CN105837682A - BMP-3 protein, and preparation method and application thereof as bone repair injection - Google Patents
BMP-3 protein, and preparation method and application thereof as bone repair injection Download PDFInfo
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Abstract
The present invention provides a method for extracting BMP-3 and preparing an injection containing BMP-3. The method extracts partially purified BMP-3, which is prepared into the injection together with collage, PEG 2000, poloxamer and PVP. The invention provides an injection using naturally extracted and partially purified BMP as base and an optimal carrier with sustained release effect and the preparation thereof. The method optimizes the process conditions in the extraction process, so as to obtain BMP with high purity, high activity and high usage rate; the method optimizes the injection carrier component and proportion; and the prepared bone repair injection has the advantages of small requirement amount, significant effect and easiness to use.
Description
[technical field]
The present invention relates to a kind of medical material and preparation method thereof, a kind of BMP-3 albumen and Bone Defect Repari injection thereof
Preparation method and application.
[background technology]
Bone morphogenetic protein i.e. Bone Morphogenetic Protein, is abbreviated as BMP, is a kind of to osteogenesis and reparation
There is the cytokine of obvious biological activity, there is prominent induced osteogenesis activity, thus have in terms of basis and clinical practice
Wide application prospect.Up to the present, it has been found that 20 kinds of BMP, to bone morphogenesis protein-2 (bone therein
Morphogenetic protein-2, BMP-2) research more.BMP-2 can purify in animal tissue, it is also possible to genetic engineering
Build reconstitution cell and express synthesis.The computer MSR Information system of Urist leader in 1979 takes the lead in having successfully been isolated purification from rabbit decalcification bone
The BMP-2 of rabbit, extracts Bovine Bone Morphoge Protnetin (bBMP) from Os Bovis seu Bubali in nineteen eighty-two, and established in 1987
A set of normal process extracting BMP from people and Os Bovis seu Bubali.
The skeletal growth factor of percutaneous injection, directly can be delivered to fracture, Cranial defect and Bone nonunion position by efficient osteoinductive agent,
There is wound little, the advantages such as indication is wide.For section comminuted fracture patient big in clinic, to fracture site after closed reduction
Inject skeletal growth factor and can be greatly improved union of fracture rate；And for bone does not connect patient, in interior fixing or that extenal fixation is perfect feelings
Under condition, take local injection skeletal growth factor, the subject range of non-operative treatment can be expanded further.As king steps on tiger at " note
The Related Experimental Study of emitting BMP and Preliminary Clinical research " bBMP, bFGF are combined with each other by a literary composition with PVP,
Make injection-type BMP (IBMP) to be used for treating delayed fracture union, being not connected to and Cranial defect.By BMP with there is thermodynamics
Reversible poloxamer (poloxamer) combines, and when temperature is relatively low, poloxamer can be dissolved in water and form colloidal sol, in vivo
Owing to temperature is higher, it is possible to form the slow release of viscogel, beneficially BMP and somatomedin, improve the induction energy of bone
Power.But BMP content in bone is few, and extracted amount is limited, uses the BMP of High Purity to be the most easily absorbed by tissue, it is difficult to play
Effect.Therefore, BMP and the problem of collocation carrier must be considered during application simultaneously.Carrier commonly used in the prior art has decalcification bone
Substrate (DBM), many empty calcium phosphate (TCP), hydroxyapatite (HA), biological active glass ceramic and fibrin etc.,
But there is such or such problem the most more or less in these carriers.
[summary of the invention]
For overcoming above-mentioned deficiency of the prior art, the invention provides a kind of based on natural extract, partially purified BMP
And it is collocated with the injection of optimum carrier with slow releasing function of optimum carrier and preparation method thereof, during being extracted by optimization
Process conditions, it is thus achieved that BMP purity high, activity is high, utilization rate is high, and optimizes injection carrier component and ratio, prepare
Bone Defect Repari injection there is the advantages such as expense is few, effect is notable, easy to use.
For achieving the above object, invention is by the following technical solutions:
The invention provides the extracting method of a kind of BMP-3, comprise the steps:
1) slightly carry:
Little bracket cortical bone after pulverizing processes the H of 10-15h, 3-10% successively through 1:1 chloroform/methanol2O2Process 10-15h,
Process 3-6min with the HCl of 0.6mol/L at room temperature, the centrifugal separation liquid obtained is lowered pH to 7.2 in 37 DEG C and is added
Enter the collagenase extraction 24h of its gross weight 0.0006%, after concentrating under reduced pressure, be dissolved in final concentration of 6mol/L urea/0.5mol/L CaCl2
Middle extraction 24h, separating liquid is the filtering with microporous membrane of 70KD through molecular cut off, dialyse, centrifugal after rough BMP, will
It redissolves in 4mol/L guanidine hydrochloride/0.5mol/L CaCl2In, filtrate, again through dialysis, is centrifuged to obtain BMP crude product；
2) purification:
A) taking 160mg BMP crude product, redissolve in the Buffer A of 10ml, under 20,000g, centrifugal 30min, takes supernatant
The heparin affinity chromatography post of 160mm × 10mm is loaded by liquid；
Described Buffer A consists of 6mol/L urea/50mmol/L Tris HCl/0.1mol/L NaCl, pH 7.0；
B) chromatographic column processes by following three kinds of buffer solution elution successively with the speed of 120cm/h: Buffer A, containing 0.15mol/L
The Buffer C of the Buffer B of the Buffer A of NaCl and the Buffer A containing 0.5mol/L NaC1 eluting respectively, collects
The protein that Buffer C elutes, it is thus achieved that BMP-3.
Additionally provide the Bone Defect Repari injection of a kind of BMP-3 extracting acquisition containing the present invention, by BMP-3, collagen protein, phosphorus
Fat and auxiliary agent composition, wherein in Bone Defect Repari injection, the concentration of BMP-3 is 1.0-2.0mg/ml, and the percent mass of collagen protein is dense
Degree is 2-5%, and the concentration of phospholipid is 0.5-1.5mg/ml, and surplus is auxiliary agent.
In the Bone Defect Repari injection that the present invention provides, auxiliary agent includes PEG, poloxamer or PVP.
Poloxamer (poloxamer) is the non-proprietary term of Pluronic F68, its trade name pluronic
(Pluronic)。
In the Bone Defect Repari injection that the present invention provides, the mass percentage concentration of PEG is 15%.
In the Bone Defect Repari injection that the present invention provides, the mass percentage concentration of poloxamer is 15.25%.
In the Bone Defect Repari injection that the present invention provides, the mass percentage concentration of PVP is 15%.
In the Bone Defect Repari injection that the present invention provides, PEG is PEG 2000, and poloxamer is poloxamer 124, poloxamer
188, poloxamer 237, poloxamer 338 or poloxamer 407.
Present invention also offers a kind of preparation method preparing Bone Defect Repari injection, apirogen water is sequentially added into BMP-3, glue
It is homogenized after former albumen, filters and eluting, eluent adds phospholipid and auxiliary agent, add loading peace after mixing and cut open in bottle, lyophilizing
After, obtain Bone Defect Repari injection.
In the preparation method of the Bone Defect Repari injection that the present invention provides, after homogenate, adding mass percentage concentration is the glutaraldehyde of 10%
Solution, at room temperature after stirring 2-4h, crossing molecular cut off is 10, and the microporous filter membrane of 000Da will be left on microporous filter membrane
BMP-3 and cross-linking agent apirogen water rinse to obtain eluent.
With immediate prior art ratio, the preparation process condition optimization that the present invention provides, the BMP purity of extraction is high, activity is high,
Utilization rate is high；Optimize the component of Bone Defect Repari injection and ratio, the Bone Defect Repari injection expense selecting the suitableeest carrier prepared is few,
Effect is notable, easy to use.
Below, the preparation technology provided by the present invention combines practical operation, is described further:
Extraction BMP-3:
Choose fresh calf bones of limbs, bracket cortical bone liquid nitrogen is met cold rear pulverizing and makes bone meal, successively through 1:1 chloroform/first
Alcohol processes 12h, 10% hydrogen peroxide oxidation 12h, at room temperature 0.6mol/L HCl treatment 5min, is centrifuged and is divided
Chaotropic regulation pH 7.2, add the liquid that obtains after the collagenase of its gross weight 0.0006% extracts 24h under 37 DEG C of aseptic conditions in
Reduced pressure at room temperature is dissolved in extracting solution after concentrating makes its final concentration of 6mol/L urea/0.5mol/L CaCl2, extract 24h, filtrate warp
The molecular weight that dams is the filtering with microporous membrane of 70KD, and filtrate obtains rough BMP after dialysing, being centrifuged, and is dissolved in refined solution
(4mol/L guanidine hydrochloride/0.5mol/L CaC12In), supernatant obtains BMP crude product again after dialysing, being centrifuged.Take 160mg
BMP crude product redissolves (Buffer A:6mol/L urea/50mmol/L Tris HC1/0.1 in the level pad of 10mL
Mol/L NaC1, pH 7.0), with the centrifugation 30min of 20000g, remove wherein insoluble matter, take filtrate 160mm
The heparin affinity chromatography post sample-adding of × 10mm.Chromatographic column is washed with following three kinds of buffer the most in order with the speed of 120cm/h
De-process, i.e. Buffer solution A, Buffer B liquid (the Buffer A containing 0.15mol/L NaCl) and with Buffer C liquid
Eluting (the Buffer A containing 0.5mol/L NaC1), collects the protein eluted respectively, through dialysis, centrifugal, lyophilizing
Rear preservation, mass metering.
The mensuration of albumen relative molecular weight:
Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Protein after purification is dissolved in containing dithiothreitol, DTT
Or do not contain in the sample buffer of DTT (DTT), 90 DEG C of heating 4min, centrifugal after be loaded with supernatant.Running gel is
The spacer gel of 50ml/L, the separation gel of 125ml/L.After electrophoresis, dye with Coomassie brilliant blue R 250, then survey it and relatively divide
Son amount.
After partially purified protein is added people's chromatographic column, eluent is with the speed eluting of 120cm/h.During with Buffer A eluting,
Unadsorbed protein the most all flows out；Using Buffer B liquid (containing 0.15mol/L NaCl) instead, eluting adsorbs more weak albumen,
PH value declines, and ion concentration increases, and has larger amount of foreign protein to flow out, referred to as B peak；With the Buffer containing 0.5mol/L NaCl
During C liquid eluting, it is thus achieved that C peak, this peak is the destination protein that we mainly collect.A section albumen and heparin affinity chromatography without affinity,
Therefore be discarded, the albumen collected in B peak, C peak steams water to four and dialyses about 24h, changes liquid therebetween 3 times.Dialysis finds, C
Peak albumen has white flock to deposit, and shows water insoluble；And B peak albumen dialysis solution still clear, all it is dissolved in water.By C peak egg
After white vacuum freeze-drying, surveying its quality is 8.23mg, nearly 20 times of purification.
BMP electrophoresis result is as in figure 2 it is shown, Proteins after purification is the most single after purification, and purity is higher, albumen MrIt is about
35×103(Fig. 2 B), after reduction treatment, albumen MrBecome 22 × 103, also it is a single band (Fig. 2 C).
Albumen is carried out component analysis, it has been acknowledged that be a kind of acidic mixed albumen, isoelectric point, IP is about 4, albumen average molecular
The mensuration of quality: using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it is used for the mensuration of protein and nucleic acid.Will
Protein after purification be dissolved in containing dithiothreitol, DTT (DTT) or do not contain DTT sample buffer in, 90 DEG C heating 3-5
Min, uses after being centrifuged and resets and add sample. and running gel is the separation gel of the spacer gel of 50mL/L, 125mL/L.After electrophoresis,
Dye with coomassie brilliant blue R250, then survey its relative molecular mass.Extract containing BMP is carried out sds gel electrophoresis analysis,
Its molecular weight is from 14 × 103To 44 × 103.Wherein heparin affinity chromatography purification and the 35 × 10 of sds gel electrophoresis announcement3Electrophoresis
Band.Relying on interionic active force eluting, through affording 3 peaks, a peak is molecular weight 35 × 103, a peak is water solublity
Albumen, a peak is referred to as non-35 × 103Albumen.This protein extraction active substances is made up of 3 parts；
(1) 35 × 10 be identified3Protein BMP-3
(2) collagen protein that a kind of water miscible albumen system is degraded.
(3) non-35 × 103Albumen, is the mixture of several albumen.
Wherein have more than 14 × 103To less than 22 × 103Albumen.These albumen are the strands of BMP, 30 × 103Albumen is BMP-2.
B peak albumen dialysis solution clear, is dissolved in water, is the collagen protein being degraded.C peak four steams water and carries out dialysis solution, in vain
The cotton-shaped deposition of color, albumen Mr is about 35 × 103, after reduction treatment, albumen Mr becomes 22 × 103, for a single band.
By heparin affinity chromatography purification and sds gel electrophoresis analysis, in protein extract, bone morphogenetic protein is BMP-3.This 30mg
Containing BMP-3 1.5mg in partially purified bmp protein, containing 1.5mg Bone Defect Repari egg in every part of injection Bone Defect Repari agent
White BMP-3.
Prepare Bone Defect Repari injection:
1. the albumen taking BMP-3 adds apirogen water, and making BMP-3 protein concentration is 1.0mg-2.0mg/ml, its collagen egg
White weight concentration is 2-5% i.e. 20mg-50mg/ml, homogenate, adds the poloxamer 407 of final concentration of 15.25% and mixes,
Canned peace in 2ml is cutd open in bottle, containing 1.5mg Bone Defect Repari protein BMP-3 in every part of injection Bone Defect Repari agent.Chilled dry
Finished product is obtained after dry.
2. taking and add apirogen water containing BMP-3 albumen, the BMP-3 protein concentration made is 1.0mg-2.0mg/ml, its glue
Former weight concentration is 2-5%, homogenate, adds the PEG 2000 of final concentration of 15% and mixes, every part of injection Bone Defect Repari agent
In containing Bone Defect Repari protein BMP-3 1.5mg.Canned peace in 2ml is cutd open in bottle, obtains finished product after lyophilization.
3. taking and add apirogen water 10ml containing BMP-3 albumen, making BMP-3 protein concentration is 1.0mg-2.0mg/ml, its
The weight concentration of collagen is 2-5%, homogenate, adds the glutaraldehyde solution 5ml mixing of 10%, after stirring 3h, crosses and retain molecule
Amount is 10, the microporous filter membrane of 000Da, and the cross-linking agent apyrogeneity that will be left in BMP-3 albumen on microporous filter membrane and collagen is washed
Getting off, add the PEG 2000 of final concentration of 15% and mix, canned cut open in bottle in 4 2ml peaces, every part of injection bone is repaiied
Containing Bone Defect Repari protein BMP-3 1.5mg in multiple agent.Finished product is obtained after lyophilization.
4. taking and add apirogen water 10ml containing BMP-3 albumen, making BMP-3 protein concentration is 1.0mg-2.0mg/ml, its
The weight concentration of collagen is 2-5%, homogenate, adds the glutaraldehyde solution 5ml mixing of 10% concentration, after stirring 3 hours, mistake
Molecular cut off is 10, the microporous filter membrane of 000Da, will be left in the cross-linking agent nothing of the BMP-3 albumen on microporous filter membrane and collagen
Pyrogen is washed, and after adding poloxamer 407 mixing of final concentration of 15.25%, the most canned peace in 4 2ml cuts open bottle
In, containing Bone Defect Repari protein BMP-3 1.5mg in every part of injection Bone Defect Repari agent.Lyophilizing obtains finished product.
5. taking BMP-3 albumen and add apirogen water 10ml, making BMP-3 protein concentration is 1.5mg/ml, the weight of its collagen
Concentration is 3%, homogenate, adds the glutaraldehyde solution 5ml mixing of 10% concentration, and after stirring 3 hours, crossing molecular cut off is
The microporous filter membrane of 10,000Da, the cross-linking agent apyrogeneity that will be left in BMP-3 albumen on microporous filter membrane and collagen washes,
Add the PVP mixing of final concentration of 15%, load 4 2ml peaces after lyophilization and cut open in bottle, every part of injection Bone Defect Repari
Agent obtains finished product containing Bone Defect Repari protein BMP-3 1.5mg.
6. taking and add apirogen water 10ml containing BMP-3 albumen, making BMP-3 protein concentration is 1.00mg/ml, its collagen
Weight concentration be 2%, add injection with phosphatidase 1 00mg be homogenized, add final concentration 15%PVP mix, canned in 4
Prop up 2ml peace and cut open in bottle, every part of injection Bone Defect Repari agent must become containing after Bone Defect Repari protein BMP-3 1.5mg lyophilization
Product.
7. taking BMP-3 albumen and add apirogen water, making BMP-3 protein concentration is 2.0mg/ml, the weight concentration of its collagen
It is 5%, adds injection phosphatidase 1 00mg, homogenate, add the poloxamer 407 of final concentration of 15.25% and mix, often
Containing Bone Defect Repari protein BMP-3 1.5mg in part injection Bone Defect Repari agent, after lyophilization, obtain finished product.
8. taking BMP-3 albumen and add apirogen water, making BMP-3 protein concentration is 1.0mg-2.0mg/ml, the weight of its collagen
Amount concentration is 2-5%, adds injection phosphatidase 1 00mg, homogenate, adds the PEG 2000 that final concentration is 15%, mixes,
Containing Bone Defect Repari protein BMP-3 1.5mg in every part of injection Bone Defect Repari agent, after lyophilization, obtain finished product.
[accompanying drawing explanation]
Fig. 1 is the Heparin Sepharose Cl-6B tomographic map of BMP；
Fig. 2 is the electrophoresis result of BMP after purification.
[detailed description of the invention]
Embodiment 1 extracts activated protein BMP-3:
Choose fresh calf bones of limbs, the lower limb cortical bone liquid nitrogen of cattle is met cold after pulverize backbone and make bone meal, through 1:1 chloroform/
Methanol processes 12h, 10% hydrogen peroxide oxidation 12h, 0.6mol/L HCl treatment 5min under room temperature, obtains after being centrifuged
Separation liquid regulation pH 7.2, add the liquid obtained after 0.0006% collagenase extracts 24h under 37 DEG C of aseptic conditions in room temperature
It is dissolved in extracting solution after concentrating under reduced pressure and makes its final concentration 6mol/L urea/0.5mol/L CaCl2, extract 24h, filtrate is through damming point
Son amount is that the filtering with microporous membrane of 70KD obtains filtrate, obtains rough BMP, redissolved in refined solution (4 after dialysing, being centrifuged
Mol/L guanidine hydrochloride/0.5mol/L CaC12), supernatant has obtained partially purified BMP again after dialysing, being centrifuged.Take 160mg
Partially purified bmp protein redissolves (Buffer A:6mol/L urea/50mmol/L in the level pad of 10ml
Tris HC1/0.1mol/L NaC1pH 7.0), with the centrifugation 30min of 20000g, remove the most insoluble material.
With supernatant, the heparin affinity chromatography post of 160mm × 10mm is loaded.Chromatographic column is washed with following three kinds of buffer the most in order
De-, i.e. contain the Buffer solution A of 0.1,0.15,0.5mol/L NaCl, Buffer B liquid (containing 0.15mol/L NaCl)
And with the Buffer C liquid eluting containing 0.5mol/L NaC1, B peak albumen dialysis solution clear, be dissolved in water, be to be degraded
Collagen protein.C peak four steams water and carries out dialysis solution, white cotton-shaped deposition, and albumen Mr is about 35 × 103, through reduction treatment
After, albumen Mr becomes 22 × 103, for a single band.By heparin affinity chromatography purification and sds gel electrophoresis analysis, egg
In white extract, bone morphogenetic protein is BMP-3.Containing BMP-3 1.5mg in the partially purified bmp protein of this 30mg,
Containing 1.5mg Bone Defect Repari protein BMP-3 in every part of injection Bone Defect Repari agent.
Embodiment 2
The albumen taking BMP-3 adds apirogen water, and making BMP-3 protein concentration is 2.0mg/ml, and the weight concentration of its collagen is
4% i.e. 40mg/ml, homogenate, add the poloxamer 407 of final concentration of 15.25% and mix, freeze-dried after finished product.
Embodiment 3
Taking and add apirogen water containing BMP-3 albumen, the BMP-3 protein concentration made is 1.0mg/ml, and the weight of its collagen is dense
Degree is 3%, homogenate, adds the PEG 2000 of final concentration of 15% and mixes, after lyophilization finished product.
Embodiment 4
Taking and add apirogen water 10ml containing BMP-3 albumen, making BMP-3 protein concentration is 1.5mg/ml, the weight of its collagen
Concentration is 2.5%, homogenate, adds the glutaraldehyde solution 5ml mixing of 10%, after stirring 3 hours, crosses and retain 10,000Da's
Microporous filter membrane, the cross-linking agent apyrogeneity that will be left in BMP-3 albumen on microporous filter membrane and collagen is washed, and adds final concentration
Be 15% PEG 2000 mix, after lyophilization finished product.
Embodiment 5
Taking and add apirogen water 10ml containing BMP-3 albumen, making BMP-3 protein concentration is 2mg/ml, the weight of its collagen
Concentration is 3.5%, homogenate, adds the glutaraldehyde solution 5ml mixing of 10% concentration, after stirring 3 hours, crosses and retain 10,000Da
Microporous filter membrane, the cross-linking agent apyrogeneity that will be left in BMP-3 albumen on microporous filter membrane and collagen is washed, and adds the denseest
After degree is poloxamer 407 mixing of 15.25%, lyophilization.
Embodiment 6
Taking BMP-3 albumen and add apirogen water 10ml, making BMP-3 protein concentration is 1.0mg/ml, the weight concentration of its collagen
It is 4.5%, homogenate, add the glutaraldehyde solution 5ml mixing of 10% concentration, after stirring 3 hours, cross and retain 10,000 dongle
Microporous filter membrane, the cross-linking agent apyrogeneity that will be left in BMP-3 albumen on microporous filter membrane and collagen wash, addition end
Concentration is the PVP mixing of 15%, and lyophilization obtains finished product.
Embodiment 7
Taking and add apirogen water 10ml containing BMP-3 albumen, making BMP-3 protein concentration is 1.5mg/ml, the weight of its collagen
Concentration is 2.5%, adds injection phosphatidase 1 00mg and is homogenized, adds final concentration and mix at 15%PVP, after lyophilization
Finished product.
Embodiment 8
Taking BMP-3 albumen and add apirogen water, making BMP-3 protein concentration is 1.5mg/ml, and the weight concentration of its collagen is
2-5%, adds injection phosphatidase 1 00mg, homogenate, adds the poloxamer 407 of final concentration of 15.25% and mix, freezing
Dried finished product.
Embodiment 9
Taking BMP-3 albumen and add apirogen water, making BMP-3 protein concentration is 1.5mg/ml, and the weight concentration of its collagen is 3%,
Adding injection phosphatidase 1 00mg, homogenate, addition final concentration mixes at the PEG 2000 of 15%, obtains finished product after lyophilization.
Embodiment 10 extracts BMP-3
By the lower limb cortical bone 5kg of cattle, the lower limb cortical bone liquid nitrogen of cattle is met cold after pulverize backbone and make bone meal, through 1:1 chloroform/
Methanol processes 12h, 10% hydrogen peroxide oxidation 12h, 0.6mol/L HCl treatment 5min under room temperature, the bone obtained after being centrifuged
Powder regulation pH 7.2 adds the liquid after 0.0006% collagenase extracts 24h under 37 DEG C of aseptic conditions and is connected in reduced pressure at room temperature concentration
The bone matrix gelatin formed with skeletal grain is dissolved in extracting solution makes final concentration 6mol/L urea/0.5mol/L CaCl extract 24h, supernatant
Liquid obtains filtrate through the filtering with microporous membrane of the molecular weight 70KD that dams, through dialysis, centrifugal after obtain rough BMP, redissolved in
Refined solution (4mol/L guanidine hydrochloride/0.5mol/L CaCl), supernatant has obtained partially purified BMP again after dialysing, being centrifuged.
Take the partially purified bmp protein of 160mg and redissolve (Buffer A:6mol/L urea/50 in the level pad of 10ml
Mmol/L Tris HC1/0.1mol/L NaCl pH 7.0), with 20, the centrifugation 30min of 000g, remove the most insoluble
Material.With supernatant, the heparin affinity chromatography post of 160mm × 10mm is loaded.Chromatographic column is respectively in order by following three kinds of bufferings
Liquid eluting, i.e. contains the Buffer solution A of 0.1,0.15,0.5mol/L NaCI, Buffer B liquid (containing 0.15mol/L NaCl)
And with the Buffer C liquid eluting containing 0.5mol/L NaCl, B peak albumen dialysis solution clear, be dissolved in water, be the glue being degraded
Former albumen.C peak four steams water and carries out dialysis solution, white cotton-shaped deposition, albumen MrIt is about 35 × 103, after reduction treatment, egg
White MrBecome 22 × 103, for a single band.By heparin affinity chromatography purification and sds gel electrophoresis analysis, protein extract
Middle bone morphogenetic protein is BMP-3.Containing BMP-3 1.5mg in the partially purified bmp protein of this 30mg.
The explanation of above example is only intended to help to understand method and the core concept thereof of the present invention.It should be pointed out that, for ability
For the those of ordinary skill in territory, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement and repaiies
Decorations, these improve and modify in the protection domain also falling into the claims in the present invention.
Claims (10)
1. an extracting method of BMP-3, comprises the steps:
1) slightly carry:
Little bracket cortical bone after pulverizing processes the H of 10-15h, 3-10% successively through 1:1 chloroform/methanol2O2Process 10-15h,
Process 3-6min with the HCl of 0.6mol/L at room temperature, the centrifugal separation liquid obtained is lowered pH to 7.2 in 37 DEG C and is added
Enter the collagenase extraction 24h of its gross weight 0.0006%, after concentrating under reduced pressure, be dissolved in final concentration of 6mol/L urea/0.5mol/L CaCl2
Middle extraction 24h, separating liquid through molecular cut off is the filtering with microporous membrane of 70KD, obtains rough BMP after dialysing, being centrifuged,
Redissolved in 4mol/L guanidine hydrochloride/0.5mol/L CaCl2In, filtrate, again through dialysis, is centrifuged to obtain BMP crude product；
2) purification:
A) taking 160mg BMP crude product, be dissolved in the Buffer A of 10ml, under 20,000g, centrifugal 30min, takes supernatant
The heparin affinity chromatography post of 160mm × 10mm is loaded；
Described Buffer A consists of 6mol/L urea/50mmol/L Tris HCl/0.1mol/L NaCl, pH 7.0；
B) chromatographic column processes with following three kinds of buffer successively with the speed of 120cm/h: Buffer A, containing 0.15mol/L NaCl
The Buffer C respectively eluting of Buffer B and Buffer A containing 0.5mol/L NaC1 of Buffer A, collect Buffer C
The protein eluted, it is thus achieved that described BMP-3.
2. the Bone Defect Repari injection of the BMP-3 obtained containing extracting method as claimed in claim 1, by BMP-3, glue
Former albumen, phospholipid and auxiliary agent composition, it is characterised in that: the concentration of BMP-3 described in Bone Defect Repari injection is 1.0-2.0mg/ml,
The mass percentage concentration of collagen protein is 2-5%, and the concentration of phospholipid is 0.5-1.5mg/ml, and surplus is auxiliary agent.
3. Bone Defect Repari injection as claimed in claim 2, it is characterised in that described auxiliary agent includes PEG, poloxamer or PVP.
4. Bone Defect Repari injection as claimed in claim 3, it is characterised in that the mass percentage concentration of described PEG is 15%.
5. Bone Defect Repari injection as claimed in claim 4, it is characterised in that described PEG is PEG 2000.
6. Bone Defect Repari injection as claimed in claim 3, it is characterised in that the mass percentage concentration of described poloxamer is
15.25%.
7. Bone Defect Repari injection as claimed in claim 6, it is characterised in that described poloxamer be poloxamer 124,
Poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407.
8. Bone Defect Repari injection as claimed in claim 3, it is characterised in that the mass percentage concentration of described PVP is 15%.
9. the preparation method of a Bone Defect Repari injection as claimed in claim 2, it is characterised in that: in apirogen water successively
It is homogenized after adding BMP-3 and collagen protein, filters and eluting, eluent adds phospholipid and auxiliary agent, add after mixing and load
Peace is cutd open in bottle, after lyophilizing, obtains described Bone Defect Repari injection.
10. preparation method as claimed in claim 9, it is characterised in that: after homogenate, adding mass percentage concentration is the penta of 10%
Dialdehyde solution, at room temperature after stirring 2-4h, crossing molecular cut off is 10, and the microporous filter membrane of 000Da will be left in microporous filter membrane
Upper BMP-3 and cross-linking agent apirogen water rinse to obtain described eluent.
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| CN201510831126 | 2015-11-25 | ||
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| CN112521638A (en) * | 2020-12-02 | 2021-03-19 | 山东省药学科学院 | Hydrogel loaded with bone morphogenetic protein, preparation method and application |
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Application publication date: 20160810 |