CN104231058B - Polypeptide and the nucleic acid and pharmaceutical composition for encoding the polypeptide - Google Patents
Polypeptide and the nucleic acid and pharmaceutical composition for encoding the polypeptide Download PDFInfo
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- CN104231058B CN104231058B CN201310228448.4A CN201310228448A CN104231058B CN 104231058 B CN104231058 B CN 104231058B CN 201310228448 A CN201310228448 A CN 201310228448A CN 104231058 B CN104231058 B CN 104231058B
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- C07—ORGANIC CHEMISTRY
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- 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
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Abstract
The invention discloses a kind of polypeptides, wherein the polypeptide has amino acid sequence shown in SEQ ID No:1；And the sequence of the polypeptide is amino acid sequence shown in SEQ ID No:2, or the segment for amino acid sequence shown in SEQ ID No:2.Polypeptide provided by the invention has the activity of higher promotion bone cell differentiation and/or proliferation.
Description
Technical field
The present invention relates to a kind of polypeptides, encode the nucleic acid of the polypeptide, the polypeptide and/nucleic acid in preparation for promoting bone thin
Application and a kind of pharmaceutical composition containing the polypeptide in the drug of born of the same parents' differentiation and/or proliferation and/or treatment osteoporosis
Object.
Background technique
Osteoporosis is the common disease of the middle-aged and the old especially postmenopausal women, frequently-occurring disease, and it is big to occupy the middle-aged and the old five
First of disease illness rate, there are about 10% people to suffer from this disease in the whole world, and with the exacerbation of aging degree, situation of falling ill is increasingly severe.
The development of the drug of bone cell differentiation and/or proliferation and exploitation is especially promoted to have become doctor in relation to treatment osteoporosis agents
One of heat subject of medicine circle.
Lactoferrin is nontoxic, harmless mainly from breast milk, has in terms of promoting bone cell differentiation and/or proliferation
Remarkable effect, therefore osteoporosis can be effectively treated, in recent years, show huge treatment and prevention bone disease
The potentiality to be exploited of disease.Existing research confirms that lactoferrin shows powerful osteogenic activity in vitro, it can effectively promote
Into the proliferation and differentiation of osteoblast.
However, lactoferrin molecules amount is about 80kDa, contain about 700 amino acid, since its biggish molecular weight makes it
It is difficult to be produced on a large scale.Also, the activity of lactoferrin still needs to be further increased.
Summary of the invention
It is an object of the invention to overcome the lactoferrin of the prior art to be difficult to be mass produced, and its activity still into
The defect that one step improves, providing one kind can be mass produced, and the higher polypeptide of activity and encode the polypeptide nucleic acid and it
Application, and the pharmaceutical composition containing the polypeptide.
To achieve the goals above, in a first aspect, the present invention provides a kind of polypeptides, wherein the polypeptide has SEQ ID
Amino acid sequence shown in No:1；And the sequence of the polypeptide is amino acid sequence shown in SEQ ID No:2, or is SEQ ID
The segment of amino acid sequence shown in No:2.
Preferably, it is SEQ that the sequence of the polypeptide, which is the sequence of amino acid sequence shown in SEQ ID No:1 or the polypeptide,
Amino acid sequence shown in ID No:2.
Second aspect, the present invention provides a kind of nucleic acid, wherein nucleic acid encode polypeptide provided by the invention.
The third aspect, the present invention provides polypeptide as described above and/or nucleic acid in preparation for promoting bone cell differentiation
And/or proliferation and/or treatment osteoporosis drug in application.
Fourth aspect, the present invention provides a kind of pharmaceutical compositions, wherein the pharmaceutical composition contains provided by the invention
Polypeptide.
The polypeptide provided by the invention and existing it can be seen from above-mentioned technical proposal and the embodiment of the present invention and comparative example
Have the advantages that the lactoferrin of technology has molecular weight short, therefore, can easily be done large-scale production, is its industrialization
It provides the foundation；Also, polypeptide provided by the invention has promotion bone cell differentiation more higher than the lactoferrin of the prior art
And/or the activity of proliferation.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the microscope photograph for injecting the mouse periosteum of polypeptide shown in SEQ ID No:1；The position that arrow is directed toward is
Osteoblast in mouse periosteum.
Fig. 2 is the microscope photograph of the mouse periosteum bone of injecting normal saline；The position that arrow is directed toward is in mouse periosteum
Osteoblast.
Fig. 3 is the microscope photograph for injecting the mouse periosteum of lactoferrin of the prior art；The position that arrow is directed toward is small
Osteoblast in mouse periosteum.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
According to the first aspect of the invention, a kind of polypeptide is provided, wherein the polypeptide has shown in SEQ ID No:1
Amino acid sequence；And the sequence of the polypeptide is amino acid sequence shown in SEQ ID No:2, or for shown in SEQ ID No:2
Amino acid sequence segment.
According to the present invention, the sequence of the polypeptide has amino acid sequence shown in SEQ ID No:1, and is SEQ ID
The segment of amino acid sequence shown in No:2 refers to that the sequence of the polypeptide is the of amino acid sequence shown in SEQID No:2
1-26,1-27,1-28,1-29,1-30,1-31,1-32,1-33,1-34,1-35,1-36,
1-37,1-38,1-39,1-40,1-41,1-42,1-43,1-44,1-45,1-46,1-47,
1-48,1-49,1-50,1-51,1-52,1-53,1-54,1-55,1-56,1-57,1-58,
1-59,1-60,1-61,1-62,1-63,1-64,1-65,1-66,1-67,1-68,1-69,
1-70,1-71,1-72,1-73,1-74,1-75,1-76,1-77,1-78,1-79,1-80,
1-81,1-82,1-83,1-84,1-85,1-86,1-87,1-88,1-89,1-90,1-91,
1-92,1-93,1-94,1-95,1-96,1-97,1-98,1-99,1-100,1-101,1-102
Position, 1-103,1-104,1-105,1-106,1-107,1-108,1-109,1-110 and 1-111 institutes
Any one in the amino acid sequence shown.
In preferred situation, the sequence of the polypeptide is amino acid sequence shown in SEQ ID No:1 or SEQ ID No:2.
According to the present invention, the source of the polypeptide is not particularly limited, for example, can be from existing albumen, for example, day
Enzyme-specific solution is carried out in right lactoferrin sequence to obtain；It can also be by the gene constructed expression carrier of coding said polypeptide, so
After be transformed into microorganism and expressed, the polypeptide is then obtained by extraction purification；Can also conventionally it consolidate
It is combined to obtain, for example, synthetic method is referred 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) disclosed in method；Synesis Company can also be entrusted to be closed
At for example, Beijing visual field Heng Yu Bioisystech Co., Ltd can be entrusted to be synthesized.
Under normal conditions, it when the amino acid number of polypeptide is greater than 100, is obtained using the method for conventional synthesis in solid state
It is more difficult.Therefore, a kind of preferred embodiment according to the present invention, the acquisition of the polypeptide for convenience, when the ammonia of polypeptide
When base acid number is 26-100, conventional synthesis in solid state can be carried out；When the amino acid number of polypeptide is greater than 100, lead to
It crosses and the gene of coding said polypeptide is carried out to conventional protokaryon and/or eukaryotic expression, this is then obtained by table by extraction purification
The polypeptide reached.Wherein, prokaryotic expression, and extract and purification of target product method it is known to those skilled in the art, tool
Body is referred to the method in " Molecular Cloning:A Laboratory guide ".
Second aspect, the invention discloses a kind of nucleic acid, wherein nucleic acid encode polypeptide provided by the invention.
A kind of preferred embodiment according to the present invention, polypeptide shown in SEQ ID No:1 is as shown in SEQ ID No:3
Nucleic acid encode obtains, and the nucleic acid encode as shown in SEQ ID No:4 of polypeptide shown in SEQ ID No:2 obtains.Art technology
Personnel are, it is understood that the nucleic acid coding sequence for other polypeptides that first aspect present invention provides can be by selecting and adding
The mode of codon is accordingly obtained according to nucleotide sequence shown in SEQ ID No:3 and SEQ ID No:4, not another herein
One enumerates.
According to the present invention, the nucleotide sequence of coding said polypeptide provided by the invention is not limited in core as described above
Nucleotide sequence mentions as long as it can encode polypeptide provided by the invention for example, encoding the present invention in above-mentioned nucleotide sequence
The codon of certain monoamino-acid in the polypeptide of confession can also be the synonym of the codon.
The third aspect, the present invention also provides polypeptide as described above and/or nucleic acid in preparation for promoting osteocyte point
Application in the drug of change and/or proliferation and/or treatment osteoporosis.
According to the present invention, the polypeptide has the effect of promoting bone cell differentiation and/or proliferation, so as to improve sclerotin
Loose symptom, therefore, polypeptide and/or nucleic acid provided by the invention, which can be applied, promotes bone cell differentiation and/or increasing in preparation
It grows and/or treats in the drug of osteoporosis.
According to the present invention, the type of the drug is not particularly limited, if its contain polypeptide provided by the invention and/
Or nucleic acid.It can also be pharmaceutical drugs for example, can be health care product.
Based on this, fourth aspect, the present invention provides a kind of pharmaceutical compositions, wherein the pharmaceutical composition contains this hair
The polypeptide of bright offer.
As long as although can promote bone cell differentiation and/or increasing containing polypeptide provided by the invention in described pharmaceutical composition
It grows, so as to achieve the effect that treat osteoporosis, but it was found by the inventors of the present invention that in the described pharmaceutical composition,
On the basis of the total weight of 1g described pharmaceutical composition, when the content of the polypeptide is 1-1000 μ g, it can significantly promote bone
The differentiation and/or proliferation of cell, to keep the better effect for treating osteoporosis obvious.The present inventor is again unexpected
It was found that when the content of the polypeptide is 1-100 μ g, can significantly promote when on the basis of the total weight of 1g described pharmaceutical composition
Into the differentiation of osteocyte；When the content of the polypeptide is 400-600 μ g, the proliferation of osteocyte can be remarkably promoted.
According to the present invention, when the purity of polypeptide in described pharmaceutical composition is more than 95 weight %, can further promote
Bone cell differentiation and/or proliferation.
Pharmaceutical composition according to the present invention, wherein also containing pharmaceutically acceptable adjuvant, for the kind of the adjuvant
Class is known to those skilled in the art, and the adjuvant is preferably physiological saline and/or glucose solution.In addition, according to difference
Needs, the concentration of the glucose can be 5-50 weight %.
The present invention will be described in detail by way of examples below.In following preparation example, embodiment and comparative example:
Lactoferrin is purchased from Yosica company, is bovine lactoferrin, and purity is 95 weight %；
MC3T3-E1 osteoblast system (U.S.'s ATCC Culture Collection Center), purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (on
Sea)；
α-MEM culture medium is purchased from U.S. Hyclone；Fetal calf serum is purchased from PAP company；
Trypsase is purchased from Sigma-Aldrich company；
BrdU cell proliferation detecting kit is purchased from U.S. Roche company；
Swiss mice ties up Animal Science Co., Ltd of tonneau China purchased from Beijing；
Bitubular electric light microscope is purchased from Olympus company of Japan, model OLYMPUS-BHT；
The mixed liquor of cell pyrolysis liquid and working solution: by the Na of 5.5mL0.1M2CO3With the NaHCO of 4.5mL0.1M3Mixing
1% Triton X-100 is added afterwards, adds 22.3mg p-nitrophenylphosphate (pNPP, purchased from Sigma-Aldrich public affairs
Department), the MgSO of 4.9mg4·7H2O；
Terminate liquid: the sodium hydroxide solution of 8 weight %.
Preparation example
(1) the commission visual field Beijing Heng Yu Bioisystech Co., Ltd is respectively synthesized polypeptide shown in SEQ ID No:1, and
1-27,1-28,1-29,1-30,1-31,1-32,1-33 of amino acid sequence shown in SEQ ID No:2
Position, 1-34,1-35,1-36,1-37,1-38,1-39,1-40,1-41,1-42,1-43,1-44
Position, 1-45,1-46,1-47,1-48,1-49,1-50,1-51,1-52,1-53,1-54,1-55
Position, 1-56,1-57,1-58,1-59,1-60,1-61,1-62,1-63,1-64,1-65,1-66
Position, 1-67,1-68,1-69,1-70,1-71,1-72,1-73,1-74,1-75,1-76,1-77
Position, 1-78,1-79,1-80,1-81,1-82,1-83,1-84,1-85,1-86,1-87,1-88
Position, 1-89,1-90,1-91,1-92,1-93,1-94,1-95,1-96,1-97,1-98,1-99
Polypeptide shown in position and 1-100, totally 75 polypeptides.
(2) it is obtained shown in polypeptide shown in SEQ ID No:2 and SEQ ID No:2 using the method for prokaryotic expression
1-101,1-102,1-103,1-104,1-105,1-106,1-107,1-108 of amino acid sequence
Position, 1-109, polypeptide shown in 1-110 and 1-111, totally 12 polypeptides.
Wherein, for the acquisition of target gene, the selection of carrier, the building and conversion, host cell of recombinant expression carrier
Selection, the inducing expression of expression vector, the method for extraction and purification of target product be not belonging to invention scope of the invention,
This is no longer described in detail, the method being specifically referred in " Molecular Cloning:A Laboratory guide ".
(3) a small amount of above-mentioned 87 polypeptides is taken to carry out Mass Spectrometric Identification, molecular weight obtained by mass spectrum and the meter obtained according to sequence
Calculation value is consistent, it was demonstrated that 87 polypeptides are polypeptide provided by the invention.And purity is more than 95 weight %.Wherein, SEQ ID
The theoretical molecular weight of polypeptide shown in No:1 is 2729.25, and it is shown in 2729.60, SEQ ID No:2 that mass spectrum, which measures molecular weight,
Theoretical molecular weight is 12152.00, and it is 12151.97 that mass spectrum, which measures molecular weight,.
Embodiment 1.1
The present embodiment is used to illustrate the effect of external the rush bone cell differentiation and proliferation of polypeptide of the invention.
(1) culture and passage of MC3T3-E1 osteoblast
Culture: with α-MEM culture medium (containing 10%(v/v) fetal calf serum of 10mL) suspension MC3T3-E1 osteoblast, and connect
Kind is placed in 37 DEG C, 5%CO in culture bottle2Culture, changes the liquid once for 24 hours, changes the liquid once within every 2 days later in incubator.
Passage: the culture solution in culture bottle is gently discarded with suction pipe to when converging, uses 10mL by osteoblast monolayer growth
The PBS buffer solution of left and right washes away culture medium remaining in culture bottle；2mL digestive juice (containing 0.25%(w/v) trypsase is added
PBS buffer solution), jog culture bottle makes trypsase be paved with cell surface, is placed in incubator, will be thin with suction pipe after 4 minutes
Born of the same parents blow down from bottle wall, suck in centrifuge tube, and 1000rpm is centrifuged 5min, collect precipitating, and cell is resuspended with culture medium, are inoculated in new
Culture bottle in continue to cultivate.It repeats this step and cell was reached into for the tenth generation.
(2) proliferation activity of MC3T3-E1 osteoblast
By the cell after cultivating and passing in step (1) with 2 × 104The concentration of a/mL is inoculated in 96 well culture plates, often
Hole is inoculated with 100 μ L, cultivates in the complete α-MEM culture medium containing 10% serum, replaces serum free medium afterwards for 24 hours, continue to cultivate
Polypeptide shown in SEQ ID No:1 is added afterwards for 24 hours, makes its final concentration of 500 μ g/mL, separately setting a blank control, (only addition is without blood
Clear culture medium does not add polypeptide of the invention), 6 parallel holes are arranged in each concentration gradient, use BrdU- after acting on 48h
ELISA method (uses 5- bromodeoxyuridine nucleosides (5-bromo-2-deoxyuridine, BrdU) cell Proliferation detection reagent
Box) measurement cell proliferation rate.Appreciation rate is indicated with calculating the opposite appreciation rate obtained relative to blank control group, the results are shown in Table 1.
(3) alkaline phosphatase (ALP) active detection
By the cell after cultivating and passing in step (1) with 4 × 105A/mL density is inoculated in 96 well culture plates, and every hole connects
In the complete medium containing 10% serum after culture to cell fusion, polypeptide shown in SEQ ID No:1 is added in 200 μ L of kind,
Make its final concentration of 500 μ g/mL.A blank control (not adding any drug in cell culture fluid) separately is set, each concentration sets 6
Parallel hole, continue culture effect 48h after, with 1%(w/v) PBS washing after, measurement ALP activity.Specific steps are as follows:
The 96 orifice plates drying that PBS is washed, the mixed liquor of 100 μ L cell pyrolysis liquids and working solution is added in every hole；Oscillator
Vibrate 1min, be placed under the conditions of 37 DEG C of constant incubators and react 30min；80 μ L terminate liquids are added in every hole, vibrate 1min, enzyme
Mark instrument measures light absorption value at 405nm wavelength；It is calculated compared with blank control group according to surveyed light absorption value and obtains the relatively living of ALP
Property, it the results are shown in Table 1.
Embodiment 1.2
The present embodiment is used to illustrate the effect of external the rush bone cell differentiation and proliferation of polypeptide of the invention.
It is thin according to the culture and passage, MC3T3-E1 skeletonization of the method progress MC3T3-E1 osteoblast in embodiment 1.1
The proliferation activity of born of the same parents detects and the active detection of ALP, unlike, the proliferation activity detection and ALP activity of osteoblast
Detection in, be added SEQ ID No:1 shown in polypeptide, make its final concentration of 10 μ g/mL.Testing result is shown in Table 1.
Embodiment 1.3
The present embodiment is used to illustrate the effect of external the rush bone cell differentiation and proliferation of polypeptide of the invention.
It is thin according to the culture and passage, MC3T3-E1 skeletonization of the method progress MC3T3-E1 osteoblast in embodiment 1.1
The proliferation activity of born of the same parents detects and the active detection of ALP, unlike, the proliferation activity detection and ALP activity of osteoblast
Detection in, be added SEQ ID No:1 shown in polypeptide, make its final concentration of 1000 μ g/mL.Testing result is shown in Table 1.
Embodiment 1.4
The present embodiment is used to illustrate the effect of external the rush bone cell differentiation and proliferation of polypeptide of the invention.
It is thin according to the culture and passage, MC3T3-E1 skeletonization of the method progress MC3T3-E1 osteoblast in embodiment 1.1
The proliferation activity of born of the same parents detects and the active detection of ALP, unlike, the proliferation activity detection and ALP activity of osteoblast
Detection in, be added SEQ ID No:1 shown in polypeptide, make its final concentration of 1 μ g/mL.Testing result is shown in Table 1.
Embodiment 1.5
The present embodiment is used to illustrate the effect of external the rush bone cell differentiation and proliferation of polypeptide of the invention.
It is thin according to the culture and passage, MC3T3-E1 skeletonization of the method progress MC3T3-E1 osteoblast in embodiment 1.1
The proliferation activity of born of the same parents detects and the active detection of ALP, unlike, the proliferation activity detection and ALP activity of osteoblast
Detection in, addition is polypeptide shown in SEQ ID No:2.Testing result is shown in Table 1.
Embodiment 1.6-1.90
For illustrating the effect of external the rush bone cell differentiation and proliferation of polypeptide of the invention.
It is thin according to the culture and passage, MC3T3-E1 skeletonization of the method progress MC3T3-E1 osteoblast in embodiment 1.1
The proliferation rate of born of the same parents detects and the active detection of ALP, unlike, the proliferation activity detection of osteoblast and ALP are active
In detection, be separately added into be amino acid sequence shown in SEQID No:2 provided by the invention 1-27,1-28,1-
29,1-30,1-31,1-32,1-33,1-34,1-35,1-36,1-37,1-38,1-39,1-
40,1-41,1-42,1-43,1-44,1-45,1-46,1-47,1-48,1-49,1-50,1-
51,1-52,1-53,1-54,1-55,1-56,1-57,1-58,1-59,1-60,1-61,1-
62,1-63,1-64,1-65,1-66,1-67,1-68,1-69,1-70,1-71,1-72,1-
73,1-74,1-75,1-76,1-77,1-78,1-79,1-80,1-81,1-82,1-83,1-
84,1-85,1-86,1-87,1-88,1-89,1-90,1-91,1-92,1-93,1-94,1-
95,1-96,1-97,1-98,1-99,1-100,1-101,1-102,1-103,1-104,1-
Polypeptide shown in 105,1-106,1-107,1-108,1-109,1-110 and 1-111.Testing result and reality
The testing result applied in example 1.1 is similar.
Comparative example 1.1
This comparative example is used to illustrate the effect of external the rush bone cell differentiation and proliferation of the lactoferrin of the prior art.
It is thin according to the culture and passage, MC3T3-E1 skeletonization of the method progress MC3T3-E1 osteoblast in embodiment 1.1
The proliferation activity of born of the same parents detects and the active detection of ALP, unlike, the proliferation activity detection and ALP activity of osteoblast
Detection in, addition is the lactoferrin of the prior art.Testing result is shown in Table 1.
Table 1
ALP is a kind of zymoprotein of osteoblast differentiation Early insulin secretion, specificity with higher, active high expression
It is considered as one of the earlier specificity label of osteoblast epimatrix maturation.ALP on the one hand can be with during bon e formation
Hydrolyse phosphate esters provide required phosphoric acid for the deposition of hydroxyapatite, on the other hand can hydrolyze pyrophosphoric acid, release it to bone
Plastidogenetic inhibiting effect, to be conducive to the deposition of paddy.The expression of ALP indicates the beginning of osteoblast differentiation,
Its content can reflect the differentiation degree and functional status of osteoblast.
As can be seen from Table 1, polypeptide provided by the invention compared with the control group, the appreciation rate of osteocyte and the activity of ALP
The raising for having obtained conspicuousness illustrates that polypeptide provided by the invention can promote the differentiation and proliferation of osteocyte.By embodiment
1.1 compared with comparative example 1.1, it can be seen that polypeptide provided by the invention can be more effectively compared with the lactoferrin of the prior art
Promote the differentiation and proliferation of osteoblast.By embodiment 1.1-1.4 can be seen that the content when polypeptide provided by the invention compared with
When low, it is mainly shown as the differentiation for promoting osteocyte, the proliferation for promoting osteocyte is mainly shown as when content is higher.
Embodiment 2.1-2.90
For illustrating the effect of internal the rush bone cell differentiation and proliferation of polypeptide of the invention.
All polypeptides prepared in preparation example are dissolved in physiological saline, are configured to the polypeptide that concentration is 500 μ g/mL respectively
Solution, in addition, it is molten to distinguish polypeptide shown in the SEQ ID No:1 that compound concentration is 10 μ g/mL, 1000 μ g/mL and 1 μ g/mL again
Liquid.
(1) grouping of experimental animal
Select closed colony CD1(ICR) Swiss mice, male, four week old.Test is divided into 91 groups, is grouped as follows:
Embodiment 2.1-2.87: polypeptide shown in the SEQ ID No:1 of 500 μ g/mL is injected respectively, shown in SEQID No:2
Polypeptide, 1-27,1-28,1-29,1-30,1-31,1-32 of amino acid sequence shown in SEQ ID No:2
Position, 1-33,1-34,1-35,1-36,1-37,1-38,1-39,1-40,1-41,1-42,1-43
Position, 1-44,1-45,1-46,1-47,1-48,1-49,1-50,1-51,1-52,1-53,1-54
Position, 1-55,1-56,1-57,1-58,1-59,1-60,1-61,1-62,1-63,1-64,1-65
Position, 1-66,1-67,1-68,1-69,1-70,1-71,1-72,1-73,1-74,1-75,1-76
Position, 1-77,1-78,1-79,1-80,1-81,1-82,1-83,1-84,1-85,1-86,1-87
Position, 1-88,1-89,1-90,1-91,1-92,1-93,1-94,1-95,1-96,1-97,1-98
Position, 1-99,1-100,1-101,1-102,1-103,1-104,1-105,1-106,1-107,1-
The pharmaceutical composition that polypeptide shown in 108,1-109,1-110 and 1-111 is prepared with physiological saline respectively；
Embodiment 2.88: the pharmaceutical composition of polypeptide shown in the SEQ ID No:1 of 10 μ g/mL of injection and physiological saline preparation
Object；
Embodiment 2.89: the medicine group of polypeptide shown in the SEQ ID No:1 of 1000 μ g/mL of injection and physiological saline preparation
Close object；
Embodiment 2.90: the pharmaceutical composition of polypeptide shown in the SEQ ID No:1 of 1 μ g/mL of injection and physiological saline preparation
Object；
Blank control: the physiological saline of injection and pharmaceutical composition same dose.
Test starts that every group of right side of mice skull surface is subcutaneously injected by predetermined dose drug for 0 day, every time 50 μ L,
Each group is injected twice daily, continuous injection 5 days.Animal is put to death in the 22nd day cervical dislocation of test.
(2) osteocyte HE in skull position dyes the detection being proliferated
Above-mentioned model mice is started the 22nd day in injection, cervical dislocation is put to death, and operative site skull is taken, and rejects Head And Face
Skin fat sets head in the 10%(v/v of Fresh) in formalin fixer it is fixed for 24 hours, reject outside skull soft group again
It knits, changes that Fresh fixative is fixed again before decalcification, to rinse sample with flowing running water for 24 hours, EDTA decalcification 50 days.It will fix later
Position entrusts Beijing Xuebang Science and Technology Ltd. to carry out HE dyeing, and using the hyperplasia of the micro- sem observation skull surface of bitubular electric light
Situation.Embodiment 2.1(inject 500 μ g/mL SEQ ID No:1 shown in polypeptide) and blank control HE picture such as Fig. 1
With shown in Fig. 2, the HE picture of embodiment 2.2-2.90 is similar with Fig. 1.
Comparative example 2.1
The present embodiment is used to illustrate the effect of internal the rush bone cell differentiation and proliferation of polypeptide of the invention.
The processing of experimental animal is carried out according to the method for embodiment 2.1 and HE is dyed and observation, unlike, this comparison
Example in inject be the prior art lactoferrin.The observation result of HE slice is as shown in Figure 3.
It can be seen that mouse and the injecting normal saline blank for injecting polypeptide provided by the invention by Fig. 1, Fig. 2 and Fig. 3
Identical control group is that bone structure is uniform, it is seen that less bone dissolves region, it was demonstrated that polypeptide provided by the invention will not be osteoclastic thin
The active information of born of the same parents.But the mouse of polypeptide provided by the invention is injected compared with the mouse of injecting normal saline, skull surface
There is apparent hyperplastic tissue to occur in periosteum position, has the mesenchymal cell, new vessels and a large amount of skeletonization of a large amount of hyperplasia thin
Born of the same parents are attached to surface (position that " ↑ " arrow is directed toward in figure is osteoblast), and the visible a large amount of area of new bone of outermost layer are in woven bone sample
Structure, new bone are connected directly with lamina externa cranii.Fig. 1 and Fig. 3 are compared as can be seen that injecting polypeptide provided by the invention
The quantity of the osteoblast of mouse neonatal is significantly larger than the newborn quantity for injecting the osteoblast of mouse of existing lactoferrin, card
Bright polypeptides exhibit provided by the invention has gone out the effect for more preferably promoting mouse bone-forming cell growth than the prior art.
And polypeptide length of the invention is short, and the polypeptide of the invention of especially less than 100 amino acid can pass through conjunction
At method largely obtain, therefore, be able to carry out large-scale production.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, it can be combined in any appropriate way.In order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (9)
1. a kind of polypeptide, which is characterized in that the sequence of the polypeptide is amino acid sequence shown in SEQ ID No:1.
2. a kind of nucleic acid, which is characterized in that nucleic acid encode polypeptide described in claim 1.
3. nucleic acid according to claim 2, wherein the sequence of the nucleic acid is nucleotide sequence shown in SEQ ID No:3.
4. polypeptide described in claim 1 and/or nucleic acid described in claim 2 or 3 are in preparation for promoting bone cell differentiation
And/or proliferation and/or treatment osteoporosis drug in application.
5. a kind of pharmaceutical composition, which is characterized in that the pharmaceutical composition contains polypeptide described in claim 1.
6. pharmaceutical composition according to claim 5, wherein on the basis of the total weight of 1g described pharmaceutical composition, institute
The content for stating polypeptide is 1-1000 μ g.
7. pharmaceutical composition according to claim 5 or 6, wherein the purity of the polypeptide is more than 95 weight %.
8. pharmaceutical composition according to claim 5, wherein the pharmaceutical composition also contains pharmaceutically acceptable auxiliary
Agent.
9. pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable adjuvant be physiological saline and/
Or glucose solution.
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Non-Patent Citations (2)
| Title |
|---|
| AAA30616.1;Seyfert,H.;《GenBank》;20051007;1 |
| CDD:a Conserved Domain Database for the fuctional annotation of proteins;Marchler-Bauer A等;《Nucleic Acids Res.》;20111231;第39卷;225-9 |
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