CN104163865A - Optimized DNA sequences of recombinant human bone morphogenetic protein-2 and coded protein thereof - Google Patents
Optimized DNA sequences of recombinant human bone morphogenetic protein-2 and coded protein thereof Download PDFInfo
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- CN104163865A CN104163865A CN201410310953.8A CN201410310953A CN104163865A CN 104163865 A CN104163865 A CN 104163865A CN 201410310953 A CN201410310953 A CN 201410310953A CN 104163865 A CN104163865 A CN 104163865A
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Abstract
Optimized DNA sequences of optimized recombinant human bone morphogenetic protein-2 and coded protein thereof are disclosed. The recombinant protein rhBMP-2SEDIDNO.1 is obtained by adding MGAKQ pentapeptide to 110 amino acids at the C end of human BMP-2 mature peptide, wherein the fifth amino acid H of the BMP-2 mature peptide is mutated into Q. By codon optimization, the optimized DNA sequences which are SEQIDNO.2 and SEQIDNO.3 are obtained by the constructed recombinant rhBMP-2 protein sequence. After recombination of adding the pentapeptide or dipeptide into the BMP-2 mature peptide, the protein expression quantity can be increased to more than 30%, subsequent purification and renaturation are easier, and a high renaturation efficiency can be obtained, thus obviously increasing the yield, allowing the biological activity to be higher, and fitting industrial production better.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of DNA sequence dna and coded protein thereof of recombinant human bone morphogenetic protein matter-2 of optimization.
Background technology
Although occurring in nature BMP is extensively present in the osseous tissue of various animals, but its content is very micro-, per kilogram weight in wet base fresh bone only contains a few microgram BMP, and different sources BMP exists difference on physico-chemical property and molecular structure, and induced osteogenesis is active and stability is also all different.Because various BMP in osseous tissue and insoluble noncollagen protein are combined closely, be difficult to therefrom isolate single any BMP molecule.Therefore, separated BMP from animal osseous tissue, process is numerous and diverse, poor repeatability, yield is low, and purity of protein is not high and inevitably there are differences.Meanwhile, the albumen of this animal-origin is used at human body, can cause that immunological rejection in various degree and potential pathogeny propagate dangerous.Therefore, rely on animal bone to extract completely, be difficult to meet the demand in experiment and clinical application.
Utilizing gene engineering method to produce people BMP-2, not only can guarantee a large amount of production, also can avoid immunological rejection, is the method that has development prospect and magnetism.From Wozney in 1988 etc., obtained the BMP-2 gene in ox bone source, and in recombination bacillus coli after successful expression, the BMP-2 that genetically engineered is produced people in a large number also just becomes possibility.
Aspect the study on the industrialization of BMP-2, be mainly that carrier for expression of eukaryon and intestinal bacteria are two kinds of gene recombination schemes of prokaryotic expression system of representative.1988, Wozney etc. obtained the BMP-2 of COS-1 eukaryotic expression; 1992, Israel DI obtained the BMP-2 of CHO eukaryotic expression; The INFUSE bone migration agent of U.S. Medtronic SofamorDanek company development and LT-CAGE Lumbar Fusion apparatus obtain FDA approval in July, 2002 and are used for the treatment of intervertebral disk retrogression disease; The InductOs (TM) of Wyeth company exploitation (rhBMP-2/ACS) obtains the approval of European Commission (EC) in September, 2002, for the treatment of the acute fracture of tibia of being grown up.The expression system of INFUSE bone migration agent and InductOs is all Chinese hamster ovary celI, and expression amount is low, and production cost is very high, and is not suitable for scale operation.Therefore, eukaryon expression is produced BMP also unrealized industrialization at home.
Aspect prokaryotic expression system, Zhao Ming, nineteen ninety-five Chen Sumin in 1994 etc. have completed the high efficient expression in intestinal bacteria of BMP-2, and expression product is inclusion body, need to carry out external renaturation.German Vallejo in 2002 etc. have realized scale operation intestinal bacteria.
Compare with eukaryotic expression system, prokaryotic expression system is easy to carry out genetic manipulation, and expression efficiency is high, and there is cell walls in extracellular, tender and lovely not as mammalian cell, nutritional requirement is low, strong to the tolerance of culture environment, be easy to cultivate, production cost is low, has very strong competitive power.Although prokaryotic expression system can not make BMP glycosylation, expression product occurs mainly with inclusion body form, and perplexed by the problems such as the difficult control of renaturation process, preparation technology's relative complex, but the expression for BMP, because glycosylation is not the requirement of BMP activity, sugar basedization is modified the biological activity that does not affect them.Therefore, prokaryotic expression system also can be used for expression and the production of BMP.
By gene engineering method, at E. coli foreign protein, be the research hot topic of biological technical field always, have the expression of various factors exogenous protein.It is a kind of method that genetically engineered field often adopts that protein is carried out to structural modification, comprises the structure of the change of natural protein inner amino acid array, the structure of truncation type and long chain type etc., and object is to improve expression, activity and the stability of target protein.Wherein improve in the method for exogenous protein expression, the optimization of gene is most important means.Therefore, this area is in the urgent need to developing the method that is applicable to the active rhBMP-2 of High-efficient Production suitability for industrialized production, based on escherichia expression system.
Summary of the invention
The DNA sequence dna and the coded protein thereof that the object of this invention is to provide a kind of recombinant human bone morphogenetic protein matter-2 of optimization, make it high efficient expression, annealing efficiency is high, output is high, active high, suitability for industrialized is produced.
The technical scheme of enforcement of the present invention is as follows:
DNA sequence dna and the coded protein thereof of recombinant human bone morphogenetic protein matter-2 of optimizing, 110 amino acid of C end that its recombinant protein rhBMP-2 is people's BMP-2 mature peptide add MGAKQ pentapeptide; Or also can be regarded as recombinant protein is that 113 amino acid of BMP-2 mature peptide C end add MG dipeptides, and wherein the five amino acid H of BMP-2 mature peptide sports Q; The aminoacid sequence SED ID as follows NO.1 of its coding:
Further, the restructuring rhBMP-2 protein sequence of structure, by codon is optimized, removes all rare codons, becomes the codon of intestinal bacteria preference; At its front end, add restriction enzyme site Nco I (CCATGG), end adds restriction enzyme site BamHI (GGATCC) simultaneously; After synthetic by full gene, be connected in pUC57 carrier; The DNA sequence dna of its optimization has two, is nucleotide sequence as follows:
SED?ID?NO.2：
SED?ID?NO.3：
Preferably, select pET28a prokaryotic expression carrier as the expression vector of restructuring BMP-2 albumen, the gene fragment described in SED ID NO.2, SED ID NO.3 is inserted in expression vector after double digestion, complete vector construction.PET28 is T7lac promotor, T7 guarantees the high efficient expression of external source insertion gene, lac is lactose operon, make gene when there is no inductor, keep lower background to express (toxic protein or hydrophobin are harmful to bacterium, and this proteinoid background is during the fermentation expressed and often caused plasmid loss or bacterium death).
Compared with prior art, beneficial effect of the present invention is:
BMP-2 mature peptide is after adding the restructuring of pentapeptide or dipeptides, and the expression amount of albumen can be increased to more than 30%, and easier in subsequent purification and renaturation process, can access higher annealing efficiency, thereby its output can obviously increase; There is higher biological activity, be more applicable to suitability for industrialized production.
Accompanying drawing explanation:
Fig. 1 is the pET28 plasmid map that the present invention adopts;
Fig. 2 is the multiple clone site of plasmid pET28;
Fig. 3 is that the present invention is with the full bacterium abduction delivering of carrier intestinal bacteria electrophorogram gene constructed described in SED ID NO.2;
Fig. 4 is that the present invention is with the full bacterium abduction delivering of carrier intestinal bacteria electrophorogram gene constructed described in SED ID NO.3;
Wherein, Fig. 3 and Fig. 4 are coomassie brilliant blue staining:
Fig. 3 shows Lane1: molecular weight standard (is followed successively by: KDa) from top to bottom; Lane2: the bacterium of not inducing; Lane3-10: (Lane3 applied sample amount is 20ul to the bacterium after induction; Lane4-10 applied sample amount is 10ul);
Fig. 4 shows Lane1: molecular weight standard (is followed successively by: KDa) from top to bottom; Lane2 and Lane10: the bacterium of not inducing; Lane3-9: the bacterium after induction (applied sample amount is followed successively by 5ul, 10ul, 15ul, 20ul, 25ul, 30ul, 35ul).
Specific embodiment
Below in conjunction with embodiment, the present invention is further described in detail.Should be noted that, below illustrating is all example, is intended to the present invention to conduct further description; Except as otherwise noted, the implication that term used in the present invention is all understood conventionally in affiliated technical field personnel is identical.
DNA sequence dna and the coded protein thereof of recombinant human bone morphogenetic protein matter-2 of 1, optimizing
110 amino acid of C end that the BMP-2 of restructuring is people's BMP-2 mature peptide add MGAKQ pentapeptide; Or also can be regarded as recombinant protein is that 113 amino acid of BMP-2 mature peptide C end add MG dipeptides, and wherein the 4th of BMP-2 mature peptide the amino acid H sports Q; Its aminoacid sequence is SED ID NO.1:
The restructuring rhBMP-2 protein sequence building, by codon is optimized, removes all rare codons, becomes the codon of intestinal bacteria preference; At its front end, add restriction enzyme site Nco I (CCATGG), end adds restriction enzyme site BamHI (GGATCC) simultaneously; The DNA sequence dna of its optimization has two, is nucleotide sequence as follows:
SED?ID?NO.2：
SED?ID?NO.3：
2, construction of expression vector, conversion colibacillus host cell, construction expression engineering bacteria and recombination and protein expression process
Select pET28a prokaryotic expression carrier as the expression vector of restructuring BMP-2 albumen, pET28 plasmid map is shown in Fig. 1, and the gene fragment described in SED ID NO.2, SED ID NO.3 is inserted in expression vector after double digestion, completes vector construction.PET28 is T7lac promotor, T7 guarantees the high efficient expression of external source insertion gene, lac is lactose operon, make gene when there is no inductor, keep lower background to express, because toxic protein or hydrophobin are harmful to bacterium, this proteinoid background is during the fermentation expressed and is often caused plasmid loss or bacterium death, and Fig. 2 shows pET28 multiple clone site.
(1) construction of expression vector
Owing to adding in advance restriction enzyme site Nco I and BamHI in gene building-up process, so BMP-2 gene fragment is connected in expression vector pET28a by these two restriction enzyme sites.Adopt Nco I and two kinds of DNA endonuclease digestion pET28a carriers of BamHI and pUC57-SED ID NO.2, pUC57-SED ID NO.3, endonuclease reaction system is:
Plasmid after enzyme is cut is separated on 1% agarose gel, and object fragment is cut down from glue, does gel reclaim object fragment with the gel recovery test kit of Axygen company.
Reclaim after DNA fragmentation, pET28a carrier segments is connected with T4DNA ligase enzyme with BMP-2 gene fragment.Ligation system is:
Linked system is carried out the reaction of 1 hour at 16 ℃.
(2) transform colibacillus host cell, construction expression engineering bacteria
After ligation completes, with heat shock conversion method by recombinant plasmid transformed in Bacillus coli cells.By connecting product, join in the competent escherichia coli cell in centrifuge tube, be positioned over 30min on ice, and water-bath is heated in 42 ℃ of water-baths, heat shock 90sec, then rapidly centrifuge tube is inserted on ice, place 1-2min. and add 1ml LB substratum, centrifuge tube is placed in shaking table, 37 ℃, 160rpm cultivates 1 hour.Centrifugal collection intestinal bacteria, are applied in the LB solid medium that adds kantlex, and flat board is put into 37 ℃ of biochemical cultivation case overnight incubation.Second day, exchanges mono-clonal for, by PCR method, identifies whether insert foreign gene, and PCR reaction system is:
The cell of getting the mono-clonal bacterium colony of part picking, joins in PCR reaction system, adds PCR pipe lid, in the enterprising performing PCR reaction of Bio-Rad PCR instrument.Response procedures is:
PCR identifies correct mono-clonal, with the order-checking of T7promoter primer, detects the exactness of sequence.Check order correct mono-clonal renewed vaccination to adding in the LB substratum of kantlex, and in shaking table 37 ℃, 250rpm cultivates, and it is standby to extract plasmid.Recombinant plasmid called after pET28a-SED ID NO.2 and pET28a-SED ID NO.3.
(3) BMP-2 expression vector transforms escherichia coli expression bacterial strain
Adopt BL21 (DE3) expression strain to express restructuring BMP-2 albumen.With heat shock method recombinant plasmid transformed in bacterium.Recombinant plasmid pET28a-SED ID NO.2 and pET28a-SED ID NO.3 are joined respectively in e. coli bl21 (DE3) competent cell in centrifuge tube, place 30 minutes on ice, and by water-bath be heated to 42 ℃ standby.Place on ice after 30 minutes, the centrifuge tube that contains e. coli bl21 (DE3) is put into 42 ℃ of water-baths, heat shock 90sec, then inserts centrifuge tube on ice rapidly, places 1-2 minute.Add 1ml LB substratum, centrifuge tube is placed in shaking table, 37 ℃, 160rpm cultivates 1 hour.Centrifugal collection e. coli bl21 (DE3), is applied in the LB solid medium that adds kantlex, and flat board is put into 37 ℃ of biochemical cultivation case overnight incubation.Second day, picking mono-clonal, by PCR method, identify whether BL21 (DE3) proceeds to recombinant plasmid, and PCR reaction system is:
The cell of getting the mono-clonal bacterium colony of part picking, joins in PCR reaction system, adds PCR pipe lid, in the enterprising performing PCR reaction of Bio-Rad PCR instrument.Response procedures is:
PCR identifies correct mono-clonal, is transferred in the LB substratum that contains kantlex, and in shaking table, 37 ℃ of 250rpm cultivate.When the OD600 of bacterium liquid reaches 0.6-0.8 by the time, bacterium liquid is taken out, add 80% aseptic glycerine to final concentration 10%, and put it into-20 ℃ and save backup.
(4) expression of restructuring BMP-2
BL21 (DE3) inoculation that contains pET28a-SED ID NO.2 and pET28a-SED ID NO.3 that glycerine is preserved is in the LB liquid nutrient medium that contains kantlex, and 37 ℃ of 250rpm are cultured to OD
600while reaching 1.0-1.5, add the IPTG of different concns to carry out abduction delivering, different induction times and temperature are carried out expressing quantity optimization, collect after thalline, through SDS-PAGE amperometry expressing quantity.
Experiment discovery, when IPTG concentration is 1.0mM, inducing temperature is 37 ℃, when induction time is 5 hours, expressing quantity is maximum.Be about 35% left and right (accompanying drawing 3 and accompanying drawing 4).
(5) renaturation and purifying
With sonioation method, by after bacteria breaking, the inclusion body that BMP-2 albumen is formed extracts, and then inclusion body washed and dissolve, Purification, the purity that finally obtains BMP-2 binary reaches more than 95%.
Technique scheme has only embodied the preferred embodiment of the present invention, can not be interpreted as the restriction of the present invention being permitted to scope, and all distortion of making according to the present invention and improvement, all belong to protection domain of the present invention.
Claims (2)
1. DNA sequence dna and the coded protein thereof of recombinant human bone morphogenetic protein matter-2 of optimizing, is characterized in that: the aminoacid sequence SED ID as follows NO.1 of its coding:
。
2. DNA sequence dna and the coded protein thereof of recombinant human bone morphogenetic protein matter-2 of optimizing, is characterized in that: the DNA sequence dna of its optimization has two, is nucleotide sequence as follows:
SED?ID?NO.2：
SED?ID?NO.3：
。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105218659A (en) * | 2015-11-03 | 2016-01-06 | 筴文奎 | Human BMP-7 mature peptide and expression thereof |
| CN105400795A (en) * | 2015-12-21 | 2016-03-16 | 南京江辉生物科技有限公司 | Method for increasing yield of recombinant human bone morphogenetic protein 2 produced from genetic engineering strains by modifying ribosome bind site |
| CN111269916A (en) * | 2020-03-17 | 2020-06-12 | 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5866364A (en) * | 1991-11-04 | 1999-02-02 | Genetics Institute, Inc. | Recombinant bone morphogenetic protein heterodimers |
| CN1484651A (en) * | 2000-11-29 | 2004-03-24 | Production of recombinant BMP-2 | |
| CN101003802A (en) * | 2006-01-18 | 2007-07-25 | 杭州北斗生物技术有限公司 | Method for preparing maturation peptide of morphogenesis protein - 2 of human bones |
| CN101787369B (en) * | 2009-01-23 | 2013-06-19 | 上海瑞邦生物材料有限公司 | DNA sequence of optimized rhBMP (recombinant human bone morphogenetic protein)-2, preparation and purpose of encoding protein thereof |
-
2014
- 2014-07-01 CN CN201410310953.8A patent/CN104163865A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5866364A (en) * | 1991-11-04 | 1999-02-02 | Genetics Institute, Inc. | Recombinant bone morphogenetic protein heterodimers |
| CN1484651A (en) * | 2000-11-29 | 2004-03-24 | Production of recombinant BMP-2 | |
| CN101003802A (en) * | 2006-01-18 | 2007-07-25 | 杭州北斗生物技术有限公司 | Method for preparing maturation peptide of morphogenesis protein - 2 of human bones |
| CN101787369B (en) * | 2009-01-23 | 2013-06-19 | 上海瑞邦生物材料有限公司 | DNA sequence of optimized rhBMP (recombinant human bone morphogenetic protein)-2, preparation and purpose of encoding protein thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105218659A (en) * | 2015-11-03 | 2016-01-06 | 筴文奎 | Human BMP-7 mature peptide and expression thereof |
| CN105218659B (en) * | 2015-11-03 | 2016-06-01 | 筴文奎 | Human BMP-7 mature peptide and expression thereof |
| CN105400795A (en) * | 2015-12-21 | 2016-03-16 | 南京江辉生物科技有限公司 | Method for increasing yield of recombinant human bone morphogenetic protein 2 produced from genetic engineering strains by modifying ribosome bind site |
| CN105400795B (en) * | 2015-12-21 | 2018-11-09 | 南京江辉生物科技有限公司 | The method that ribosome bind site improves engineering strain production rhBMP2 yield is transformed |
| CN111269916A (en) * | 2020-03-17 | 2020-06-12 | 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 |
| 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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Application publication date: 20141126 |