CN104231058A - Polypeptide, nucleic acid coding polypeptide and pharmaceutical composition - Google Patents

Polypeptide, nucleic acid coding polypeptide and pharmaceutical composition Download PDF

Info

Publication number
CN104231058A
CN104231058A CN201310228448.4A CN201310228448A CN104231058A CN 104231058 A CN104231058 A CN 104231058A CN 201310228448 A CN201310228448 A CN 201310228448A CN 104231058 A CN104231058 A CN 104231058A
Authority
CN
China
Prior art keywords
polypeptide
seq
pharmaceutical composition
nucleic acid
sequence shown
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.)
Granted
Application number
CN201310228448.4A
Other languages
Chinese (zh)
Other versions
CN104231058B (en
Inventor
郭慧媛
郑丽敏
文鹏程
任发政
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Agricultural University
Original Assignee
China Agricultural University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by China Agricultural University filed Critical China Agricultural University
Priority to CN201310228448.4A priority Critical patent/CN104231058B/en
Publication of CN104231058A publication Critical patent/CN104231058A/en
Application granted granted Critical
Publication of CN104231058B publication Critical patent/CN104231058B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses polypeptide. The polypeptide contains an amino acid sequence shown as SEQ ID NO:1; a sequence of the polypeptide is the amino acid sequence shown as SEQ ID NO:2 or segments of the amino acid sequence shown as SEQ ID NO:2. The polypeptide provided by the invention has high activity for promoting osteocyte differentiation and/or proliferation.

Description

The nucleic acid of polypeptide and this polypeptide of coding and pharmaceutical composition
Technical field
The present invention relates to a peptide species, the nucleic acid of this polypeptide of encoding, described polypeptide and the application of/nucleic acid in the medicine for the preparation of promotion bone cell differentiation and/or propagation and/or treatment osteoporosis, and a kind of pharmaceutical composition containing this polypeptide.
Background technology
Osteoporosis is common disease, the frequently-occurring disease of middle-aged and old especially postmenopausal women, occupies first of the elderly's five large disease morbiditys, and the whole world about has the people of 10% to suffer from this disease, and along with increasing the weight of of aging degree, morbidity situation is increasingly severe.Associated therapy osteoporosis agents especially promotes Study and Development one of heat subject becoming the world of medicine of the medicine of bone cell differentiation and/or propagation.
Lactoferrin is mainly from breast milk, it is nontoxic, harmless, and the remarkable effect had in promotion bone cell differentiation and/or propagation, therefore, it is possible to effectively treat osteoporosis, in recent years, the potentiality to be exploited of huge treatment and prevention skeletal diseases is shown.Existing research confirms that lactoferrin shows powerful osteogenic activity in vitro, and it can promote osteoblastic proliferation and growth effectively.
But lactoferrin molecules amount is about 80kDa, containing 700 amino acid of having an appointment, because its larger molecular weight makes it be difficult to be produced on a large scale.Further, the activity of lactoferrin still needs to be improved further.
Summary of the invention
The lactoferrin that the object of the invention is to overcome prior art is difficult to scale operation, and its activity still treats the defect improved further, there is provided one can scale operation, and the nucleic acid of the more much higher peptide of activity and this polypeptide of encoding and their application, and the pharmaceutical composition containing this polypeptide.
To achieve these goals, first aspect, the invention provides a peptide species, and wherein, this polypeptide has the aminoacid sequence shown in SEQ ID No:1; And the sequence of this polypeptide is the aminoacid sequence shown in SEQ ID No:2, or it is the fragment of the aminoacid sequence shown in SEQ ID No:2.
Preferably, the sequence of this polypeptide is the aminoacid sequence shown in SEQ ID No:1, or the sequence of this polypeptide is the aminoacid sequence shown in SEQ ID No:2.
Second aspect, the invention provides a kind of nucleic acid, wherein, and this nucleic acid encoding polypeptide provided by the invention.
The third aspect, the invention provides polypeptide as above and/or the application of nucleic acid in the medicine for the preparation of promotion bone cell differentiation and/or propagation and/or treatment osteoporosis.
Fourth aspect, the invention provides a kind of pharmaceutical composition, and wherein, this pharmaceutical composition contains polypeptide provided by the invention.
As can be seen from technique scheme and embodiments of the invention and comparative example, the lactoferrin of polypeptide provided by the invention and prior art has the short advantage of molecular weight, therefore, it is possible to easily produce on a large scale, for its industrialization provides the foundation; Further, polypeptide provided by the invention has the promotion bone cell differentiation higher than the lactoferrin of prior art and/or the activity of propagation.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the microscope photograph of the mouse periosteum of the polypeptide shown in injection SEQ ID No:1; The position of arrow points is the scleroblast in mouse periosteum.
Fig. 2 is the microscope photograph of the Mouse Bone membrane bone of injecting normal saline; The position of arrow points is the scleroblast in mouse periosteum.
Fig. 3 is the microscope photograph of the mouse periosteum of the lactoferrin of injection prior art; The position of arrow points is the scleroblast in mouse periosteum.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
According to a first aspect of the invention, provide a peptide species, wherein, this polypeptide has the aminoacid sequence shown in SEQ ID No:1; And the sequence of this polypeptide is the aminoacid sequence shown in SEQ ID No:2, or it is the fragment of the aminoacid sequence shown in SEQ ID No:2.
According to the present invention, the sequence of described polypeptide has the aminoacid sequence shown in SEQ ID No:1, and is the fragment of the aminoacid sequence shown in SEQ ID No:2, refers to the 1-26 position of the sequence of described polypeptide for the aminoacid sequence shown in SEQID No:2, 1-27 position, 1-28 position, 1-29 position, 1-30 position, 1-31 position, 1-32 position, 1-33 position, 1-34 position, 1-35 position, 1-36 position, 1-37 position, 1-38 position, 1-39 position, 1-40 position, 1-41 position, 1-42 position, 1-43 position, 1-44 position, 1-45 position, 1-46 position, 1-47 position, 1-48 position, 1-49 position, 1-50 position, 1-51 position, 1-52 position, 1-53 position, 1-54 position, 1-55 position, 1-56 position, 1-57 position, 1-58 position, 1-59 position, 1-60 position, 1-61 position, 1-62 position, 1-63 position, 1-64 position, 1-65 position, 1-66 position, 1-67 position, 1-68 position, 1-69 position, 1-70 position, 1-71 position, 1-72 position, 1-73 position, 1-74 position, 1-75 position, 1-76 position, 1-77 position, 1-78 position, 1-79 position, 1-80 position, 1-81 position, 1-82 position, 1-83 position, 1-84 position, 1-85 position, 1-86 position, 1-87 position, 1-88 position, 1-89 position, 1-90 position, 1-91 position, 1-92 position, 1-93 position, 1-94 position, 1-95 position, 1-96 position, 1-97 position, 1-98 position, 1-99 position, 1-100 position, 1-101 position, 1-102 position, 1-103 position, 1-104 position, 1-105 position, 1-106 position, 1-107 position, 1-108 position, 1-109 position, any one in aminoacid sequence shown in 1-110 position and 1-111 position.
In preferred situation, the sequence of this polypeptide is the aminoacid sequence shown in SEQ ID No:1 or SEQ ID No:2.
According to the present invention, the source of described polypeptide has no particular limits, and such as, from existing albumen, such as, can carry out specific enzymes solution and obtain in native lactoferrin sequence; Also by the gene constructed expression carrier of coding said polypeptide, then can be transformed in microorganism and express, then obtain described polypeptide by extraction purification; Also can conventionally obtain by solid phase synthesis, such as, synthetic method can with reference to Systemic screening of milk protein-derived ACE inhibitors through a chemically synthesised tripeptide library(Ren F.Z.et al, Food Chemistry, 2011,128 (3), 761-768) method disclosed in; Synesis Company can also be entrusted to synthesize, such as, the Heng Yu visual field, Beijing Bioisystech Co., Ltd can be entrusted to synthesize.
Under normal circumstances, when the amino acid number of polypeptide is greater than 100, the method for conventional solid phase synthesis is adopted to obtain comparatively difficulty.Therefore, according to one of the present invention preferred embodiment, the conveniently acquisition of described polypeptide, when the amino acid number of polypeptide is 26-100, can carry out conventional solid phase synthesis; When the amino acid number of polypeptide is greater than 100, by the gene of coding said polypeptide being carried out conventional protokaryon and/or eukaryotic expression, then obtain this polypeptide be expressed by extraction purification.Wherein, prokaryotic expression, and the method extracting also purification of target product is conventionally known to one of skill in the art, specifically can with reference to the method in " Molecular Cloning: A Laboratory guide ".
Second aspect, the invention discloses a kind of nucleic acid, wherein, and this nucleic acid encoding polypeptide provided by the invention.
According to one of the present invention preferred embodiment, the polypeptide shown in SEQ ID No:1 is obtained by the nucleic acid encoding shown in SEQ ID No:3, and the polypeptide shown in SEQ ID No:2 is obtained by the nucleic acid encoding shown in SEQ ID No:4.Those skilled in the art it is understood that, the nucleic acid coding sequence of other polypeptide that first aspect present invention provides can by selecting and adding the mode of codon, nucleotide sequence according to SEQ ID No:3 and SEQ ID No:4 is corresponding to be obtained, and will not enumerate at this.
According to the present invention, the nucleotide sequence of coding said polypeptide provided by the invention is not limited in nucleotide sequence as above, the polypeptide provided by the invention as long as it can be encoded, such as, a certain amino acid whose codon of encoding in above-mentioned nucleotide sequence in polypeptide provided by the invention can also be the synonym of this codon.
The third aspect, present invention also offers polypeptide as above and/or the application of nucleic acid in the medicine for the preparation of promotion bone cell differentiation and/or propagation and/or treatment osteoporosis.
According to the present invention, described polypeptide has the effect promoting bone cell differentiation and/or propagation, thus osteoporotic symptom can be improved, therefore, polypeptide provided by the invention and/or nucleic acid can be applied in preparation and promote in the medicine of bone cell differentiation and/or propagation and/or treatment osteoporosis.
According to the present invention, the kind of described medicine has no particular limits, as long as it contains polypeptide provided by the invention and/or nucleic acid.Such as, can be healthcare products, can also be pharmaceutical drugs.
Based on this, fourth aspect, the invention provides a kind of pharmaceutical composition, and wherein, this pharmaceutical composition contains polypeptide provided by the invention.
As long as although bone cell differentiation and/or propagation can be promoted containing polypeptide provided by the invention in described pharmaceutical composition, thus the effect for the treatment of osteoporosis can be reached, but the present inventor finds, when in described pharmaceutical composition, with the gross weight of pharmaceutical composition described in 1g for benchmark, when the content of described polypeptide is 1-1000 μ g, differentiation and/or the propagation of osteocyte can be promoted significantly, thus make the effect for the treatment of osteoporosis more obviously good.The present inventor finds again unexpectedly, when with the gross weight of pharmaceutical composition described in 1g for benchmark, when the content of described polypeptide is 1-100 μ g, significantly can promote the differentiation of osteocyte; When the content of described polypeptide is 400-600 μ g, the propagation of osteocyte significantly can be promoted.
According to the present invention, when the purity of polypeptide in described pharmaceutical composition is more than 95 % by weight, bone cell differentiation and/or propagation can be promoted further.
According to pharmaceutical composition of the present invention, wherein also containing pharmaceutically acceptable assistant agent, the kind for described assistant agent is conventionally known to one of skill in the art, and described assistant agent is preferably physiological saline and/or glucose solution.In addition, according to different needs, the concentration of described glucose can be 5-50 % by weight.
Below will be described the present invention by embodiment.In following preparation example, embodiment and comparative example:
Lactoferrin, purchased from Yosica company, be Bovinelactoferrin, purity is 95 % by weight;
MC3T3-E1 scleroblast system (ATCC DSMZ of the U.S.), purchased from Chinese Academy of Sciences's Shanghai cell bank (Shanghai);
α-MEM substratum purchased from American Hyclone; Foetal calf serum, purchased from PAP company;
Trypsinase, purchased from Sigma-Aldrich company;
BrdU cell proliferation detecting kit, purchased from American Roche company;
Swiss mice, purchased from Beijing Animal Science company limited of dimension tonneau China;
Bitubular electric light microscope, purchased from Japanese Olympus company, model is OLYMPUS-BHT;
The mixed solution of cell pyrolysis liquid and working fluid: by the Na of 5.5mL0.1M 2cO 3with the NaHCO of 4.5mL0.1M 3add the Triton X-100 of 1% after mixing, then add 22.3mg p-nitrophenylphosphate (pNPP, purchased from Sigma-Aldrich company), the MgSO of 4.9mg 47H 2o;
Stop buffer: the sodium hydroxide solution of 8 % by weight.
Preparation example
(1) the Heng Yu visual field, Beijing Bioisystech Co., Ltd is entrusted to synthesize the polypeptide shown in SEQ ID No:1 respectively, and the 1-27 position of the aminoacid sequence shown in SEQ ID No:2, 1-28 position, 1-29 position, 1-30 position, 1-31 position, 1-32 position, 1-33 position, 1-34 position, 1-35 position, 1-36 position, 1-37 position, 1-38 position, 1-39 position, 1-40 position, 1-41 position, 1-42 position, 1-43 position, 1-44 position, 1-45 position, 1-46 position, 1-47 position, 1-48 position, 1-49 position, 1-50 position, 1-51 position, 1-52 position, 1-53 position, 1-54 position, 1-55 position, 1-56 position, 1-57 position, 1-58 position, 1-59 position, 1-60 position, 1-61 position, 1-62 position, 1-63 position, 1-64 position, 1-65 position, 1-66 position, 1-67 position, 1-68 position, 1-69 position, 1-70 position, 1-71 position, 1-72 position, 1-73 position, 1-74 position, 1-75 position, 1-76 position, 1-77 position, 1-78 position, 1-79 position, 1-80 position, 1-81 position, 1-82 position, 1-83 position, 1-84 position, 1-85 position, 1-86 position, 1-87 position, 1-88 position, 1-89 position, 1-90 position, 1-91 position, 1-92 position, 1-93 position, 1-94 position, 1-95 position, 1-96 position, 1-97 position, 1-98 position, polypeptide shown in 1-99 position and 1-100 position, totally 75 polypeptide.
(2) method of prokaryotic expression is adopted to obtain the polypeptide shown in SEQ ID No:2, and the 1-101 position of the aminoacid sequence shown in SEQ ID No:2,1-102 position, 1-103 position, 1-104 position, 1-105 position, 1-106 position, 1-107 position, 1-108 position, 1-109 position, the polypeptide shown in 1-110 position and 1-111 position, totally 12 polypeptide.
Wherein, for the acquisition of goal gene, the selection of carrier, the method of the selection of the structure of recombinant expression vector and conversion, host cell, the abduction delivering of expression vector, the extraction and purification of target product does not belong to invention scope of the present invention, in this not go into detail, specifically can with reference to the method in " Molecular Cloning: A Laboratory guide ".
(3) above-mentioned 87 polypeptide taken a morsel carry out Mass Spectrometric Identification, and mass spectrum gained molecular weight conforms to the calculated value obtained according to sequence, prove that described 87 polypeptide are polypeptide provided by the invention.And purity is all more than 95 % by weight.Wherein, the theoretical molecular of the polypeptide shown in SEQ ID No:1 is 2729.25, and it be the theoretical molecular shown in 2729.60, SEQ ID No:2 is 12152.00 that mass spectrum records molecular weight, and it is 12151.97 that mass spectrum records molecular weight.
Embodiment 1.1
The external short bone cell differentiation of the present embodiment for illustration of polypeptide of the present invention and the effect of propagation.
(1) the osteoblastic cultivation of MC3T3-E1 with go down to posterity
Cultivate: α-MEM substratum (containing the 10%(v/v) foetal calf serum with 10mL) suspension MC3T3-E1 scleroblast, and be inoculated in culturing bottle, be placed in 37 DEG C, 5%CO 2cultivate in incubator, 24h changes liquid once, within later every 2 days, changes liquid once.
Go down to posterity: the nutrient solution in culturing bottle, to when converging, discards with suction pipe by scleroblast monolayer growth gently, wash away substratum remaining in culturing bottle with the PBS damping fluid of about 10mL; Add the tryptic PBS damping fluid of 2mL Digestive system (containing 0.25%(w/v)), jog culturing bottle makes trypsinase be paved with cell surface, be placed in incubator, after 4 minutes, with suction pipe, cell is blown down from bottle wall, suck in centrifuge tube, the centrifugal 5min of 1000rpm, collecting precipitation, uses substratum re-suspended cell, is inoculated in new culturing bottle and continues to cultivate.Repeat this step and cell was reached the tenth generation.
(2) the osteoblastic proliferation activity of MC3T3-E1
To cultivate in step (1) and cell after going down to posterity with 2 × 10 4the concentration of individual/mL is inoculated in 96 well culture plates, 100 μ L are inoculated in every hole, cultivate in containing the complete α-MEM substratum of 10% serum, serum free medium is changed after 24h, the polypeptide shown in SEQ ID No:1 is added after continuing to cultivate 24h, its final concentration is made to be 500 μ g/mL, a blank is separately established (only to add serum free medium, do not add polypeptide of the present invention), each concentration gradient arranges 6 parallel holes, BrdU-ELISA method is adopted (to use 5-bromodeoxyuridine nucleosides (5-bromo-2-deoxyuridine after effect 48h, BrdU) cell proliferation detecting kit) measure cell proliferation rate.Appreciation rate represents to calculate relative to blank group the relative appreciation rate obtained, and the results are shown in Table 1.
(3) detection that alkaline phosphatase (ALP) is active
To cultivate in step (1) and cell after going down to posterity with 4 × 10 5individual/mL density is inoculated in 96 well culture plates, and 200 μ L are inoculated in every hole, after being cultured to cytogamy, adding the polypeptide shown in SEQ ID No:1, makes its final concentration be 500 μ g/mL in the perfect medium containing 10% serum.Separately establish a blank (not adding any medicine in cell culture fluid), each concentration establishes 6 parallel holes, after continuing cultivation effect 48h, with 1%(w/v) after PBS washing, measure ALP active.Concrete operation step is as follows:
Dried by 96 washed for PBS orifice plates, every hole adds the mixed solution of 100 μ L cell pyrolysis liquids and working fluid; Oscillator vibrates 1min, be placed on 37 DEG C of constant incubator conditions under react 30min; Every hole adds 80 μ L stop buffers, vibration 1min, and microplate reader measures light absorption value in 405nm wavelength place; Compare with blank group the relative reactivity calculating and obtain ALP according to surveyed light absorption value, the results are shown in Table 1.
Embodiment 1.2
The external short bone cell differentiation of the present embodiment for illustration of polypeptide of the present invention and the effect of propagation.
Carry out the osteoblastic cultivation of MC3T3-E1 according to the method in embodiment 1.1 and go down to posterity, the osteoblastic proliferation activity of MC3T3-E1 detects and the detection of ALP activity, unlike, in the detection of osteoblastic proliferation activity detection and ALP activity, add the polypeptide shown in SEQ ID No:1, make its final concentration be 10 μ g/mL.Detected result is in table 1.
Embodiment 1.3
The external short bone cell differentiation of the present embodiment for illustration of polypeptide of the present invention and the effect of propagation.
Carry out the osteoblastic cultivation of MC3T3-E1 according to the method in embodiment 1.1 and go down to posterity, the osteoblastic proliferation activity of MC3T3-E1 detects and the detection of ALP activity, unlike, in the detection of osteoblastic proliferation activity detection and ALP activity, add the polypeptide shown in SEQ ID No:1, make its final concentration be 1000 μ g/mL.Detected result is in table 1.
Embodiment 1.4
The external short bone cell differentiation of the present embodiment for illustration of polypeptide of the present invention and the effect of propagation.
Carry out the osteoblastic cultivation of MC3T3-E1 according to the method in embodiment 1.1 and go down to posterity, the osteoblastic proliferation activity of MC3T3-E1 detects and the detection of ALP activity, unlike, in the detection of osteoblastic proliferation activity detection and ALP activity, add the polypeptide shown in SEQ ID No:1, make its final concentration be 1 μ g/mL.Detected result is in table 1.
Embodiment 1.5
The external short bone cell differentiation of the present embodiment for illustration of polypeptide of the present invention and the effect of propagation.
Carry out the osteoblastic cultivation of MC3T3-E1 according to the method in embodiment 1.1 and go down to posterity, the osteoblastic proliferation activity of MC3T3-E1 detects and the detection of ALP activity, unlike, in the detection of osteoblastic proliferation activity detection and ALP activity, what add is the polypeptide shown in SEQ ID No:2.Detected result is in table 1.
Embodiment 1.6-1.90
For illustration of the external short bone cell differentiation of polypeptide of the present invention and the effect of propagation.
Carry out the osteoblastic cultivation of MC3T3-E1 according to the method in embodiment 1.1 and go down to posterity, the detection of the osteoblastic proliferation rate detection of MC3T3-E1 and ALP activity, unlike, in the detection of osteoblastic proliferation activity detection and ALP activity, the 1-27 position for the aminoacid sequence shown in SEQID No:2 provided by the invention added respectively, 1-28 position, 1-29 position, 1-30 position, 1-31 position, 1-32 position, 1-33 position, 1-34 position, 1-35 position, 1-36 position, 1-37 position, 1-38 position, 1-39 position, 1-40 position, 1-41 position, 1-42 position, 1-43 position, 1-44 position, 1-45 position, 1-46 position, 1-47 position, 1-48 position, 1-49 position, 1-50 position, 1-51 position, 1-52 position, 1-53 position, 1-54 position, 1-55 position, 1-56 position, 1-57 position, 1-58 position, 1-59 position, 1-60 position, 1-61 position, 1-62 position, 1-63 position, 1-64 position, 1-65 position, 1-66 position, 1-67 position, 1-68 position, 1-69 position, 1-70 position, 1-71 position, 1-72 position, 1-73 position, 1-74 position, 1-75 position, 1-76 position, 1-77 position, 1-78 position, 1-79 position, 1-80 position, 1-81 position, 1-82 position, 1-83 position, 1-84 position, 1-85 position, 1-86 position, 1-87 position, 1-88 position, 1-89 position, 1-90 position, 1-91 position, 1-92 position, 1-93 position, 1-94 position, 1-95 position, 1-96 position, 1-97 position, 1-98 position, 1-99 position, 1-100 position, 1-101 position, 1-102 position, 1-103 position, 1-104 position, 1-105 position, 1-106 position, 1-107 position, 1-108 position, 1-109 position, polypeptide shown in 1-110 position and 1-111 position.Detected result in detected result and embodiment 1.1 is similar.
Comparative example 1.1
The external short bone cell differentiation of this comparative example for illustration of the lactoferrin of prior art and the effect of propagation.
Carry out the osteoblastic cultivation of MC3T3-E1 according to the method in embodiment 1.1 and go down to posterity, the osteoblastic proliferation activity of MC3T3-E1 detects and the detection of ALP activity, unlike, in the detection of osteoblastic proliferation activity detection and ALP activity, the lactoferrin for prior art added.Detected result is in table 1.
Table 1
ALP is a kind of zymoprotein of osteoblast differentiation Early insulin secretion, has higher specificity, one of high expression level earlier specificity mark being considered to scleroblast epimatrix maturation of its activity.ALP on the one hand can hydrolyse phosphate esters in osteoplastic process, and the deposition for hydroxyapatite provides required phosphoric acid, can be hydrolyzed tetra-sodium on the other hand, removes the restraining effect that it is formed osteocyte, thus is conducive to the deposition of paddy.The expression level of ALP indicates the beginning of osteoblast differentiation, and its content can reflect osteoblastic differentiation degree and functional status.
As can be seen from Table 1, polypeptide provided by the invention is compared with control group, and the appreciation rate of osteocyte and the activity of ALP all obtain the raising of significance, illustrates that polypeptide provided by the invention can promote differentiation and the propagation of osteocyte.By embodiment 1.1 compared with comparative example 1.1, can find out, polypeptide provided by the invention can more effectively promote osteoblastic Differentiation and proliferation compared with the lactoferrin of prior art.Can be found out by embodiment 1.1-1.4, when the content of polypeptide provided by the invention is lower, main manifestations is the differentiation promoting osteocyte, and when content is higher, main manifestations is the propagation promoting osteocyte.
Embodiment 2.1-2.90
For illustration of the effect of urging bone cell differentiation and propagation in the body of polypeptide of the present invention.
The all polypeptide prepared in preparation example are dissolved in physiological saline, are mixed with the polypeptide solution that concentration is 500 μ g/mL respectively, in addition, more respectively compound concentration be 10 μ g/mL, the polypeptide solution shown in the SEQ ID No:1 of 1000 μ g/mL and 1 μ g/mL.
(1) grouping of laboratory animal
Select closed colony CD1(ICR) Swiss mice, male, surrounding age.Test is divided into 91 groups, is grouped as follows:
Embodiment 2.1-2.87: the polypeptide shown in SEQ ID No:1 injecting 500 μ g/mL respectively, polypeptide shown in SEQID No:2, the 1-27 position of the aminoacid sequence shown in SEQ ID No:2, 1-28 position, 1-29 position, 1-30 position, 1-31 position, 1-32 position, 1-33 position, 1-34 position, 1-35 position, 1-36 position, 1-37 position, 1-38 position, 1-39 position, 1-40 position, 1-41 position, 1-42 position, 1-43 position, 1-44 position, 1-45 position, 1-46 position, 1-47 position, 1-48 position, 1-49 position, 1-50 position, 1-51 position, 1-52 position, 1-53 position, 1-54 position, 1-55 position, 1-56 position, 1-57 position, 1-58 position, 1-59 position, 1-60 position, 1-61 position, 1-62 position, 1-63 position, 1-64 position, 1-65 position, 1-66 position, 1-67 position, 1-68 position, 1-69 position, 1-70 position, 1-71 position, 1-72 position, 1-73 position, 1-74 position, 1-75 position, 1-76 position, 1-77 position, 1-78 position, 1-79 position, 1-80 position, 1-81 position, 1-82 position, 1-83 position, 1-84 position, 1-85 position, 1-86 position, 1-87 position, 1-88 position, 1-89 position, 1-90 position, 1-91 position, 1-92 position, 1-93 position, 1-94 position, 1-95 position, 1-96 position, 1-97 position, 1-98 position, 1-99 position, 1-100 position, 1-101 position, 1-102 position, 1-103 position, 1-104 position, 1-105 position, 1-106 position, 1-107 position, 1-108 position, 1-109 position, the pharmaceutical composition that polypeptide shown in 1-110 position and 1-111 position is prepared with physiological saline respectively,
Embodiment 2.88: inject pharmaceutical composition prepared by the polypeptide shown in SEQ ID No:1 of 10 μ g/mL and physiological saline;
Embodiment 2.89: inject pharmaceutical composition prepared by the polypeptide shown in SEQ ID No:1 of 1000 μ g/mL and physiological saline;
Embodiment 2.90: inject pharmaceutical composition prepared by the polypeptide shown in SEQ ID No:1 of 1 μ g/mL and physiological saline;
Blank: the physiological saline of injection and pharmaceutical composition same dose.
Test and within 0 day, start to carry out subcutaneous injection by predetermined dose medicine to often organizing right side of mice skull surface, each 50 μ L, each group injects twice every day, continuously injection 5 days.The 22nd day cervical dislocation in test puts to death animal.
(2) detection of breeding is carried out in skull position osteocyte HE dyeing
By above-mentioned model mice in injection beginning the 22nd day, cervical dislocation is put to death, get operative site skull, rejecting head-face skin fat, puts head in freshly prepared 10%(v/v) fix 24h in formalin stationary liquid, again reject the outer soft tissue of skull, change Fresh fixative and fix 24h again, before decalcification, with flowing tap water sample, EDTA decalcification 50 days.Entrusted by fixed position Xuebang, Beijing Science and Technology Ltd. to carry out HE dyeing afterwards, and adopt the proliferative conditions of bitubular electric light microscopic examination skull surface.Embodiment 2.1(injects the polypeptide shown in SEQ ID No:1 of 500 μ g/mL) and blank HE picture as depicted in figs. 1 and 2, the HE picture of embodiment 2.2-2.90 and Fig. 1 similar.
Comparative example 2.1
The present embodiment is for illustration of the effect of urging bone cell differentiation and propagation in the body of polypeptide of the present invention.
Carry out process and the HE dyeing of laboratory animal according to the method for embodiment 2.1 and observe, unlike, the lactoferrin for prior art injected in this comparative example.The observations of HE section as shown in Figure 3.
Can be found out by Fig. 1, Fig. 2 and Fig. 3, inject the mouse of polypeptide provided by the invention identical with injecting normal saline blank group be that bone structure is even, visible less bone dissolves region, proves that polypeptide provided by the invention can not the active information of osteoclast.But inject the mouse of polypeptide provided by the invention compared with the mouse of injecting normal saline, its skull surface has obvious hyperplastic tissue to occur in periosteum position, the mesenchymal cell of a large amount of hyperplasia, new vessel and a large amount of scleroblasts is had to be attached to surface (in figure, the position of " ↑ " arrow points is scleroblast), the visible a large amount of area of new bone of outermost layer, in woven bone spline structure, new bone is directly connected with lamina externa cranii.Fig. 1 and Fig. 3 is compared and can find out, the osteoblastic quantity of injecting the mouse neonatal of polypeptide provided by the invention much larger than the osteoblastic newborn quantity of the mouse of the existing lactoferrin of injection, will prove that polypeptides exhibit provided by the invention has gone out the effect of the better promotion mouse bone-forming cell growth than prior art.
And polypeptide length of the present invention is short, being especially less than 100 amino acid whose polypeptide of the present invention can be obtained, therefore, it is possible to produce on a large scale in a large number by the method for synthesis.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each the concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode.In order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.

Claims (10)

1. a peptide species, is characterized in that, this polypeptide has the aminoacid sequence shown in SEQ ID No:1; And the sequence of this polypeptide is the aminoacid sequence shown in SEQ ID No:2, or it is the fragment of the aminoacid sequence shown in SEQ ID No:2.
2. polypeptide according to claim 1, wherein, the sequence of described polypeptide is the aminoacid sequence shown in SEQ ID No:1 or SEQ ID No:2.
3. a nucleic acid, is characterized in that, this polypeptide described in nucleic acid encoding claim 1 or 2.
4. nucleic acid according to claim 3, wherein, the sequence of this nucleic acid is the nucleotide sequence shown in SEQ ID No:3 or SEQ ID No:4.
5. the polypeptide described in claim 1 or 2 and/or the application of the nucleic acid described in claim 3 or 4 in the medicine for the preparation of promotion bone cell differentiation and/or propagation and/or treatment osteoporosis.
6. a pharmaceutical composition, is characterized in that, this pharmaceutical composition contains the polypeptide described in claim 1 or 2.
7. pharmaceutical composition according to claim 6, wherein, with the gross weight of pharmaceutical composition described in 1g for benchmark, the content of described polypeptide is 1-1000 μ g.
8. the pharmaceutical composition according to claim 6 or 7, wherein, the purity of described polypeptide is more than 95 % by weight.
9. pharmaceutical composition according to claim 6, wherein, this pharmaceutical composition is also containing pharmaceutically acceptable assistant agent.
10. pharmaceutical composition according to claim 9, wherein, described pharmaceutically acceptable assistant agent is physiological saline and/or glucose solution.
CN201310228448.4A 2013-06-08 2013-06-08 Polypeptide and the nucleic acid and pharmaceutical composition for encoding the polypeptide Active CN104231058B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310228448.4A CN104231058B (en) 2013-06-08 2013-06-08 Polypeptide and the nucleic acid and pharmaceutical composition for encoding the polypeptide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310228448.4A CN104231058B (en) 2013-06-08 2013-06-08 Polypeptide and the nucleic acid and pharmaceutical composition for encoding the polypeptide

Publications (2)

Publication Number Publication Date
CN104231058A true CN104231058A (en) 2014-12-24
CN104231058B CN104231058B (en) 2019-01-22

Family

ID=52220045

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310228448.4A Active CN104231058B (en) 2013-06-08 2013-06-08 Polypeptide and the nucleic acid and pharmaceutical composition for encoding the polypeptide

Country Status (1)

Country Link
CN (1) CN104231058B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105254750A (en) * 2015-10-16 2016-01-20 上海交通大学 Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007043900A1 (en) * 2005-10-14 2007-04-19 Auckland Uniservices Limited Use of lactoferrin fragments and hydrolysates

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007043900A1 (en) * 2005-10-14 2007-04-19 Auckland Uniservices Limited Use of lactoferrin fragments and hydrolysates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MARCHLER-BAUER A等: "CDD:a Conserved Domain Database for the fuctional annotation of proteins", 《NUCLEIC ACIDS RES.》 *
SEYFERT,H.: "AAA30616.1", 《GENBANK》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105254750A (en) * 2015-10-16 2016-01-20 上海交通大学 Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof
CN105254750B (en) * 2015-10-16 2019-01-08 浙江辉肽生命健康科技有限公司 A kind of biologically active polypeptide FGYSGAFKCL and its preparation and application

Also Published As

Publication number Publication date
CN104231058B (en) 2019-01-22

Similar Documents

Publication Publication Date Title
Chen et al. Parathyroid hormone and its related peptides in bone metabolism
CN103667182B (en) A kind of mesenchymal stem cells MSCs is in vitro to induction method and the inducing culture of osteoblast differentiation
CN110577931B (en) Intermittent hypoxia treatment stem cell source exosome and application thereof in myocardial tissues
JP2009028056A (en) Monoclonal antibody specific for marrow-derived mesenchymal cell
CN108348555A (en) Methods for cell expansion and therapeutic combination
KR101001117B1 (en) Composition for coating support for preparation of cell sheet, support for preparation of cell sheet and process for producing cell sheet
CN108823172A (en) The preparation and application of giant panda luteotropin β subunit monoclonal antibody
Qi et al. An oriented‐collagen scaffold including Wnt5a promotes osteochondral regeneration and cartilage interface integration in a rabbit model
EP1664280A1 (en) Skin regeneration system
CN103814124A (en) In vitro cardiovascular model
US20140200174A1 (en) Peptide bfp4 for promoting osteogenesis or vascularization and use thereof
CN1865436B (en) Method of separating derma epidermis stem cell
CN103966167A (en) Cell culture composition used for primary culture of tumor cells and application thereof
CN107353333A (en) A kind of bone protectiveness molecule Semaphorine3A and its pharmacy application
CN107073067A (en) Use the treatment of the new cornea of laminin
RU2012156861A (en) CHIMERS OF VITRONECTIN: KERATINOCYTES GROWTH FACTOR
CN107446022B (en) Polypeptide PIP-14 capable of antagonizing RNA binding activity of PARP1 protein and application thereof
CN104231058A (en) Polypeptide, nucleic acid coding polypeptide and pharmaceutical composition
CN112725268A (en) Composition for promoting osteogenic differentiation of adipose-derived stem cells and application thereof
CN114836377B (en) Stem cell in vitro osteogenic induction differentiation method
CN112430269B (en) Composition for promoting osteogenic differentiation of umbilical cord blood stem cells and application thereof
CN107384862A (en) The preparation method and kit of the schwann cell in MSCs sources
CN110354254A (en) Antler polypeptide is preparing the purposes in the drug for treating bone and flesh tumor metastasis
CN104861076B (en) A kind of fused polypeptide and its application in skeletonization and vascularization is promoted
CN103387605A (en) RTN4B polypeptide and monoclonal antibody thereof, hybridoma cell strain generating monoclonal antibody, and preparation and application of RTN4B polypeptide, monoclonal antibody and hybridoma cell strain

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