CN1215171C - Process for preparing truncated recombinant human bone morphorgenetic protein-2 mature peptide - Google Patents
Process for preparing truncated recombinant human bone morphorgenetic protein-2 mature peptide Download PDFInfo
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Abstract
The present invention relates to a method for producing truncation type recombinant human bone morphogenetic protein-2 mature peptide (rhBMP-2-108) by constructing gene engineering bacteria. The method obtains recombinant human bone morphogenetic protein-2 mature peptide having high expression amount by constructing gene engineering bacteria containing truncation type human BMP-2 genes, the recombinant human bone morphogenetic protein-2 mature peptide is easy to purify and renature, so the recombinant human bone morphogenetic protein-2 mature peptide has high biological activity. The amount of the truncation type human BMP-2 produced by the gene engineering bacteria of the present invention reaches 30 to 50% of the amount of the total soluble proteins of the bacteria. The recombinant protein separation and purification technology established by the present invention is simple, convenient and practical, is beneficial to mass production of BMP-2 for medical care and lays a foundation for being used in clinic and benefiting patients.
Description
The present invention relates to a kind of structure genetic engineering bacterium and produce the method for truncated recombinant human bone morphogenesis protein-2 mature peptide (rhBMP-2-108), belong to biological technical field.
The ground substance of bone of nineteen sixty-five U.S. doctor Urist discovery decalcification is induced the dystopy osteogenesis, he thinks to contain in the ground substance of bone of decalcification and a kind ofly can induce new osteoplastic material, and with its called after Delicious peptide (Bone Morphogenetic Protein, BMP).He leader's in 1988 research group at first isolates 4 kinds of people's different BMP in the world.The gene that nearest people have successfully cloned other 15 people BMP again, these all provide good material for the research of BMP.Except that BMP-1, they all are transforming growth factor TGF-beta superfamily members, and are no matter on gene or protein structure, still all closely similar on biological function each other.
The biological action of BMP does not have tangible species specificity, has the ability of striding kind of induced osteogenesis.Such as, the BMP of implantation 0.1mg/kg body weight in the mouse muscle of thigh, cartilage can appear in 2～3 weeks.The generating capacity of new bone and the implantation amount of BMP are proportionate.Clinically, if the damaged scope of bone is bigger, generally healing voluntarily adopts usually and transplants reparation from body bone, allograph bone or allosome decalcified bone matrix and some biomaterial.In this case, because the intensity of the speed of bone defect repair and reparation bone is different, and the problems such as rejection of microbiological contamination and allosome decalcification bone, treatment of diseases is all brought influence.Therefore, damagedly be subjected to general attention with what recombinant human B MP repaired bone.Bolande finds, if BMP is mixed implantation with the decalcified bone matrix powder, will improve repair ability.This mixture is used for the rabbit ulna and does the damaged of 2cm length, and its effect obviously is better than simple decalcification bone or autologous bone transplanting control group.Though occurring in nature BMP extensively is present in the osseous tissue of various animals, content is extremely low, is example with people BMP-2, and the per kilogram bone only contains tens～several hectogammas, is difficult to satisfy the clinical application needs.Utilizing engineered method scale operation that bioactive BMP is arranged is extremely attractive method.Since the Genetics Institute of the U.S. in 1988 cloned 4 people's the cDNA of BMP, the foreign scholar cloned 11 new BMP again.Wherein deep to the research of the structure of recombinant human BMP-2 (rhBMP-2) and function.
Genetics Institute company changes people's people BMP-2 cDNA in the eucaryon engineering cell (as COS and Chinese hamster ovary celI) expression.But the rhBMP-2 cost of producing with Chinese hamster ovary celI is very high and output is lower, the clinical practice needs that cost an arm and a leg and be not suitable for China.It is imperative to seek new method.
It is low to the purpose of this invention is to provide a kind of cost, can form the method that truncated recombinant human bone morphogenesis protein-2 mature peptide (rh BMP-2-108) is produced in industrialization.
Technical scheme of the present invention is: make up truncated-type human BMP-2 gene (BMP-2-108) with recombinant DNA technology, and efficiently express this gene in intestinal bacteria, obtained bioactive rhBMP-2-108 protein.Method of the present invention may further comprise the steps:
1) nucleotide sequence of 107 amino-acid residues of synthetic coding people's BMP-2 carboxyl terminal:
Natural human BMP-2 mature peptide is made up of 114 amino-acid residues.Experimental results show that segmental the losing of being made up of the alkaline amino acid residue of two groups of three bunchiness do not influence its biological activity before the N of people BMP-2 mature peptide holds first halfcystine.A potential glycosylation site is arranged on the 55th the asparagicacid residue, and glycosylation is relevant with its biological half-life, and irrelevant with activity.For high-efficiency earth's surface intelligent BMP-2 mature peptide more in the intestinal bacteria system, the gene coded sequence that has at first synthesized 107 amino-acid residues of its carboxyl terminal, and before the codon of first amino-acid residue, added the recognition site of initiator codon ATG and restriction enzyme, second amino acids Arginine residue sported lysine residue and in the end add the recognition site of terminator codon TAA and restriction enzyme after the codon of an amino-acid residue, make up truncated-type human BMP-2 gene
SEQ?ID：NO：1
Length: 324bp
Type: thymus nucleic acid
Chain number: two strands
Geometry: linearity
Source: synthetic
Feature: the sequence of 107 amino-acid residues of coding people BMP-2 mature peptide carboxyl terminal
The gene coded sequence of people BMP-2-108:
5’ATG?AAA?AAA?CTG?AAA?TCC?TCC?TGC?AAA?AGA?CAT?CCG?CTG?TAT
GTG?GAT?TTT?AGC?GAT?GTG?GGC?TGG?AAC?GAT?TGG?ATT?GTG?GCG
CCG?CCG?GGC?TAT?CAT?GCG?TTT?TAT?TGC?CAT?GGC?GAA?TGC?CCG?TTT
CCG?CTG?GCG?GAT?CAT?CTG?AAC?TCC?ACC?AAC?CAT?GCG?ATT?GTG
CAG?ACC?CTG?GTG?AAC?TCT?GTG?AAC?AGC?AAA?ATT?CCG?AAA?GCG
TGC?TGT?GTG?CCG?ACC?GAA?CTG?AGC?GCG?ATT?TCG?ATG?CTG?TAT
CTG?GAT?GAA?AAC?GAA?AAA?GTG?GTG?CTG?AAA?AAC?TAT?CAG?GAT?ATG
GTG?GTG?GAA?GGT?TGT?GGC?TGT?CGC?TAA?3’
The amino acid residue sequence of people BMP-2-108:
MKKLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLN
STNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKWLKNYQDMVVE
GCGCR
2) structure contains the BMP-2-108 expression carrier:
Synthetic BMP-2-108 gene is cut with relevant restriction enzyme respectively with containing with the carrier of this gene same restrictions restriction endonuclease recognition site, and enzyme Qie Wendu is 25～38 ℃, and the enzyme time of cutting is 1～5 hour.Then enzyme is cut product and carried out 1～2% agarose gel electrophoresis, from gel, reclaim and cmy vector and two dna fragmentations of gene with ordinary method.Again two fragments are placed T4DNA ligase enzyme reaction buffer simultaneously, add the T4DNA ligase enzyme, under 4～22 ℃, carry out for the time 1 hour～1 week ligation, wherein employed expression vector or induce the carrier that inserts genetic expression for chemical reagent, or for induce the carrier that inserts genetic expression by temperature variation.
The restriction enzyme enzyme recognition site that is added in synthetic gene head, last two ends can be Ndel and BamHI, perhaps also can be Ncol and HindIII, the expression vector that the former is correlated with can be with available from the chemical reagent induction type carrier of the pET series that contains Ndel and two restriction enzyme sites of BamHI of U.S. Novagen company or the temperature-induced type carrier of pJLA series.Latter's expression vector can be selected the pTrc99A carrier that contains Ncol and two restriction enzyme sites of HindIII available from U.S. Promega company for use.Restriction enzyme and enzyme cutting buffering liquid used in the experiment are TAKARA company product.
3) adopt the molecular cloning operative technique, prepare colibacillary competent cell: directly dip in aseptic platinum filament and get frozen intestinal bacteria, in the line of LB planar surface, in 25～38 ℃ of cultivations 10～20 hours down with the Calcium Chloride Method of routine.1 is separated in good single colony inoculation to the 50～100 milliliter LB nutrient solution, under 25～38 ℃, be cultured to OD with 150～300 times/minute hunting speeds
600=0.3～1.0.Culture is loaded in the centrifuge tube, put 5～20 minutes on ice.Subsequently, with culture in 2～6 ℃ centrifugal 5～20 minutes, speed is 1500～2000g.The precipitation of centrifugal gained is used 10～20 milliliters of ice-cold 0.1mol/L CaCl again
2After solution suspends, descended centrifugal 5～7 minutes at 2～6 ℃ for the second time, speed is 1000～1500g.Centrifugal gained precipitation is used 10～20 milliliters of ice-cold 0.1mol/L CaCl again
2Solution suspends, and places on ice 28～32 minutes.Descended centrifugal 5～7 minutes at 2～6 ℃ for the third time then, speed is 1000～1500g.Centrifugal gained is precipitated with 2～5 milliliters of ice-cold 0.1mol/L CaCl
2Solution suspends.Amount by every pipe 100～200 microlitres is sub-packed in suspension in the aseptic centrifuge tube at last, behind ferrule, freezes immediately in-70 ℃.
4) with the above-mentioned the 2nd) the ligation thing and the 3rd of step gained) go on foot the competence bacterium mixing of gained, placed 10～50 minutes on ice, then in 42 ℃ water-bath, placed 30～150 seconds, ice bath is 1～10 minute again, the LB substratum that adds 0.5～5 milliliter then, temperature was bathed 20～120 minutes in 28～38 ℃ water-baths or incubator.Subsequently, the competent cell that 150～250 microlitres are transformed is applied to and contains on the corresponding antibiotic LB flat board, flat board is placed under the room temperature after liquid is absorbed, be inverted dull and stereotyped, after in 25～38 ℃ of incubators, cultivating 10～30 hours, can occur the recombinating bacterium colony of bacterium is finished the step of converting of plasmid;
Microbiotic can be used kantlex, ammonia benzyl mould etc., at different carriers, selects corresponding microbiotic for use, is carrier as adopting the pET9a in the pET series, then uses kantlex, and employing pET-11b is a carrier, then uses penbritin.
5) from by picking list bacterium colony on the solid plate of the recombination bacillus coli that transformed, be inoculated in the test tube that 1～10 milliliter of LB nutrient solution is housed, the middling speed shaking culture is 6～15 hours under 25～38 ℃ temperature, and the thalline to results adopts conventional alkaline lysis extracting plasmid DNA again;
6) above-mentioned extractive DNA is identified with digestion with restriction enzyme that enzyme Qie Wendu is 30～37 ℃, the enzyme time of cutting is 1～3 hour.The endonuclease bamhi that should contain 300～400bp (BMP-2-108) and 5～6kb (carrier DNA) in the correct recon.All clones that can cut out above-mentioned big or small endonuclease bamhi are the clone who has transformed new gene (rhBMP-2-108), make up genetic engineering bacterium achieving success (this genetically engineered bacterial classification has been kept at Chinese typical culture collection center, preserving number CCTCC NO.M201011).
7) culturing gene engineering bacteria in containing the LB substratum of glucose: by 1% inoculum size genetic engineering bacterium is inserted in 5～10 milliliters the LB substratum, with 100～200 times/component velocity shaking culture 2～3 hours.To induce the carrier that inserts genetic expression with chemical reagent, culture temperature is 30～32 ℃; To induce the carrier that inserts genetic expression by temperature variation, culture temperature is 28～30 ℃.
8) abduction delivering of rhBMP-2-108 gene.Carrying out 7) after the step, the ratio in 1% is inoculated into culture in the Erlenmeyer flask that contains 200～500 milliliters of LB substratum, with 200～300 times/minute hunting speeds, according under the specified culture temperature of step 7) genetic engineering bacterium being cultured to OD
600=1.2～1.4, incubation time is 10～12 hours.Then, continue to cultivate in the LB substratum in the big jar of access.Inoculum size be 5: 1 (nutrient solution: seed liquor), pH7.0.For induce the genetic engineering bacterium that inserts genetic expression with chemical reagent, culture temperature is 30～32 ℃, 100～600 rev/mins of hunting speeds cultivate that to add final concentration in 4 hours be that the IPTG of 1mmol/L induced 3～4 hours, then at 8000～10000 rev/mins of centrifugal collection thalline; Then culture temperature is raised to 42～45 ℃ rapidly for the genetic engineering bacterium of inducing insertion genetic expression by temperature variation, and under concuss, continues to cultivate 3～4 hours, then at 8000～10000 rev/mins of centrifugal collection thalline.
9) with physical method or chemical reagent lysing cell, the extracting recombinant protein through renaturation, separates, and behind the purifying, obtains product truncated recombinant human bone morphogenesis protein-2 mature peptide of the present invention (rhBMP-2-108)
Test shows that the present invention is easier to purifying and renaturation, thereby higher biological activity is arranged by making up the high recombinant human bone morphogenesis protein-2 mature peptide of expression amount that truncated-type human BMP-2 gene obtains.Comparatively speaking, both at home and abroad some laboratories and production department be when carrying out related work, but because of further people's total length BMP-2 of renaturation and purifying intestinal bacteria generation, makes work only rest on laboratory level, can't enter the pharmaceutical stage, more be difficult to form industrialization.The amount of people's truncation type BMP-2 that genetic engineering bacterium of the present invention produced reaches 30～50% of the total soluble protein amount of bacterium, the recombinant protein separating and purifying technology that the present invention sets up is simple and practical, help the therapeutic medical BMP-2 of mass production,, benefit patient and lay the foundation for it is used for clinically.
Description of drawings:
Fig. 1 contains synthetic truncated-type human bone morphogenesis protein-2 mature peptide gene, IPTG abduction delivering type carrier (pET 11b-BMP-2-108)
Fig. 2 contains synthetic truncated-type human bone morphogenesis protein-2 mature peptide gene, temperature-induced expression type carrier (pJLA503-BMP-2-108)
Fig. 3 is that NdeI and two digestion with restriction enzyme of BamHI of pET11b-BMP-2-108 identify that M1 and M2 are molecular weight standard among the figure, and 1～6 is different samples; A is carrier segments (5.7Kb), the gene fragment (324bp) of B for inserting
Fig. 4 is the polyacrylamide gel electrophoresis figure of truncated recombinant human bone morphogenesis protein-2.M is molecular weight standard (is followed successively by from top to bottom 97.4,66,43,31,20.1 and 14.4Kd) among the figure, and C is without IPTG inductive control sample, and 1-5 is through IPTG inductive sample, and arrow indication place is a BMP-2-108 albumen
Fig. 5 is the Western blot figure of recombinant human bone morphogenesis protein-2, and arrow indication place is BMP-2-108
Fig. 6 is the capillary electrophoresis analysis figure of the recombinant human bone morphogenesis protein-2 of purifying, shows that its purity surpasses 97%, abscissa be the time (minute), ordinate is an absorbance value.
Further specify the present invention by the following examples.
Embodiment 1: the method for producing truncated-type human BMP-2 mature peptide with the chemical reagent inducible expression vector
Method of the present invention comprises the steps:
1) nucleotide sequence of 107 amino-acid residues of synthetic coding people's BMP-2 carboxyl terminal:
The gene coded sequence that has at first synthesized 107 amino-acid residues of its carboxyl terminal, and before the codon of first amino-acid residue, added the recognition site of initiator codon ATG and restriction enzyme Ndel, second amino acids Arginine residue sported lysine residue and in the end add the recognition site of terminator codon TAA and restriction enzyme BamHI after the codon of an amino-acid residue.Be built into truncated-type human BMP-2 gene
SEQ?ID：NO：1
Length: 324bp
Type: thymus nucleic acid
Chain number: two strands
Geometry: linearity
Source: synthetic
Feature: the sequence of 107 amino-acid residues of coding people BMP-2 mature peptide carboxyl terminal
The gene order of people BMP-2-108:
5’ATG?AAA?AAA?CTG?AAA?TCC?TCC?TGC?AAA?AGA?CAT?CCG?CTG?TAT
GTG?GAT?TTT?AGC?GAT?GTG?GGC?TGG?AAC?GAT?TGG?ATT?GTG?GCG
CCG?CCG?GGC?TAT?CAT?GCG?TTT?TAT?TGC?CAT?GGC?GAA?TGC?CCG?TTT
CCG?CTG?GCG?GAT?CAT?CTG?AAC?TCC?ACC?AAC?CAT?GCG?ATT?GTG
CAG?ACC?CTG?GTG?AAC?TCT?GTG?AAC?AGC?AAA?ATT?CCG?AAA?GCG
TGC?TGT?GTG?CCG?ACC?GAA?CTG?AGC?GCG?ATT?TCG?ATG?CTG?TAT
CTG?GAT?GAA?AAC?GAA?AAA?GTG?GTG?CTG?AAA?AAC?TAT?CAG?GAT?ATG
GTG?GTG?GAA?GGT?TGT?GGC?TGT?CGC?TAA?3’
The amino acid residue sequence of people BMP-2-108:
MKKLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLN
STNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVE
GCGCR
2) structure contains the BMP-2-108 expression carrier
With chemical reagent inducible expression vector pET-11b, with synthetic BMP-2 gene, (initiating terminal contains Nde I restriction enzyme site, and clearing end contains Bam HI restriction enzyme site), handle through Nde I and Bam HI double digestion respectively with pET-11b, the enzyme tangent condition is 37 ℃ of incubations 3 hours.Then enzyme is cut product with 1～2% agarose gel electrophoresis, electrophoresis 30 minutes, 70 volts of voltages.With above-mentioned two dna fragmentations of Bio101 purification kit purifying.Its process is: downcut the purpose fragment with clean scalpel, put into two 1.5 milliliters of centrifuge tubes respectively.Add 200～500 microlitre Nal, place warm water (45～55 ℃) to glue to melt fully.With granulated glass sphere (Glassmilk is the commodity in the Bio101 purification kit) vortex 1 minute, get 7 microlitres and add above-mentioned two pipes, to place on ice 5 minutes, every 1-2 minute mixing is once.With two pipes under 13000g centrifugal 5 seconds.Remove supernatant, again throw out is resuspended to 500 microlitre New Wash liquid.Then again under 13000g centrifugal 5 seconds.Remove supernatant again.Repeat above-mentioned steps secondary (repeating to suspend centrifugal three times).Last careful exhaustion supernatant.Air drying 5～10 minutes.To precipitate with 17～50 microlitre TE dissolving under 13000g centrifugal 2 minutes.Careful sucking-off supernatant moves to respectively in two new 1.0ml centrifuge tubes.From above-mentioned two pipe DNA, draw an amount of the adding in the 1.0ml centrifuge tube.Comprising dna fragmentation 1～17 microlitre, T4 dna ligase (MBI company) 1 μ l, 10 times of concentration DNA connect damping fluid 2 microlitres, and adding water to cumulative volume is 20 microlitres.Centrifugal slightly behind the mixing, in 22 ℃ carry out for the time 1 hour ligation.Constructed pET-11b-BMP-2-108 sees accompanying drawing 1.
3) adopt molecule clone technology, prepare the competent cell of e. coli bl21 (DE3) with the calcium chloride method of routine: directly dip in aseptic platinum filament and get frozen e. coli bl21 (DE3), in the line of LB planar surface, under 37 ℃ in cultivation 16 hours.1 is separated in good single colony inoculation to 50 milliliter LB nutrient solution, under 37 ℃, be cultured to OD with 250 times/minute velocity fluctuations
600=0.3～0.8.Culture is sub-packed in 2 50 milliliters the centrifuge tube, put 10 minutes on ice.Then, in 4 ℃ centrifugal 7 minutes, speed is 1600g.Centrifugal precipitation is with 10 milliliters of ice-cold CaCl
2Solution dissolving, 4 ℃ centrifugal 5 minutes, centrifugal speed is 1100g.Centrifugal gained precipitation is used 10 milliliters of ice-cold CaCl again
2Solution dissolving was placed 30 minutes on ice, 4 ℃ centrifugal 5 minutes, be 1100g from speed.Again centrifugal gained precipitation is used ice-cold CaCl for the third time
2The solution dissolving, consumption is 2 milliliters.Be added in 1.5 milliliters of aseptic centrifuge tubes by every pipe 200 microlitres, freeze immediately in-70 ℃.
CaCl
2Solution formula (60mmol/L CaCl
2, 15% (V/V) glycerine, 10mmol/LPIPES, pH7.0): anhydrous CaCl
23.33g, PIPES 1.5126g, glycerine 75ml, H
2O 350ml adds 10mol/L NaOH adjust pH to 7.0 after the mixing, constant volume is in 500ml.
4) get the above-mentioned the 2nd) ligation thing 10 microlitres and the 3rd of step gained) the competence bacterium 100 microlitres mixing gently that goes on foot gained, placed 50 minutes on ice, then in 42 ℃ water-bath, placed 90 seconds, put ice bath again 1～2 minute, the LB substratum that adds 0.8 milliliter then, temperature was bathed 60 minutes under 37 ℃ of conditions.Subsequently, the competent cell that 100 microlitres are transformed is applied to and contains on the antibiotic LB flat board of penbritin, and flat board is placed under the room temperature after liquid is absorbed, be inverted dull and stereotyped, cultivate 10～30 hours in 37 ℃ of incubators after, the bacterium colony of the bacterium that can occur recombinating is finished the conversion of plasmid;
5) picking list bacterium colony from the solid plate of the recombination bacillus coli that transforms is inoculated in the test tube that 3 milliliters of LB nutrient solutions are housed, and the middling speed shaking culture is 10 hours under 37 ℃ of temperature, and the thalline to results adopts conventional alkaline lysis extracting plasmid DNA again; This routine extractive process is as follows: the culture that will cultivate 15 hours changes in 1.5 milliliters of centrifuge tubes, centrifugal 30 seconds of 12000g.Abandon supernatant, bacterial precipitation is slightly dry.In the ice-cold Sol I of 100 microlitres, vortex clears precipitation entirely with pellet resuspended.Add the Sol II that 200 microlitres are newly joined, put upside down 6-8 time, centrifuge tube places on ice.Add the ice-cold Sol III of 150 microlitres again, put upside down 6-8 time, centrifuge tube placed 30 minutes on ice.Centrifugal 10 minutes of 12000g shifts in 1.5 milliliters of new centrifuge tubes of supernatant to.Add 400 microlitre phenol chloroformic solutions (phenol: chloroform: primary isoamyl alcohol=25: 24: 1), in supernatant, the vortex mixing, centrifugal 5 minutes of 12000g shifts in 1.5 milliliters of new centrifuge tubes of supernatant to.Add the dehydrated alcohol that is equivalent to 2 times of volumes of supernatant, put upside down mixing, placed 30 minutes for-20 ℃.Centrifugal 10 minutes of 12000g.Abandon supernatant, add 1 milliliter of 70% ethanol, centrifugal 2 minutes of 12000g.Carefully remove supernatant, the dry air precipitation.The TE that contains Pancreatic RNase (20 mcg/ml) with 30 microlitres is dissolution precipitation again, after 37 ℃ of digestion half an hour, be stored in-20 ℃ standby.
Solution formula:
1.Sol I:125ml 200mmol/L glucose, 12.5ml 1mol/L TrisCl (pH8.0), 10ml 0.5mol/L EDTA (pH8.0), constant volume is in 500ml, autoclaving, final concentration: 50mM glucose, 25mM TrisCl (pH8.0), 10mM EDTA (pH8.0).
2.Sol II:10mol/L NaOH 60 μ l, 10%SDS 300 μ l, ddH2O 2640 μ l or 10mol/L NaOH 20 μ l, 10%SDS 100 μ l, ddH2O 880 μ l
3.Sol III (3mol/L NaAc (pH4.8)): anhydrous Na Ac 24.6g, with 70ml ddH2O dissolving, transfer pH to 4.8 with about 20ml glacial acetic acid, be settled to 100ml.
4.TE:5ml 1mol/L TrisCl (pH8.0), 1ml 0.5mol/L EDTA (pH8.0), constant volume be in 500ml, autoclaving, final concentration: 10mM TrisCl (pH8.0), 1mM EDTA (pH8.0).
6) above-mentioned extractive DNA is identified with digestion with restriction enzyme that enzyme Qie Wendu is 30～37 ℃, the enzyme time of cutting is 1～3 hour.The endonuclease bamhi that should contain 300～400bp (BMP-2-108) and 5～6kb (carrier DNA) in the correct recon.All clones that can cut out above-mentioned big or small endonuclease bamhi are the clone who has transformed new gene (rhBMP-2-108), make up genetic engineering bacterium achieving success (bacterial classification has been kept at Chinese typical culture collection center, preserving number CCTCC NO.M201011).Accompanying drawing 3 is cut evaluation figure for its enzyme, and M1 and M2 are molecular weight standard among the figure, and 1～6 is different samples; A is carrier segments (5.7Kb), the gene fragment (324bp) of B for inserting.
7) culturing gene engineering bacteria in containing the LB substratum of glucose: in the LB substratum of the inoculum size by 1% with 5～10 milliliters of genetic engineering bacterium accesses, cultivated 2～3 hours for 100～200 rev/mins with rotating speed.Culture temperature is 30～32 ℃.
8) abduction delivering of rhBMP-2-108 gene.Carrying out 7) after the step, the ratio in 1% is inoculated into culture in the Erlenmeyer flask that contains 200～500 milliliters of LB substratum, with 200～300 rev/mins of rotating speeds, under 30～32 ℃ genetic engineering bacterium is cultured to OD
600=1.2～1.4, incubation time is 10～12 hours.Then, continue to cultivate in the LB substratum in the big jar of access.Inoculum size is 5: 1 (nutrient solutions: seed liquor), pH7.0, culture temperature is 30～32 ℃, 100～600 rev/mins of stirring velocitys, cultivate that to add final concentration in 4 hours be that the IPTG of 1mmol/L induced 3～4 hours, at 8000～10000 rev/mins of centrifugal collection thalline, polyacrylamide gel electrophoresis identifies that explanation has produced this protein (seeing accompanying drawing 4) then;
9) with physical method or the cracking of chemical reagent inducing cell.In stirring and dissolving in ratio adding Tris-Cl (50mmol/L)-EDTA (mmol/L) damping fluid of 1: 5, the PMSF that adds 1: 100 100mmol/L makes that final concentration is 1mmol/L the freezing wet thallus of collecting.Centrifugal collecting precipitation after carrying out ultrasonic bacteria breaking cleans once the inclusion body of collecting precipitation again with Triton X-100.Urea with 1mol/L cleans the freezing preservation of collecting precipitation once more then.Wherein the pH of Tris-EDTA damping fluid is 8.0.About 5～6 hours of whole process need time, under 4 ℃, carry out, 12500 rev/mins of centrifugal rotational speeds, with resolution of precipitate in lysate (Gu.HCl, 7mol/L; Tris, 50mmol/L; EDTA-Na, 1mmol/L; DTT 10mmol/L), is stirred to the complete cracking of inclusion body on magnetic stirring apparatus.With the inclusion body lysate that stirs under 18000g centrifugal 20 minutes, abandon precipitation, supernatant liquor is used for renaturation.Then, further pass through Q post and CM column chromatography purification, just obtain product recombinant human bone morphogenesis protein-2 of the present invention, capillary electrophoresis identifies that purity is (to see accompanying drawing 5, Fig. 6) more than 97%.
The method that the temperature-induced type expression vector of embodiment 2 usefulness is produced truncated-type human BMP-2 mature peptide
1) with embodiment 1 the 1st) step identical.
2) make up the temperature-induced type expression vector that contains the BMP-2-108 gene, working method only changes used expression vector pET-11b into pJLA503 with embodiment 1.The pJLA503-BMP-2-108 that is built into (seeing accompanying drawing 2).
3) with embodiment 1 the 3rd) step identical.
4) with embodiment 1 the 4th) step identical.
5) with embodiment 1 the 5th) step identical.
6) with embodiment 1 the 6th) step identical.
7) culturing gene engineering bacteria in containing the LB substratum of glucose, its method is with embodiment 1, and only culture temperature is 28～30 ℃.
8) abduction delivering of rhBMP-2-108 gene is carrying out 7) after the step, the ratio in 1% is inoculated into culture in the Erlenmeyer flask that contains 200-500 milliliter LB substratum, with 200-300 rev/min of rotating speed, under 28～30 ℃ genetic engineering bacterium is cultured to OD
600=1.2～1.4, incubation time is 10～12 hours.Then, continue to cultivate in the LB substratum in the big jar of access.Inoculum size be 5: 1 (nutrient solution: seed liquor), pH7.0 is raised to 42～45 ℃ rapidly with culture temperature, and continue to cultivate under concuss 3～4 hours, then, at 8000～10000 rev/mins of centrifugal collection thalline.
9) with embodiment 1 the 6th) step identical.
The biological experiment proof has the activity that promotes that bone is repaired with the truncated-type human bone morphogenesis protein-2 mature peptide that the inventive method makes.Biological activity determination method is as follows:
1, cultured cell in vitro is surveyed and lived: the method for pressing people such as Thies is measured alkaline phosphatase activities with the strain of mouse W-20-17 stem cell.The alkaline phosphatase activities unit definition: alkaline phosphatase (AP) refers to the enzyme activity unit number that contains in every milligram of protein than vigor, be expressed as U/mg, an enzyme activity unit is defined as: at 37 ℃, per minute transforms the required enzyme amount of 1umol p-nitrophenyl phosphoric acid (pNPP) under the pH10.5 condition.Detect proof: alkaline phosphatase specific activity of enzyme and BMP-2 dosage are dependency, and promptly the dosage with BMP-2 increases, and illustrate that this product has clear and definite biological activity.
2, dystopy skeletonization experiment in the body: prepare mouse quadriceps muscle of thigh bag model according to universal method in the world.Give 0.5mg rhBMP-2-108 in each mouse quadriceps muscle of thigh lacuna, the type i collagen (available from Sigma company) that adds equivalent is as excipient.Control group is only implanted the 0.5mg type i collagen.After three weeks, carry out the X-ray detection.The result shows that control group does not have dystopy skeletonization phenomenon, and the laboratory animal of implantation rhBMP-2-108 all has tangible dystopy bone-forming effect.
In the radius extirpation experiment that reaches 2cm with rhBMP-2-108 reparation dog, also obtain satisfied effect, implant 4～12 weeks behind the material contain rhBMP-2-108 in the bone defect, through the X-X-ray test X, find that embedded material is absorbed fully, defect produces new bone.
Claims (1)
1. produce the method for truncated recombinant human bone morphogenesis protein-2 mature peptide, it is characterized in that this method may further comprise the steps:
1) nucleotide sequence of 107 amino-acid residues of synthetic coding recombinant human bone morphogenesis protein-2 mature peptide, and before first amino-acid residue codon, added the recognition site of initiator codon ATG and restriction enzyme, second amino acids Arginine residue sported lysine residue and in the end add the recognition site of terminator codon TAA and restriction enzyme after the codon of an amino-acid residue, constitute the gene of truncated recombinant human bone morphogenesis protein-2 mature peptide
SEQ?ID：NO：1
Length: 324bp
Type: thymus nucleic acid
Chain number: two strands
Geometry: linearity
Source: synthetic
Feature: the sequence of 107 amino-acid residues of coding recombinant human bone morphogenesis protein-2 mature peptide
5′ATG?AAA?AAA?CTG?AAA?TCC?TCC?TGC?AAA?AGA?CAT?CCG?CTG?TATGTG?GAT?TTT?AGC?GAT?GTG?GGC?TGG?AAC?GAT?TGG?ATT?GTG?GCGCCG?CCG?GGC?TAT?CAT?GCG?TTT?TAT?TGC?CAT?GGC?GAA?TGC?CCG?TTTCCG?CTG?GCG?GAT?CAT?CTG?AAC?TCC?ACC?AAC?CAT?GCG?ATT?GTGCAG?ACC?CTG?GTG?AAC?TCT?GTG?AAC?AGC?AAA?ATT?CCG?AAA?GCGTGC?TGT?GTG?CCG?ACC?GAA?CTG?AGC?GCG?ATT?TCG?ATG?CTG?TATCTG?GAT?GAA?AAC?GAA?AAA?GTG?GTG?CTG?AAA?AAC?TAT?CAG?GAT?ATGGTG?GTG?GAA?GGT?TGT?GGC?TGT?CGC?TAA 3′
Below the human bone morphogenesis protein-2 mature peptide is represented with english abbreviation BMP-2-108
The amino acid residue sequence of people BMP-2-108:
MKKLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR
2) structure contains the BMP-2-108 expression carrier, synthetic BMP-2-108 gene and the carrier that contains with this gene same restrictions restriction endonuclease recognition site are cut with the relevant limit restriction endonuclease respectively, enzyme Qie Wendu is 25～38 ℃, the enzyme time of cutting is 1～5 hour, then enzyme is cut product and carried out 1～2% agarose gel electrophoresis, from gel, reclaim and cmy vector and two dna fragmentations of gene with ordinary method, again two fragments are placed T4DNA ligase enzyme reaction buffer simultaneously, add the T4DNA ligase enzyme, under 4～22 ℃, carry out for the time 1 hour-1 week ligation, wherein employed expression vector or for inducing the carrier that inserts genetic expression with chemical reagent or for induce the carrier that inserts genetic expression by temperature variation;
3) adopt the molecular cloning operative technique, Calcium Chloride Method with routine prepares colibacillary competent cell: directly dip in aseptic platinum filament and get frozen intestinal bacteria, rule at the LB planar surface, cultivated 10～20 hours down in 25～38 ℃, separate good single colony inoculation in 50-100 milliliter LB nutrient solution with 1, under 25～38 ℃, be cultured to OD with 150～300 times/minute hunting speeds
600=0.3-1.0 is loaded on culture in the centrifuge tube, put 5～20 minutes on ice, subsequently, with culture in 2～6 ℃ centrifugal 5～20 minutes, speed is 1500～2000g, and the precipitation of centrifugal gained is used 10～20 milliliters of ice-cold 0.1mol/L CaCl again
2After solution suspends, descended centrifugal 5～7 minutes at 2～6 ℃ for the second time, speed is 1000～1500g, and centrifugal gained precipitation is used 10～20 milliliters of ice-cold 0.1mol/L CaCl again
2Solution suspends, and places on ice 28～32 minutes, descends centrifugal 5～7 minutes at 2～6 ℃ for the third time then, and speed is 1000～1500g, and centrifugal gained is precipitated with 2～5 milliliters of ice-cold 0.1mol/L CaCl
2Solution suspends, and the amount by every pipe 100～200 microlitres is sub-packed in suspension in the aseptic centrifuge tube at last, behind ferrule, freezes immediately in-70 ℃;
4) with the above-mentioned the 2nd) the ligation thing and the 3rd of step gained) go on foot the competence bacterium mixing of gained, placed 10～50 minutes on ice, then in 42 ℃ water-bath, placed 30～150 seconds, ice bath is 1～10 minute again, the LB substratum that adds 0.5～5 milliliter then, temperature was bathed 20～120 minutes in 28～38 ℃ water-baths or incubator, subsequently, the competent cell that 150～250 microlitres are transformed is applied to and contains on the corresponding antibiotic LB flat board, flat board is placed under the room temperature after liquid is absorbed, be inverted dull and stereotyped, in 25～38 ℃ of incubators, cultivate 10～30 hours after, can occur the recombinating bacterium colony of bacterium is so far finished the conversion process of plasmid;
5) picking list bacterium colony on the colibacillary solid plate after transform, be inoculated in the test tube that 1～10 milliliter of LB nutrient solution is housed, the middling speed shaking culture is 6～15 hours under 25～38 ℃ temperature, and the thalline to results adopts conventional alkaline lysis extracting plasmid DNA again;
6) above-mentioned extractive DNA is identified with digestion with restriction enzyme, enzyme Qie Wendu is 30～37 ℃, the enzyme time of cutting is 1～3 hour, the endonuclease bamhi that should contain the carrier DNA of the BMP-2-108 gene of 300～400bp and 5～6kb in the correct recon, all clones that can cut out above-mentioned big or small endonuclease bamhi are the clone who has transformed the BMP-2-108 gene, make up genetic engineering bacterium and succeed;
7) culturing gene engineering bacteria in containing the LB substratum of glucose: in the LB substratum of the inoculum size by 1% with 5～10 milliliters of genetic engineering bacterium accesses, cultivated 2～3 hours for 100～200 times/minute with hunting speed, to induce the carrier that inserts genetic expression with chemical reagent, culture temperature is 30～32 ℃, to induce the carrier that inserts genetic expression by temperature variation, culture temperature is 28～30 ℃;
8) abduction delivering of BMP-2-108 gene, carrying out 7) after the step, the ratio in 1% is inoculated into culture in the Erlenmeyer flask that contains 200～500 milliliters of LB substratum, with 200～300 times/minute hunting speed, under the specified culture temperature of step 7), genetic engineering bacterium is cultured to OD
600=1.2～1.4, incubation time is 10～12 hours, then, insert in the LB substratum in big jar and continue to cultivate, inoculum size is to add 1 part of seed liquor in 5 parts of nutrient solutions, pH7.0, for induce the genetic engineering bacterium that inserts genetic expression with chemical reagent, culture temperature is 30～32 ℃, 100～600 rev/mins of stirring velocitys, cultivate that to add final concentration in 4 hours be that the IPTG of 1mmol/L induced 3～4 hours, then at 8000～10000 rev/mins of centrifugal collection thalline, then culture temperature is raised to 42～45 ℃ rapidly for the genetic engineering bacterium of inducing insertion genetic expression by temperature variation, and under concuss, continues to cultivate 3～4 hours, then at 8000～10000 rev/mins of centrifugal collection thalline;
9) with physical method or chemical reagent lysing cell, the extracting recombinant protein through renaturation, separates, and behind the purifying, obtains product truncated recombinant human bone morphogenesis protein-2 mature peptide of the present invention.
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| CN100410374C (en) * | 2004-12-02 | 2008-08-13 | 中国科学院遗传与发育生物学研究所 | Method for expressing human bone morphogenetic protein and its special expression vector |
| CN100410371C (en) * | 2006-05-13 | 2008-08-13 | 暨南大学 | Expression method of recombination human bone formation protein-2 |
| US11078248B2 (en) | 2010-03-19 | 2021-08-03 | Lifenet Health | BMP peptides and methods of use |
| CN106749606B (en) * | 2016-12-29 | 2018-06-22 | 广州领晟医疗科技有限公司 | A kind of peptide for repairing cartilage and/or treating osteoarthritis |
| CN111269916B (en) * | 2020-03-17 | 2023-06-13 | 珠海深泓鑫生物科技有限公司 | Human bone morphogenetic protein 2 coding gene suitable for escherichia coli expression |
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