CN1412200A - Method for preparing recombinant human bone morphogenetic protein-7 mature peptide dimer - Google Patents

Abstract

The method for preparing recombinant human bone morphogenetic protein-7 mature peptide dimer includes the following-steps: 1). coding nucleotide sequence SEQ ID No.1 of rh BMP-7 mature peptide, it has SEQ ID NO.2 protein sequence; 2). constituting expression vector containing BMP-7; 3). transferring the expression vector being in step 2 nito host cell and forming the transformation cell capable of producing human BMP-7 protein, and making detection; 4). culturing the transformatino cell; and 5). using physical method or chemical reagent to crack the cell, extracting recombinant protein, and making monochain BMP-7 undergo the processes of renaturation, separation and purification so as to obtain the invented product.

Description

The method for preparing the recombinant human bone morphogenetic protein-7 mature peptide dimer

Technical field

The invention belongs to the medical biotechnology field, specifically, relate to, especially in intestinal bacteria, prepare in a large number, and obtain the method for the recombinant human bone morphogenetic protein-7 mature peptide dimer of high biological activity at prokaryotic cell prokaryocyte

Background technology

The orthopaedics illness is the human common disease of puzzlement, no matter at ordinary times traffic or work accident, still all occupies significant proportion in the war wound incident.The aging of human society, the orthopaedic disease that makes factors such as preventing and treating osteoporosis cause become the serious problem of paying close attention to of each side that is subjected to.And odontopathy runs through the illness in people's all one's life especially, particularly experiences the grinding in years, and the damaged pain of bringing of dentine is made us palpitaition.So under the urgent expectation of medical personnel and pharmaceutical industry, biotechnology worker is at the medicine with new technology and novel method development treatment orthopaedics and tooth section disease.

(Bone Morphogenetic Protein BMP) is a kind of somatomedin with dystopy osteogenesis to Delicious peptide.Since the Urist discovery BMP of the nineteen sixty-five U.S., successfully cloned about 19 kinds people BMP gene so far, created condition for developing new orthopaedics therapy medicine.Wherein, BMP-7 not only has the effect that impels bone growth, promotes the function of dentine growth in addition.Therefore, it has special status in the treatment of orthopaedics and dental disorder.

Studies have shown that in a large number BMP-7 exists with the former form of the propetide of being made up of 431 amino-acid residues in people's cell.In its ripening process, this precursor molecule is cut into the Qian Yuan district of 292 amino-acid residues and the mature peptide fragment of 139 amino-acid residues by eukaryotic special crack enzyme.Research is also found, is comprised that the bmp protein of BMP-7 must form homology or heterodimer, and have only their dimer that activity is just arranged that monomer does not have biologic activity.Have only eukaryotic cell just to contain the Furin enzyme,, could produce sophisticated BMP-7 protein so have only BMP-7 gene transfection eukaryotic cell with total length.Up to now, produce people BMP-7 with biotechnology and only just succeed in eukaryotic cell system, make that the cost of this product of preparation is very high, output also is restricted.As go out to have bioactive people BMP-7 with the prokaryotic organism systems produce, just can reduce cost widely, easy operating process and raising output, this is an extremely attracting research topic.Though the people is arranged for a long time, as famous biologist Mr. Hou Yunde of China etc., at first at expression in escherichia coli the mature peptide of people BMP-7,, resulting is monomer, and can not show biologic activity.So, have only with intestinal bacteria to have produced bioactive BMP-7 mature peptide dimer, could realize the medicative inexpensive people BMP-7 of scale operation, satisfy the needs of clinical treatment.

Summary of the invention

The present invention will provide a kind of with what intestinal bacteria produced the dimeric method of bioactive recombinant human bone morphogenetic protein-7 (rhBMP-7) mature peptide to be arranged.

Method provided by the invention may further comprise the steps:

1) the nucleotide sequence SEQ ID NO.1 of coding rhBMP-7 mature peptide, it has SEQ ID NO.2 protein sequence;

2) structure contains the BMP-7 expression carrier;

3) with step 2) in expression vector change host cell over to, formation can produce the proteinic transformant of people BMP-7, and it is detected;

4) culturing step 3) in the transformant cell;

5) with physical method or chemical reagent lysing cell, the extracting recombinant protein has obtained bioactive dimer with strand BMP-7 renaturation, separation, purifying.

The encode nucleotide sequence of rhBMP-7 mature peptide of the present invention sees SEQ ID NO.1 for details, and it has SEQID NO.2 protein sequence

Nucleotide sequence (SEQ ID NO.1) for coding rhBMP-7 mature peptide, before first amino acid code, added the recognition site of initiator codon ATG and restriction enzyme, in the end add the recognition site of terminator codon TAA and restriction enzyme after amino acid

With eukaryotic cell hobby in the nucleotide sequence of coding rhBMP-7 mature peptide, and the codon that intestinal bacteria need not or seldom be used is fully changed into the codon of intestinal bacteria hobby,

Among the present invention, the restriction enzyme enzyme recognition site that is added in synthetic gene head, last two ends can be an II class restriction enzyme enzyme recognition site arbitrarily.

Among the present invention, can select for use with the BMP-7 gene on the recognition site that adds have the commercially available carrier system of identical restriction enzyme enzyme recognition site.

Among the present invention, term " host cell " is a prokaryotic cell prokaryocyte, and prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.

Among the present invention, be the Guanidinium hydrochloride of 5-7mol/L with the lysate of chemical reagent lysing cell, the Tris of 50mmol/L and the EDTA of 1mmol/L, pH8-9.

Be redox agent with strand BMP-7 renaturation solutions employed among the present invention---Sleep-promoting factor B/reduced glutathion of 0.1-1mmol/L; The NaCl of solubility promoter---0.1-2mol/L; Metal chelator---EDTA of 1-10mmol/L and other additives---glycerine about 0.5-5%, polyoxyethylene glycol etc.

Prepare the recombinant human bone morphogenetic protein-7 mature peptide dimer with the inventive method, can reduce cost greatly, improve output, easy and simple to handle.

Description of drawings

Fig. 1 contains synthetic human bone morphogenetic protein-7 mature peptide gene, IPTG abduction delivering type carrier (pET 11b-BMP-7)

Fig. 2 is that NdeI and two digestion with restriction enzyme of BamHI of pET 11b-BMP-7 identify that M is a molecular weight standard among the figure, and 1-10 is different sample; A, B are respectively 500bp, 250bp molecular weight size, and C is carrier segments (5.7Kb), the gene fragment (423bp) of D for inserting

Fig. 3 is the monomeric polyacrylamide gel electrophoresis figure of recombinant human bone morphogenetic protein-7.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 arrow indication place is the BMP-7 monomer.C is without IPTG inductive control sample, and 1-5 is through IPTG inductive sample.

Fig. 4 is the dimeric polyacrylamide gel electrophoresis figure of recombinant human bone morphogenetic protein-7.5 are molecular weight standard (be followed successively by from top to bottom 97.4,66,43,31,20.1 and 14.4Kd) among the figure.1 is the BMP-7 dimer behind the purifying, and 2 is the BMP-7 inclusion body, and 3 is the BMP-7 whole bacterial protein of abduction delivering, and 4 for not inserting host's mycoprotein of goal gene, and 5 is the molecular weight of albumen standard, and 6 is the BMP-7 recombinant protein, and A is a disome, and B is a monomer.

Fig. 5 is for to be implanted into the new bone that forms behind recombinant human bone morphogenetic protein-7 dimer at two mouse flesh bags.

Embodiment

Following specific examples only is used to illustrate the present invention, and is not used in the restriction scope of the invention.

The method for preparing the recombinant human bone morphogenetic protein-7 mature peptide dimer of the present invention comprises:

1) the nucleotide sequence SEQ ID NO.1 of coding rhBMP-7 mature peptide, it has SEQ ID NO.2 protein sequence;

Coding rhBMP-7 mature peptide nucleotide sequence (SEQ ID NO.1), before first codon, add the recognition site of initiator codon ATG and restriction enzyme, in the end added the recognition site of terminator codon TAA and restriction enzyme after amino acid code;

The eukaryotic cell hobby in the rhBMP-7 mature peptide nucleotide sequence of will encoding, and intestinal bacteria fully need not or few codon that uses change into the codon of intestinal bacteria hobby, as the CCC of proline(Pro); Arginic AGA, AGG and CGG; Changes such as the GGA of glycine and GGG,

2) structure contains the BMP-7 expression carrier;

Use expression vector pET-11b, with synthetic BMP-7 gene, (initiating terminal contains Nde I restriction enzyme site, and clearing end contains Bam HI restriction enzyme site), and to 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 20 minutes, 100 volts of voltages.The test kit that reclaims DNA with the Bio101 gel reclaims above-mentioned two dna fragmentations.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 Bi0101 purification kit) vortex 1 minute, respectively get 7 microlitres and add above-mentioned two pipes, to place 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 dissolving, and place 37 ℃ of water-baths to place 5 minutes with 17～50 microlitre TE.Under 13000g centrifugal 2 minutes.Careful sucking-off supernatant moves to respectively in two new 1.5ml centrifuge tubes.From above-mentioned two pipe DNA, draw an amount of the adding in the 1.5ml 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-7 sees accompanying drawing 1.

3) with step 2) in expression vector change host cell over to, formation can produce the proteinic transformant of people BMP-7, and it is detected;

This example, the preparation host cell is meant the employing molecule clone technology, prepares the competent cell of e. coli bl21 (DE3) with the calcium chloride method of routine.Get above-mentioned 2) ligation thing 10 microlitres of step gained and competent cell 100 microlitres mixing gently, 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;

Picking list bacterium colony from the solid plate of the recombination bacillus coli that transforms, be inoculated in the test tube that 3 milliliters of LB (containing penbritin) nutrient solution is housed, the middling speed shaking culture is 10-16 hour under 37 ℃ of temperature, and the thalline to results adopts conventional alkaline lysis extracting plasmid DNA again; This routine extractive process is as follows: culture is changed in 1.5 milliliters of centrifuge tubes over to centrifugal 30 seconds of 12000g.Abandon supernatant, bacterial precipitation is slightly dry.In the Sol I of 100 microlitres ice precooling, 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 SolIII of 150 microlitres ice precooling again, put upside down 6-8 time.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), 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 supernatant 2-2.5 times volume, put upside down mixing.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).

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 400～500bp (BMP-7) 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-7), make up genetic engineering bacterium and succeed.Accompanying drawing 2 is cut evaluation figure for its enzyme, and M is a molecular weight standard among the figure, and 1～10 is different samples; A, B are respectively 500bp, 250bp molecular weight size, and C is carrier segments (5.7Kb), the gene fragment (423bp) of D for inserting.

4) be fit under the condition of expressing human BMP-7 albumen mature peptide culturing step 3) in the transformant cell;

Culturing gene engineering bacteria in containing the LB of glucose (containing penbritin) substratum: in LB (the containing penbritin) 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 ℃.

In 1% ratio, culture is inoculated in the Erlenmeyer flask that contains 200～500 milliliters of LB substratum again,, under 30～32 ℃, genetic engineering bacterium is cultured to OD with 200～300 rev/mins of rotating speeds 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, then at 8000～10000 rev/mins of centrifugal collection thalline, polyacrylamide gel electrophoresis identifies that explanation has produced this protein (seeing accompanying drawing 3), the BMP-7 mature peptide of strand accounts for more than 30% of bacterial protein content, and the BMP-7 mature peptide of strand accounts for more than 85% of inclusion body total protein content.

5) with physical method or chemical reagent lysing cell, the extracting recombinant protein has obtained bioactive dimer with strand BMP-7 renaturation, separation, purifying;

In stirring and dissolving in ratio adding Tris-Cl (50mmol/L)-EDTA (1mmol/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 behind the broken bacterium of ultrasonic wave 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 resulting inclusion body place lysate (Guanidinium hydrochloride of 5-7mol/L, the Tris of 50mmol/L and the EDTA of 1mmol/L, pH8-9) in, at room temperature placed 20 minutes, with the cracking inclusion body; Then, carry out renaturation after the concentration that is diluted to BMP-7 is about the 10-100 mcg/ml, make it to form dimer.Sleep-promoting factor B/the reduced glutathion that in diluent, adds 0.1-1mmol/L; 0.1-2mmol/L NaCl; The EDTA of 1-10mmol/L and the glycerine of 0.5-5% etc. then, place under 4-10 ℃ (or in room temperature) and spend the night.At any time detect the situation that dimer forms with non-reducing SDS-PAGE.

After reaction, can obtain pure product recombinant human bone morphogenetic protein-7 mature peptide dimer (see figure 4) of the present invention through the chromatography column purifying again except that desolvating by dialysis.

The biological experiment proof has the activity that promotes that bone is repaired with the recombinant human bone morphogenetic protein-7 mature peptide dimer that the inventive method makes.After implantation contained 1 milligram of dimeric material of recombinant human B MP-7 mature peptide in mouse flesh bag, dystopy skeletonization amount reached 340-602 milligram (see figure 5).

The explanation of SEQ ID NO.1 and SEQ ID NO.2.SEQ ID NO.1：423bp：：：：cDNA：：BMP-7139SEQ ID NO.1 ( BMP-7 ) ：5’ ATG TCC ACT GGT AGC AAA CAG CGT TCT CAG AAC CGT TCCAAA ACG CCG AAA AAC CAG GAA GCC CTG CGT ATG GCT AAC GTTGCT GAA AAC AGC AGC AGC GAC CAG AAA CAG GCT TGC AAA AAACAC GAA CTG TAC GTT AGC TTC CGT GAC CTG GGT TGG CAG GACTGG ATC ATC GCT CCG GAA GGT TAC CAC GCT TAC TAC TGC GAAGGT GAA TGC GCT TTC CCG CTG AAC TCC TAC ATG AAC GCT ACTAAC CAC GCT ATC GTT CAG ACT CTG GTT CAC TTC ATC AAC CCG GAAACT GTT CCG AAA CCG TGC TGC GCT CCG ACT CAG CTG AAC GCTATC TCC GTT CTG TAC TTC GAC GAC AGC TCC AAC GTT ATC CTG AAAAAA TAC AGA AAC ATG GTT GTT CGT GCT TGC GGT TGC CAC TAG 3’SEQ ID NO.2：139：：：：BMP-7139SEQ ID NO.2 ( BMP-7 ) ：MSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYHAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYEFDDSSNVILKKYRNMVVRACGCH

Claims (4)

1. method for preparing the recombinant human bone morphogenetic protein-7 mature peptide dimer is characterized in that this method may further comprise the steps:

1) the nucleotide sequence SEQ ID NO.1 of coding rhBMP-7 mature peptide, it has SEQ ID NO.2 protein sequence;

2) structure contains the BMP-7 expression carrier;

3) with step 2) in expression vector change host cell over to, formation can produce the proteinic transformant of people BMP-7, and it is detected;

4) culturing step 3) in the transformant cell;

5) with physical method or chemical reagent lysing cell, the extracting recombinant protein has obtained bioactive dimer with strand BMP-7 renaturation, separation, purifying.

2. by the described method for preparing the recombinant human bone morphogenetic protein-7 mature peptide dimer of claim 1, it is characterized in that the lysate with the chemical reagent lysing cell is the Guanidinium hydrochloride of 5-7mol/L, the Tris of 50mmol/L and the EDTA of 1mmol/L, pH8-9.

3. by the described method for preparing the recombinant human bone morphogenetic protein-7 mature peptide dimer of claim 1, it is characterized in that with strand BMP-7 renaturation solutions employed be redox agent---Sleep-promoting factor B/reduced glutathion of 0.1-1mmol/L; The NaCl of solubility promoter---0.1-2mol/L; Metal chelator---EDTA of 1-10mmol/L and other additives---glycerine about 0.5-5%, polyoxyethylene glycol etc.

4. by the described method for preparing the recombinant human bone morphogenetic protein-7 mature peptide dimer of claim 1, it is characterized in that said host cell is intestinal bacteria.

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