CN1782076A - Method for expressing human bone morphogenetic protein and its special expression vector - Google Patents
Method for expressing human bone morphogenetic protein and its special expression vector Download PDFInfo
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- CN1782076A CN1782076A CN 200410096493 CN200410096493A CN1782076A CN 1782076 A CN1782076 A CN 1782076A CN 200410096493 CN200410096493 CN 200410096493 CN 200410096493 A CN200410096493 A CN 200410096493A CN 1782076 A CN1782076 A CN 1782076A
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- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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Abstract
The present invention discloses a method of expressing human bone morphogenetic protein (BMP) and its special expression vector. The plant expression vector for human BMP is pBI121 plasmid of 35S promoter of cauliflower mosaic virus with human BMP mature peptide coding gene inserted in its downstream. The method of expressing human BMP of the present invention is to introduce the said plant expression vector for human BMP into tobacco to obtain transgenic tobacco and culture transgenic tobacco cell so as to express to obtain human BMP. The said method can express human BMP accounting for 0.02 % of the holoprotein.
Description
Technical field
The present invention relates to a kind of method and dedicated expression vector therefor thereof of expressing human Delicious peptide.
Background technology
The histoorgan engineering is the culture technique of utilizing cell in external Artificial Control cytodifferentiation, breeds and grow into the tissue that needs, the in-vivo tissue of the afunction that be used for repairing because accidental injury etc. causes.In cell proliferation, in growth and the directed differentiation process, need multiple somatomedin that it is regulated and control.(bone morphogenetic protein, BMP) formation to the bone object official plays important regulatory role to people's Delicious peptide.BMP belongs to TGF-beta superfamily (Urist, M.R., Lietze, A., Mizutani, H., Takagi, K., Triffitt, J.T., Amstutz, J., DeLange, R., Termine, J., Finerman, G.A., (1982) .Abovine low molecular weight bone morphogenetic protein (BMP) fraction.Clin.Orthop.162,219-232), found that at present (BMP-1～BMP-13), that bone-inducing activity is the strongest is BMP2 to 13 kinds of BMP.BMP2 is at first by Wozney (Wozney, J.M., Rosen, V., Celeste, A.J., Mitsock, L.M., Whitters, M.J., Kriz, R., Hewick, R., Wang, E.A. (1988) .Novelregulators of bone formation:Molecular clones and activities.Science 242.1528-1534) clone comes out from people's cDNA library.The biological activity of BMP2 is to induce bone forming, promotes the reparation of bone injury, induces undifferentiated mesenchymal cell to skeletonization, chondroblast differentiation, simultaneously, the growth of histoorgans such as embryo's appendage bud and the heart, eye, kidney, nerve is also had important regulating effect.The unique biological activity of BMP2 makes it not only have irreplaceable effect in histoorgan engineering research field, and multiple bone treatment of diseases such as fracture, damaged, the osteoporosis of bone, periodontopathy are had huge using value.The common expression amount of BMP2 is extremely low, though adopt biochemical method to extract natural BMP2 from various osseous tissues, the reason that yields poorly with animal proteinum antigenicity etc. makes it can not satisfy the needs of histoorgan engineering research and clinical requirement far away.Use engineered method can obtain the people BMP2 albumen of q.s.Bone morphogenesis protein-2 is synthetic with precursor forms in vivo, removes signal peptide and propetide through proteolytic cleavage, obtains the mature peptide be made up of 114 amino-acid residues, and carries out glycosylation modifiedly can becoming sophisticated people BMP2.At present people BMP gene obtains expressing (Hammonds, R.G.Jr., Schwall, R. in eukaryotic cell such as COS, CHO, insect and intestinal bacteria, Dudley, A., Berkemeier, L., Lai, C., Lee, J., Cunningham, N., Reddi, A.H., Wood, W.I., Mason, A.J. (1991), Bone-inducing activity of mature BMP-2b produced froma hybrid BMP-2a/2b precursor.Mol.Endocrinol.Jan; 5 (1), 149-155; Ishida, N., Tsujimoto, M., Kanaya, T., Shimamura, A., Tsuruoka, N., Kodama, S., Katayama, T., Oikawa, S., Matsui, M., Nakanishi, T., et al. (1994), Expressionand characterization of human bone morphogenetic protein-2 in silkworm larvaeinfected with recombinant Bombyx mori nuclear polyhedrosis virus.J.Biochem (Tokyo) .Feb; 115 (2): 279-85), but yield poorly, cost is high or active shortcoming such as low has restricted its application.
Though, bacterium, yeast, insect, mammalian cell and transgenic animal can produce pharmaceutical protein as expression system, but plant has a lot of potential advantages as the bio-reactor of producing pharmaceutical protein: 1) can translate post-treatment and protein glycosylation accurately to eukaryotic protein, and bacterium usually produces insoluble polymer during the fermentation, reduce protein-active, improved into the product cost; 2) do not contain the human cause of disease of potential in the plant, its production system is safer, and bacterium itself is human pathogenic agent, and animal virus has potential hazard to the mankind; 3) be easy to enlarge production and storage transportation, reduce production costs; 4) can simplify the link of protein extraction and purifying by effective means; 5) a lot of human or animals' gene in primary object, be subjected to the whole regulation and control of cell to influence expression amount extremely low, efficiently expressed even also be difficult in zooblast or the body by engineered method, and these proteic accumulation may injure animal or zooblast, are expected to overcome this shortcoming with transgenic plant as expression system.
In recent years, continuous development and maturation along with plant transgenic technology, increasing vaccine gene, antibody gene, be transferred in the different plants with the pharmaceutical protein gene and express, as: the HBsAg gene changes in the tobacco and obtains expressing (Mason, H.S., Ball, J.M., Shi, J.J., Jiang, X., Estes, M.K., and Arntzen, C.J. (1996) .Expression of Norwalk virus capsid protein intransgenic tobacco and potato and its oral immunogenicity in mice.Proc.Natl.Assoc.Sci.93,5335-5340); People's beta-interferon, protoheme, serum albumin all obtain expressing (Cramer in tobacco, C., Boothe, J.G., Oishi, K.K. (1999) .Transgenic plantsfor therapeutic proteins:linking upstream and downstream technologies.Current Topics in Microbiology and Immunology.240,95-118).
Summary of the invention
An object of the present invention is to provide a kind of can be in tobacco the plant expression vector of expressing human Delicious peptide.
Human bone morphorgenetic protein plant expression vector provided by the present invention is the pBI121 plasmid that is inserted with the mature peptide encoding gene of human bone morphorgenetic protein in the 35S promoter downstream of cauliflower mosaic virus.
The pBI121 plasmid can be available from CLONTECH company.
The mature peptide of human bone morphorgenetic protein has the amino acid residue sequence of sequence 1 in the sequence table; The mature peptide encoding gene of described human bone morphorgenetic protein can be the nucleotide sequence with sequence 2 in the sequence table.
In order to improve the activity of the BMP2 after the expression, the encoding sequence of 12 amino acid whose heparin lands of N end of the mature peptide of disappearance human bone morphorgenetic protein in the encoding gene of the mature peptide of described human bone morphorgenetic protein obtains having the encoding gene from the mature peptide of the human bone morphorgenetic protein of 5 ' end the 37th to the 345th bit base of sequence 2.
For the ease of the purifying of human bone morphorgenetic protein, 5 of the mature peptide encoding gene of described human bone morphorgenetic protein ' end is connected with histidine-tagged (His-tag).
For the ease of the purifying of human bone morphorgenetic protein and the screening of transgenic plant, described human bone morphorgenetic protein mature peptide encoding gene the 35S promoter of cauliflower mosaic virus and and the GRD beta-glucuronidase gene between and the GRD beta-glucuronidase gene form fusion gene.
For the ease of human bone morphorgenetic protein and the excision of the GRD beta-glucuronidase in the GRD beta-glucuronidase fusion rotein in purge process the present invention expressed, 3 of described human bone morphorgenetic protein mature peptide encoding gene ' end is connected with enteropeptidase identification restriction enzyme site.
In order to improve expression efficiency, the 35S promoter of the cauliflower mosaic virus of described plant expression vector is substituted by two 35S promoters.
Described pair of 35S promoter has the nucleotide sequence of sequence 3 in the sequence table.
In order to improve the intravital mRNA translation efficiency of plant, 3 of described promotor ' end is connected with AMV enhancement sequences (the RNA4 leader sequence of plant virus alfalfa mosaic virus).
Described AMV enhancement sequences has the nucleotide sequence of sequence 4 in the sequence table.
3 of described pair of 35S promoter ' end closely connects the AMV enhancement sequences and obtains two 35S-AMV, and it has the nucleotide sequence of sequence 5 in the sequence table.
The plant expression vector of described human bone morphorgenetic protein specifically can be 11ds2 (Fig. 1 b) and pBIb2-11, and (Fig. 1 a); Be preferably 11ds2.。
Second purpose of the present invention provide a kind of can be in tobacco the method for expressing human Delicious peptide.
The method of expressing human Delicious peptide provided by the present invention is to obtain transgene tobacco in the plant expression vector importing tobacco with above-mentioned human bone morphorgenetic protein, expresses obtaining human bone morphorgenetic protein.
The plant expression vector of described human bone morphorgenetic protein can import tobacco by agriculture bacillus mediated leaf disc transformation method.
The equal expressing human Delicious peptide of transgene tobacco whole plant, the highest with the expression amount of blade, the best moment of results human bone morphorgenetic protein is that plant grows to 20-40cm height (transgenosis seedling to the root media approximately 2-4 month).The cultural method of described transgene tobacco is identical with the cultural method of transgene tobacco not.
The present invention utilizes plant binary expression vector PBI 121 to make up the human bone morphorgenetic protein plant expression vector of the mature peptide encoding gene that contains human bone morphorgenetic protein, utilization is easy to breed tobacco with gene transformation as genetically modified host expresses human bone morphorgenetic protein, but this method expressing human Delicious peptide is 0.02% (a quality percentage composition) of whole protein.
Description of drawings
Fig. 1 a is the physical map of carrier segments of the mature peptide encoding gene that contains human bone morphorgenetic protein of plant expression vector pBIb2-11
Fig. 1 b is the physical map of carrier segments of the mature peptide encoding gene that contains human bone morphorgenetic protein of plant expression vector 11ds2 carrier
Fig. 2 transforms resistant plant for 11ds2 and pBIb2-11 transforms the result of the genomic dna of part plant in the resistant plant colony with primer 1 and primer 2 PCR detection
Fig. 3 transforms resistant plant for 11ds2 and pBIb2-11 transforms the result of the genomic dna of part plant in the resistant plant colony with primer 1 and primer 3 PCR detection
Fig. 4 is the active detected result mean level (ML) of 11ds2 colony and the control population pBIb2-11 GUS of a colony column diagram relatively
Fig. 5 is the active RT-PCR detected result that detects higher plant of GUS
Fig. 6 is the active Western-blot detected result that detects higher plant of GUS
Embodiment
Experimental technique in following examples is ordinary method if no special instructions.
The structure of embodiment 1, plant expression vector pBIb2-11 and 11ds2
1, design of primers: computer aided design (CAD) people BMP2 forward primer and reverse primer, in forward primer, introduce the His-tag of 6 Histidines, according to the achievement in research (Joshi of Joshi, C.P., Zhou, H., Huang, X., Chiang, V.L. (1997), Context sequences of translation initiation codonin plants.Plant.Mol.Biol.Dec; 35 (6): 993-1001) meet the initiator codon of Kozak rule and sequence and XbaI enzyme cutting site on every side.Introduce the proteolytic enzyme restriction enzyme site of enteropeptidase and the restriction enzyme site of BamHI in the reverse primer.
HBMP2 forward primer (Prb2-2a): 5 ' CG
TCTAGATAMCA
GCT
XbaI Kozak
CATCATCATCATCATCAT AGCTGTAAGAGACAC 3’
His-tag
HBMP2 reverse primer (Prb2-2b): 5 ' GC
GGATCC CTTGTCATCGTCATC
The restriction enzyme site of BamHI enteropeptidase
GCGACACCCACAACCC
Two 35SAMV forward primers: 5 ' ACGCC
AAGCTTGCATGCCTGC 3 '
HindIII
Two 35SAMV reverse primers: 5 ' AC
TCTAGATGGAAGTATTTGAAAGAAAATTAAAAATAAAAAG
XbaI
2, the structure of plant expression vector pBIb2-11
Extract total RNA of people, reverse transcription is cDNA, as template, carries out pcr amplification under the guiding of the forward and reverse primer of hBMP2.Wherein, reaction system is: two each 20pmol/L of primer, 2mmol/L dNTPs 3 μ l, Taq enzyme 2U, 10 * Taq enzyme buffer liquid (QIAGEN), 3 μ l, cumulative volume is 30 μ l.Reaction conditions is 94 ℃ of pre-sex change 10min down; 94 ℃ of following sex change 30s then, 67 ℃ of annealing 45s down, 72 ℃ are extended 1min down, circulate 35 times; 72 ℃ are fully extended 5min down.Amplified production carries out 1.0% agarose gel electrophoresis, and the result obtains a band about 360bp.Amplified band is cloned into the pGEM-T carrier after reclaiming, carry out gene sequencing, and the result shows that the human bone morphorgenetic protein mature peptide encoding gene that amplification obtains is 5 ' end the 37th to the 345th bit base from SEQ ID NO:2.Pcr amplification product connects on the pBI121 of same double digestion behind XbaI and BamHI double digestion after reclaiming.With the connection product transformed into escherichia coli JM109 competent cell that obtains.After the dull and stereotyped cultivation screening of the LB that contains kantlex, extract plasmid and carry out the PCR evaluation with the forward and reverse primer of hBMP2, the result shows the band that obtains about 360bp, with this plasmid called after pBIb2-11 (Fig. 1 b).PBIb2-11 is checked order, the result show insertion human bone morphorgenetic protein mature peptide coding gene sequence (for sequence 2 from 5 ' end the 37th to the 345th bit base) correct.
3, the structure of plant expression vector 11ds2
With the two 35S promoters that contain cauliflower mosaic virus and the plasmid pGPTV (Kohler of AMV enhancer sequence, R.H., Zipfel, W.R., Webb, W.W., Hanson, M.R. (1997), The green fluorescentprotein as a marker to visualize plant mitochondria in vivo.PlantJ.Mar; 11 (3): 613-21.) be template, under the guiding of the forward and reverse primer of two 35SAMV, carry out pcr amplification.Wherein, reaction system is: two each 20pmol/L of primer, 2mmol/L dNTPs 3 μ l, Taq enzyme 2U, 10 * Taq enzyme buffer liquid (QIAGEN), 3 μ l, cumulative volume is 30 μ l.Reaction conditions is 94 ℃ of pre-sex change 10min down; 94 ℃ of following sex change 30s then, 67 ℃ of annealing 45s down, 72 ℃ are extended 1min down, circulate 35 times; 72 ℃ are fully extended 5min down.Amplified production carries out 1.0% agarose gel electrophoresis, and the result obtains the band about a treaty 700bp.Amplified band is cloned into the pGEM-T carrier after reclaiming, carry out gene sequencing, and the result shows that two 35S-AMV that amplification obtains have the nucleotide sequence of SEQ ID NO:5 in the sequence table.Pcr amplification product connects on the pBIb2-11 of same double digestion behind HindIII and XbaI double digestion after reclaiming.With the connection product transformed into escherichia coli JM109 competent cell that obtains.After the dull and stereotyped cultivation screening of the LB that contains kantlex, extract plasmid and carry out the PCR evaluation with the forward and reverse primer of two 35SAMV, the result shows the band that obtains about about 700bp, with this plasmid called after 11ds2 (Fig. 1 b).11ds2 is checked order, and the result shows that two 35S-AMV sequences of insertion are correct.
1, the acquisition of transfer-gen plant
According to document (Wang Guanlin, Fang Hongjun, philosophy and technique Beijing Science Press 1998 of plant genetic engineering) leaf disc transformation method by Agrobacterium LBA4404 mediation is with pBIb2-11 or 11ds2 transformation of tobacco NC89, on the MS substratum that contains 500mg/L Pyocianil and 100mg/L kantlex, screen, the result obtains the 11ds2 transformed plant (11ds2 colony) that 126 strains are taken root on the resistance substratum, 90 strain pBIb2-11 transformed plants (pBIb2-11 colony).Genomic dna with above-mentioned 216 strains is a template respectively, utilize the forward and reverse primer amplification human bone morphorgenetic protein of hBMP2 mature peptide encoding gene, utilize hBMP2 forward primer and Prpb-1b amplification human bone morphorgenetic protein mature peptide encoding gene and merged the fusogenic peptide part that GUS N holds 44bp, the result shows that this 216 strain resistant plant utilizes all the increase positive fragment (Fig. 2) of 367bp of the forward and reverse primer of hBMP2, utilizes all the increase positive fragment (Fig. 3) of 411bp of hBMP2 forward primer and Prpb-1b.Fig. 2 and Fig. 3 have shown the amplification of part plant.This presentation of results, the fusion gene of BMP2 and GUS correctly are connected in the genome of the plant that also successfully has been incorporated into the process antibiotic-screening on the carrier.Wherein, Prpb-1b is gus gene 5 ' the end reverse primer of amplification upstream, and its sequence is: 5 ' GTTGGGGTTTCTACAGGAC 3 '.
2, the active detection of GUS in the transgenic plant
Carrying out the GUS active level when plant that the antibiotic-screening in the step 1 goes out grows to 20-40cm detects and histochemical stain, method by people such as Jefferson is carried out (Jefferson, R.A., Kavanagh, T.A., Bevan, M.W. (1987), GUS fusions: β-Glucuronidase as a Sensitive and Versatile GeneFusion Marker in Higher Plants.EMBO.J.6:3901-3907).Result such as table 1 and shown in Figure 4 show that the active integral level of the GUS of 11ds2 colony apparently higher than the GUS of pBIb2-11 colony activity level (P＜0.01), on average exceed 3.94 times, and the active highest level of the GUS of 11ds2 colony exceeds 5.92 times than pBIb2-11 colony.Among Fig. 4,1 is the mean level (ML) that the GUS of 11ds2 colony expresses, and 2 is the mean level (ML) that the control group pBIb2-11 GUS of colony expresses.
The comparison of the active detected result of table 1. 11ds2 colony and the control population pBIb2-11 GUS of colony
| Colony's plant number | Detect the active plant number of GUS | Proportion | The mean level (ML) (pmol MU/mg/min) that the GUS of colony expresses | The highest level (pmol MU/mg/min) that GUS expresses in the colony | |
| pBIb2-11 | 90 | 21 | 23.3％ | 798.8 | 1409.3 |
| 11ds2 | 126 | 57 | 45.2％ | 3143.6 | 8346.5 |
3, RT-PCR and Western-Blot detect
(1) RT-PCR detects
RNeasy Plant Kit with QIAGEN company carries out RT-PCR detection (used primer is the forward and reverse primer of hBMP2) by its experimental program that provides to the higher plant of part GUS detection of active in the 11ds2 colony, the result as shown in Figure 5, show in five higher plant genomes of the active detection of GUS the fragment of the 367bp that all increases.Reclaim fragment and the order-checking of this 367bp, the result shows that the BMP2 gene order of this fragment and insertion is in full accord.This presentation of results, the BMP2 gene can correctly be transcribed on transcriptional level.Among Fig. 5,1,3,5,7,9 swimming lanes are five higher plant of the active detection of GUS, and 2,4,6,8 swimming lanes are the contrast that four strains in above-mentioned five plant do not add reversed transcriptive enzyme.
(2) Western-Blot detects
Get the blade of the higher plant of part GUS detection of active in the 11ds2 colony, extract the plant total protein according to a conventional method, with anion-exchange column (Hiprep 16/10 QXL
TM, Amersham), His-tag post (Chelatingsepharose
TMFast flow, Amersham) and molecular sieve column (Hiload 16/60 superdex
TM75 prepgrade Amersham) carry out purifying, and it is 91% human bone morphorgenetic protein that the result obtains purity, and its expression amount is 0.02% (a quality percentage composition) of whole protein.
The albumen that purifying is obtained with BMP2 monoclonal antibody (purchasing the company in SIGMA) carries out the Western-blot detection, and the result shows that BMP2 albumen obtains to express as shown in Figure 6 in transfer-gen plant.Among Fig. 6,1-8 is the higher plant of GUS detection of active in the 11ds2 colony, and 0 is not transgene tobacco contrast.
Sequence table
<160>5
<210>1
<211>114
<212>PRT
＜213〉Genus Homo people (Homo sapiens)
<400>1
Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg
1 5 10 15
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile
20 25 30
Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro
35 40 45
Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln
50 55 60
Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val
65 70 75 80
Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu
85 90 95
Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly
100 105 110
Cys Arg
<210>2
<211>345
<212>DNA
＜213〉Genus Homo people (Homo sapiens)
<400>2
caagccaaac acaaacagcg gaaacgcctt aagtccagct gtaagagaca ccctttgtac 60
gtggacttca gtgacgtggg gtggaatgac tggattgtgg ctcccccggg gtatcacgcc 120
ttttactgcc acggagaatg cccttttcct ctggctgatc atctgaactc cactaatcat 180
gccattgttc agacgttggt caactctgtt aactctaaga ttcctaaggc atgctgtgtc 240
ccgacagaac tcagtgctat ctcgatgctg taccttgacg agaatgaaaa ggttgtatta 300
aagaactatc aggacatggt tgtggagggt tgtgggtgtc gctag 345
<210>3
<211>634
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
acgccaagct tgcatgcctg caggtccgat tgagactttt caacaaaggg taatatccgg 60
aaacctcctc ggattccatt gcccagctat ctgtcacttt attgtgaaga tagtggaaaa 120
ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggccatcg ttgaagatgc 180
ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga 240
agacgttcca accacgtctt caaagcaagt ggattgatgt gatggtccga ttgagacttt 300
tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt 360
tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg 420
aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac 480
gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg 540
tgatatctcc actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc 600
ctctatataa ggaagttcat ttcatttgga gagg 634
<210>4
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
ttattttt aattttcttt caaatacttc ca 32
<210>5
<211>672
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
acgccaagct tgcatgcctg caggtccgat tgagactttt caacaaaggg taatatccgg 60
aaacctcctc ggattccatt gcccagctat ctgtcacttt attgtgaaga tagtggaaaa 120
ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggccatcg ttgaagatgc 180
ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga 240
agacgttcca accacgtctt caaagcaagt ggattgatgt gatggtccga ttgagacttt 300
tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt 360
tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg 420
aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac 480
gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg 540
tgatatctcc actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc 600
ctctatataa ggaagttcat ttcatttgga gaggagatct ttttattttt aattttcttt 660
caaatacttc ca 672
Claims (10)
1, human bone morphorgenetic protein plant expression vector is the pBI121 plasmid that is inserted with the mature peptide encoding gene of human bone morphorgenetic protein in the 35S promoter downstream of cauliflower mosaic virus.
2, plant expression vector according to claim 1 is characterized in that: the encoding sequence of 12 amino acid whose heparin lands of N end of the mature peptide of disappearance human bone morphorgenetic protein in the encoding gene of the mature peptide of described human bone morphorgenetic protein.
3, plant expression vector according to claim 2 is characterized in that: 5 of the mature peptide encoding gene of described human bone morphorgenetic protein ' end is connected with histidine-tagged.
4, plant expression vector according to claim 2, it is characterized in that: described human bone morphorgenetic protein mature peptide encoding gene the 35S promoter of cauliflower mosaic virus and and the GRD beta-glucuronidase gene between and the GRD beta-glucuronidase gene form fusion gene.
5, plant expression vector according to claim 2 is characterized in that: 3 of described human bone morphorgenetic protein mature peptide encoding gene ' end is connected with enteropeptidase identification restriction enzyme site.
6, according to each described plant expression vector among the claim 1-5, it is characterized in that: the 35S promoter of the cauliflower mosaic virus of described plant expression vector is substituted by two 35S promoters.
7, plant expression vector according to claim 6 is characterized in that: 3 of described promotor ' end is connected with the AMV enhancement sequences.
8, plant expression vector according to claim 7 is characterized in that: the plant expression vector of described human bone morphorgenetic protein is 11ds2 and pBIb2-11.
9, a kind of method of expressing human Delicious peptide is to obtain transgene tobacco in the plant expression vector importing tobacco with each described human bone morphorgenetic protein among the claim 1-7, expresses obtaining human bone morphorgenetic protein.
10, method according to claim 9 is characterized in that: the plant expression vector of described human bone morphorgenetic protein imports tobacco by agriculture bacillus mediated leaf disc transformation method.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776198A (en) * | 2011-05-09 | 2012-11-14 | 扬州泰达生物科技有限公司 | Method for expressing recombinant human bone morphogenetic protein in insect cell |
| US20150096070A1 (en) * | 2008-09-22 | 2015-04-02 | Monsanto Technology Llc | Chimeric promoters and their uses in plants |
| CN102604964B (en) * | 2007-07-19 | 2015-08-19 | 加利福尼亚大学董事会 | Grain yield is increased by the minimizing of the Ethylene Signal Transduction of target |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1062017C (en) * | 1995-09-01 | 2001-02-14 | 中国科学院遗传研究所 | Plant expression carrier plasmid of phylaxin gene |
| CN1215171C (en) * | 2001-04-21 | 2005-08-17 | 杭州华东医药(集团)公司基因技术研究所 | Process for preparing truncated recombinant human bone morphorgenetic protein-2 mature peptide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604964B (en) * | 2007-07-19 | 2015-08-19 | 加利福尼亚大学董事会 | Grain yield is increased by the minimizing of the Ethylene Signal Transduction of target |
| US20150096070A1 (en) * | 2008-09-22 | 2015-04-02 | Monsanto Technology Llc | Chimeric promoters and their uses in plants |
| CN102776198A (en) * | 2011-05-09 | 2012-11-14 | 扬州泰达生物科技有限公司 | Method for expressing recombinant human bone morphogenetic protein in insect cell |
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