2018-05-22 | 广西中医药大学 | Micromolecule polypeptide and application thereof |
Citations (2)
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| CN101173004A (en) * | 2007-10-30 | 2008-05-07 | 沈子龙 | Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof |
| JP2011072294A (en) * | 2009-10-02 | 2011-04-14 | Hokkaido Univ | New antibacterial peptide |
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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 |
| JP2011072294A (en) * | 2009-10-02 | 2011-04-14 | Hokkaido Univ | New antibacterial peptide |
Non-Patent Citations (2)
| Title |
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| PASCALE FEHLBAUM ET AL: "Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides", 《PROC. NATL. ACAD. SCI. USA》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107892712A (en) * | 2017-12-29 | 2018-04-10 | 广西中医药大学 | Micromolecule polypeptide and application thereof |
| CN107936093A (en) * | 2017-12-29 | 2018-04-20 | 广西中医药大学 | Micromolecule polypeptide and application thereof |
| CN108059656A (en) * | 2017-12-29 | 2018-05-22 | 广西中医药大学 | Micromolecule polypeptide and application thereof |
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| Publication number | Publication date |
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| CN104327165B (en) | 2017-07-28 |
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Effective date of registration: 20200617 Address after: 230000 413a, building 14, electromechanical Industrial Park, 767 Yulan Avenue, hi tech Zone, Hefei City, Anhui Province Patentee after: Hefei rike Biotechnology Co., Ltd Address before: 13 No. 530213 the Guangxi Zhuang Autonomous Region Nanning Qingxiu District five Avenue Patentee before: Guangxi University of Chinese Medicine |