CN100355787C - Activated collagen bone repair material and special fusion active bone repair factor thereof - Google Patents
Activated collagen bone repair material and special fusion active bone repair factor thereof Download PDFInfo
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Abstract
The invention discloses an activated collagen bone repair material and a special fusion active bone repair factor thereof. The amino acid residue sequence of the special fusion active bone repair factor is shown as SEQ ID NO: 2, respectively. The activated collagen bone repair material is collagen loaded with the special fusion active bone repair factor. The preparation method is simple and convenient, can be applied to large-scale industrial production, and plays a great role in the medical field, particularly in the repair of bone injury.
Description
Technical field
The present invention relates to activatory collagen protein bone renovating bracket material and special-purpose fusion-activity bone reparative factor thereof.
Background technology
The bone that is caused by reasons such as wound, infection, tumour and heteroplasia is damaged to be the problem that orthopaedics all will face clinical every day.People's bone polymorphism protein BMP 2 has very strong bone-inducting active.It is the glycosylated protein of 396 amino-acid residues of a total length, comprises one section signal peptide of being made up of 19 amino-acid residues, pro-region that is made up of 263 amino-acid residues and the mature peptide of being made up of 114 amino-acid residues.Mature peptide contains 7 halfcystines and a N-glycosylation site, its functional form is the homodimer that forms by a pair of disulfide linkage contact, each single intravital all the other six halfcystine forms three intrachain disulfide bond (Wozney, J.M.et al.Novelregulators of bone formation:molecular clones and activities.Science 242,1528-34,1988).But natural B MP2 content seldom, and separation and purification BMP2 has very big limitation from human or animal's ground substance of bone.At present, people's research emphatically prepare reorganization BMP2 by engineered method, to satisfy clinical and needs fundamental research.In addition, function class be similar to BMP2 other can induced tissue the factor of regeneration and trauma repair also comprise: BMP3, PDGF, FGF, EGF, TGF, VEGF, NGF, NT3/4 etc.
Collagenic material is the bone renovating material of using always.Collagen is the main organic composition of bone, and type i collagen and cross filament structure thereof are the abundantest albumen in the osteocyte epimatrix.Collagen structure has inducing action to mineral deposition, and the site of mineral deposition is contained on its surface, can effectively cause and control mineralisation process, and the promotion bone forming also brings out to implant.Traditional collagen carrier is the molecular structure prevention BMP2 composition disperse by tropocollagen molecule, keep host target cell BMP2 concentration on every side, also require its aperture can not be too big so on the one hand, be unfavorable for osteoblastic growth again but the aperture is too small, this just needs the certain collagen aperture of control, to bringing certain difficulty on the technology.On the other hand, use the carrier of present biomaterial as BMP2, the consumption of BMP2 is very big, often will reach milligram level (Kirker-Head, C.A., Gerhart, T.N., Armstrong, R., Schelling, S.H.﹠amp; Carmel, L.A.Healingbone using recombinant human bone morphogenetic protein 2 and copolymer.ClinOrthop Relat Res, 205-17 (1998); Kokubo, S.et al.Bone regeneration byrecombinant human bone morphogenetic protein-2 and a novel biodegradablecarrier in a rabbit ulnar defect model.Biomaterials 24,1643-51 (2003)).Because BMP2 is very expensive in the market, only sigma company is used for the BMP2 of experimental study, per 10 μ g prices reach 500 dollars, domestic patient seldom can afford the cost of use of BMP2, in addition, several milligrams BMP2 is equivalent to from the extractive BMP2 summation of a head of cattle, and use BMP2 so in large quantities, security is also very troubling.
Summary of the invention
The purpose of this invention is to provide a kind of activatory collagen bone renovating material and special-purpose fusion-activity bone reparative factor thereof.
Special-purpose fusion-activity bone reparative factor provided by the present invention is collagen binding domains (collagen binding domain, the fusion rotein that CBD) obtains that aminoterminal (N end) at reparative factor or carboxyl terminal (C end) merge the vWF factor; The albumen that the collagen binding domains of the described vWF factor is made up of 10-30 amino-acid residue, its conserved sequence are the SEQ ID № in the sequence table: 1.
Described reparative factor can be BMP2, BMP3, PDGF, FGF, EGF, TGF, VEGF, NGF or NT3/4 etc. can induced tissue the factor of regeneration and trauma repair, be preferably BMP2.
SEQ ID № in the sequence table: 1 is made up of 10 amino-acid residues.
Wherein, the fusion-activity bone reparative factor of holding the collagen binding domains of the fusion vWF factor to obtain at the N of BMP2 can have SEQ ID № in the sequence table: 2 amino acid residue sequence, SEQ ID № in the sequence table: 2 are made up of 162 amino-acid residues, and the conserved sequence of its vWF factor collagen binding domains is from aminoterminal 22-31 amino acids residue; The dna sequence dna of this fusion-activity bone reparative factor of encoding is the SEQ ID № in the sequence table: 3, and the SEQ ID № in the sequence table: 3 by 486 based compositions.
Above-mentioned fusion rotein can be according to the ordinary method preparation in genetically engineered field.
Second purpose of the present invention provides a kind of activatory collagen bone renovating material.
Activatory collagen bone renovating material provided by the present invention is the collagen protein that is loaded with above-mentioned special-purpose fusion-activity bone reparative factor.
The heap(ed) capacity of described special-purpose fusion-activity bone reparative factor is 1-4000pmol albumen/mg collagen protein.
The invention provides a kind of activatory collagen bone renovating material and special-purpose fusion-activity bone reparative factor thereof.This special use fusion-activity bone reparative factor is to merge collagen land CBD by engineered method at the aminoterminal or the carboxyl terminal of reparative factors such as BMP2, to strengthen combining of reparative factor and collagen, between reparative factor and collagen protein, set up a kind of stronger physical-chemistry force, reparative factors such as BMP2 can be fixed on the collagen scaffold, thereby significantly improve the repair ability of bone injury, also can significantly reduce simultaneously the consumption of reparative factor such as BMP2, and reduce application risk.Preparation method of the present invention is easy, can be applicable to large-scale industrial production, and will particularly play a great role in the reparation of bone injury in the trauma repair field.
The invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is the 15%SDS-PAGE detected result to purified expressing protein
Fig. 2 is the 15%SDS-PAGE detected result to the expressing protein of purified back renaturation
The statistics of natural B MP2 that Fig. 3 increases progressively gradually for concentration and rhBMP2-v and collagen protein binding capacity
Fig. 4 is the cytoactive detected result of rhBMP2-v
Fig. 5 induces the gross examination of skeletal muscle result of three experimental group embedding things of experiment for the dystopy bone that carries out with natural B MP2 and rhBMP2-v
Fig. 6 induces Histological section's observations of three experimental group rat embedded materials of experiment for the dystopy bone that carries out with natural B MP2 and rhBMP2-v
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The preparation and the activity identification thereof of embodiment 1, special-purpose fusion-activity bone reparative factor
One, the preparation of special-purpose fusion-activity bone reparative factor
1, special-purpose fusion-activity bone reparative factor construction of prokaryotic expression vector
According to the cDNA sequence of known people BMP2 (hBMP2) (be for GenBank number: 650) design pcr amplification primer, and in forward primer, introduce the vWF factor collagen binding domains (CBD) " WREPSFCALS " (the SEQ ID № in the sequence table: encoding sequence 1), primer sequence is as follows:
hBMP2F1：
5’-TACCGGTAGCGCGGGCAGTGCTGCGGGTTCTGGCGGTGTCGACCAAGCCAAACAC-3’
hBMP2F2：5’-CGCCATATGTGGCGTGAACCGAGCTTCATGGCTCTGAGCGGTACCGGTAGC-3’
hBMP2R：5’-CCGCTCGAGCTATTAACGACAACCACAACC-3’
With people BMP2 cDNA total length is template, carries out pcr amplification under the guiding of primer hBMP2F1 and hBMP2R, and 30 μ l PCR reaction systems are: each 1pmol/ μ l of forward and reverse primer, dNTPs 200 μ mol/ μ l, Taq enzyme 3ul; The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min.After reaction finishes, amplified production is carried out 1.5% agarose gel electrophoresis detect, the result obtains the dna fragmentation of an about 400bp of length through pcr amplification.To this fragment reclaim and purifying after, template as the PCR second time, under the guiding of primer hBMP2F2 and hBMP2R, carry out the pcr amplification second time again, PCR reaction system and reaction conditions are the same, after amplification finishes, the PCR product is carried out 1.5% agarose gel electrophoresis to be detected, the result obtains the band of an about 430bp of length, to its reclaim and purifying after, with behind restriction enzyme Nde I and the Xho I double digestion with through the prokaryotic expression carrier pET-28a of same enzyme double digestion (Novagen) under 16 ℃, use T
4Dna ligase connects 12-24 hour, to connect product transformed into escherichia coli DH5 α competent cell, screening positive clone, the upgrading grain, this expression vector multiple clone site district is checked order, insertion sequence conforms to expected sequence as a result, has SEQ ID № in the sequence table: 3 nucleotide sequence, SEQ ID № in the sequence table: 3 by 486 based compositions, SEQ ID № in the code sequence tabulation: 2 amino acid residue sequence, comprise the CBD district, linker district and hBMP2 mature peptide coding region, before inserting the district, also merge one section Histidine affinity tag, the conserved sequence in its CBD district is SEQ ID № in the sequence table: 2 from aminoterminal 22-31 amino acids residue, and the linker district is from aminoterminal 34-46 amino acids residue, and the hBMP2 mature peptide is from aminoterminal 49-162 amino acids residue, show and obtained the correct recombinant plasmid that contains special-purpose fusion-activity bone reparative factor encoding sequence, called after pET-28a-BMP2-v.
2, the prokaryotic expression of special-purpose fusion-activity bone reparative factor
Prokaryotic expression carrier pET-28a-BMP2-v transformed into escherichia coli BL21 (DE3) competent cell with step 1 structure, the positive monoclonal that screens is transferred in the LB liquid nutrient medium, cultivated 12-24 hour for 37 ℃, transfer in 100mL LB liquid nutrient medium with 2% inoculative proportion again, cultivate 3 hours to OD for 37 ℃
600Value reaches 0.8, and adding final concentration is the IPTG of 1mM, and the same terms continued inducing culture 4 hours down.After cultivating end, centrifugal collection thalline is collected thalline with PBS washing back recentrifuge, with the resuspended thalline of 10mL PBS, ultrasonication thalline, lysate is carried out 15%SDS-PAGE detect, detected result shows and has obtained protein crude extract that expressing protein exists with the form of inclusion body.
3, the purifying and the renaturation of special-purpose fusion-activity bone reparative factor
At first with step 2 through the centrifugal collection inclusion body of the thalline of ultrasonication.The protein solution that dilution refolding is obtained carries out the ultrafiltration and concentration processing, then the protein solvent system is replaced cryopreservation after the lyophilize with 50mM MES damping fluid (available from GIBCO company).Purified and expressing protein renaturation are carried out 15%SDS-PAGE to be detected, (swimming lane M is Marker to detected result as shown in Figure 1, swimming lane 1 is the whole bacterial protein of the e. coli bl21 (DE3) as negative control, swimming lane 2 and swimming lane 3 are respectively the whole bacterial protein that transforms the transformant that carrier pET-28a-BMP2 and pET-28a-BMP2-v are arranged, swimming lane 4 and swimming lane 5 are respectively the purified pET-28a-BMP2 and the expression product (under the reductive condition) of pET-28a-BMP2-v transformant), the purification result of swimming lane 4 and swimming lane 5 shows that the target protein of expression has reached suitable purity.The renaturation effect of recombinant protein is (swimming lane 1 is that purifying is after the expression product of the pET-28a-BMP2-v transformant of renaturation) as shown in Figure 2, because the activity form of BMP2 is the dimeric forms that forms by a pair of disulfide linkage, under non-reduced condition, can see its dimer band, the detected result of Fig. 2 shows that the dimer that renaturation forms has reached 30%, the special-purpose fusion-activity bone reparative factor called after rhBMP2-v that this is purified.
Two, the collagen of special-purpose fusion-activity bone reparative factor is in conjunction with activity identification
Detect the binding capacity of equivalent collagen to difference amount natural B MP2 and special-purpose fusion-activity bone reparative factor, concrete grammar is: the special-purpose fusion-activity bone reparative factor of natural B MP2 that concentration is increased progressively gradually and step 1 preparation is carried on the collagem membrane with the collagenic material preparation of equivalent, fully after the absorption, washing away through PBS can not bonded albumen, specially can obtain the protein binding amount by quantitative color reaction by measuring with fusion tag bonded antibody is produced, (X-coordinate is the albumen heap(ed) capacity to statistics as shown in Figure 3, ordinate zou is the light absorption value of measuring in 405 nanometers), under identical albumen heap(ed) capacity situation, the reservation amount of the rhBMP2-v that obtains through transformation is apparently higher than natural B MP2 (rhBMP-2), and promptly the former joint efficiency is apparently higher than the latter.Experimental result shows that the BMP2 that process is transformed significantly strengthens than natural B MP2 the binding ability of collagen.
Three, the cytoactive of special-purpose fusion-activity bone reparative factor detects
The effect of BMP2 pair cell mainly is to promote cell to osteoblast differentiation.Hiraki finds by somatic cell culture, BMP2 has the intense stimulus effect to skeletonization like cell MC3T3-E1, can make the active 5-20 of the increasing times (Hiraki of ALPase (alkaline phosphatase) of this cell with 100ng/mL BMP2, Y.et al.Bone morphogeneticproteins (BMP-2 and BMP-3) promote growth and expression of the differentiatedphenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro.J Bone Miner Res 6,1373-85 (1991) .), in addition, it also has transdifferentiation consumingly to sarcoplast C2C12, can suppress it and transfer to break up to the scleroblast direction to the differentiation of myocyte's direction.Now to use mouse muscle-forming cell be C2C12 (available from consonance medical university preclinical medicine institute cell bank) detects the external activity of the special-purpose fusion-activity bone reparative factor of step 1 preparation, method is: the rhBMP2-v with different concns stimulates the C2C12 cell, act on the activity that detects ALPase after three days, that positive controls adopts is the rhBMP-2 (available from Sigma company) that is produced by Chinese hamster ovary celI, the experiment contrast group is the natural B MP2 (rhBMP-2) that does not have the collagen binding domains, (X-coordinate is a protein concentration to the result as shown in Figure 4, ordinate zou is the ALPase activity), rising along with the rhBMP2-v concentration dose, the ALPase activity also increases, demonstrate dose-dependent effect, and through statistical study, all there are utmost point significant difference (P＜0.05) in each experimental group and control group, and above-mentioned experimental result shows that the special-purpose fusion-activity bone reparative factor through dilution refolding has higher biological activity.
Detect the method that the BMP2 bone-inducing activity adopts classical dystopy skeletonization mostly in the body, be about in the BMP2 implantable bone tissue ectopic tissue in addition, implant the back different time and do inspections such as Histological section, the photograph of X-line, observe its bone-inducing activity (Wozney in this tissue, J.M.et al.Novel regulators of bone formation:molecular clones and activities.Science 242,1528-34 (1988).; Urist, M.R.Bone:formation by autoinduction.1965.Clin Orthop Relat Res, 4-10 (2002) .).
Present embodiment adopts the carry out determination of activity of above-mentioned identical method to the special-purpose fusion-activity bone reparative factor of natural B MP2 and embodiment 1 preparation, and concrete experimental technique and result are as follows:
At first get ox bone, reject soft tissue, after the degreasing decalcification, be made into the cubes of 1cm * 0.5cm * 0.5cm, handle freeze-dried back through acid and enzyme again.
Experiment divides three groups: natural B MP2 (rhBMP-2) group, rhBMP2-v group and control group.Get the decalcification osso-albumin material after the above-mentioned lyophilize, every mg collagen loads 200pmol albumen, and control group does not load any albumen, freeze-dried back.
Select adult male SD rats for use, the different proteic collagenic material pieces of the above-mentioned loading that the subcutaneous embedding in back prepares.After 4 weeks of embedding, take out the embedding thing, cut into slices and carry out HE dyeing, gross examination of skeletal muscle result is (scale: 5 millimeters) as shown in Figure 5, A figure among Fig. 5 is the collagenic material piece that does not load the implantation of any proteic control group implantation, the part degraded of visible embedding thing, integral thinned deliquescing; B figure is the collagenic material piece of the loading natural B MP2 of experimental group implantation, because rhBMP-2 itself has dystopy and lures the bone ability, it is carried on the collagenic material, stoped the diffusion of rhBMP-2 composition ground to a certain extent by collagen structure, started certain ossific process, having induced a small amount of mesenchymal cell is cytodifferentiation to bone, thereby secretes a small amount of collagen stroma and calcium ion deposition on material, apparent performance is that the embedding thing is thicker than negative control group, and its degradation speed is also slack-off; C figure is the collagenic material piece of the loading rhBMP2-v of experimental group implantation, transformed rhBMP2-v and collagen have special keying action, reactive force is stronger, implant and be difficult for diffusion dilution, all the time keep higher concentration at action site, inducing a large amount of mesenchymal cells is cytodifferentiation to bone, the bone that is differentiated to form is that cell can secrete a large amount of new collagen stromas and calcium ion deposition again on original collagenic material, the rhBMP2-v that original collagenic material degraded discharges has part may be combined on the collagen of new formation again, the degradation speed that its apparent performance is a material block is slack-off, and size variation is not obvious.And because the sedimentation of a large amount of calcium ions, material is harder.Histological section's observations of above-mentioned three experimental group embedded materials as shown in Figure 6, the A figure among Fig. 6 does not load any proteic contrast, without any osteoplastic sign, the morphological specificity of collagenic material just; B figure is the experimental group that loads natural B MP2, and the skeletonization effect is also not obvious; (scale: 50 μ m) be the experimental group that loads rhBMP2-v, as seen begin to have osseous tissue to form at the edge of collagenic material, the arrow indication is newly-generated bone structure to C, D figure.Above-mentioned experimental result shows that the dystopy of the activated collagenic material that loads special-purpose fusion-activity bone reparative factor lures the bone effect obviously to be better than contrast.
Sequence table
<160>3
<210>1
<211>10
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Trp?Arg?Glu?Pro?Ser?Phe?Cys?Ala?Leu?Ser
1 5 10
<210>2
<211>162
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
1 5 10 15
Arg?Gly?Ser?His?Met?Trp?Arg?Glu?Pro?Ser?Phe?Met?Ala?Leu?Ser?Gly
20 25 30
Thr?Gly?Thr?Gly?Ser?Ala?Gly?Ser?Ala?Ala?Gly?Ser?Gly?Gly?Val?Asp
35 40 45
Gln?Ala?Lys?His?Lys?Gln?Arg?Lys?Arg?Leu?Lys?Ser?Ser?Cys?Lys?Arg
50 55 60
His?Pro?Leu?Tyr?Val?Asp?Phe?Ser?Asp?Val?Gly?Trp?Asn?Asp?Trp?Ile
65 70 75 80
Val?Ala?Pro?Pro?Gly?Tyr?His?Ala?Phe?Tyr?Cys?His?Gly?Glu?Cys?Pro
85 90 95
Phe?Pro?Leu?Ala?Asp?His?Leu?Asn?Ser?Thr?Asn?His?Ala?Ile?Val?Gln
100 105 110
Thr?Leu?Val?Asn?Ser?Val?Asn?Ser?Lys?Ile?Pro?Lys?Ala?Cys?Cys?Val
115 120 125
Pro?Thr?Glu?Leu?Ser?Ala?Ile?Ser?Met?Leu?Tyr?Leu?Asp?Glu?Asn?Glu
130 135 140
Lys?Val?Val?Leu?Lys?Asn?Tyr?Gln?Asp?Met?Val?Val?Glu?Gly?Cys?Gly
145 150 155 160
Cys?Arg
<210>3
<211>486
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
atgggcagca?gccatcatca?tcatcatcac?agcagcggcc?tggtgccgcg?cggcagccat 60
atgtggcgtg?aaccgagctt?catggctctg?agcggtaccg?gtagcgcggg?cagtgctgcg 120
ggttctggcg?gtgtcgacca?agccaaacac?aaacagcgga?aacgccttaa?gtccagctgt 180
aagagacacc?ctttgtacgt?ggacttcagt?gacgtggggt?ggaatgactg?gattgtggct 240
cccccggggt?atcacgcctt?ttactgccac?ggagaatgcc?cttttcctct?ggctgatcat 300
ctgaactcca?ctaatcatgc?cattgttcag?acattggtca?actctgttaa?ctctaagatt 360
cctaaggcat?gctgtgtccc?gacagaactc?agtgctatct?cgatgctgta?ccttgacgag 420
aatgaaaagg?ttgtattaaa?gaactatcag?gacatggttg?tggagggttg?tggttgtcgt 480
taatag 486
Claims (4)
1, fusion-activity bone reparative factor, its amino acid residue sequence is shown in SEQ ID NO:2.
2, the encoding gene of the described fusion-activity bone of claim 1 reparative factor, its base sequence is shown in SEQ ID NO:3.
3, activatory collagen bone renovating material is the collagen protein that is loaded with the described fusion-activity bone of claim 1 reparative factor.
4, activatory collagen bone renovating material according to claim 3 is characterized in that: the heap(ed) capacity of described special-purpose fusion-activity bone reparative factor is 1-4000pmol albumen/mg collagen protein.
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| CN1230402A (en) * | 1998-12-21 | 1999-10-06 | 冶金工业部钢铁研究总院 | Production of composite preparation of bone morphogenesis protein and collagen particle |
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| CN1230402A (en) * | 1998-12-21 | 1999-10-06 | 冶金工业部钢铁研究总院 | Production of composite preparation of bone morphogenesis protein and collagen particle |
Non-Patent Citations (2)
| Title |
|---|
| 人vWF因子A3区基因在大肠杆菌中表达及其生物学活性的研究 祝怀平等.中国试验血液杂志，第12卷第2期 2004 * |
| 骨形态发生蛋白载体材料的研究和应用现状 孙明林等.国外医学生物医学工程分册，第24卷第3期 2001 * |
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