CN1869072A - Nerve growth factor combined with collagen specificity and its coading gene and application - Google Patents
Nerve growth factor combined with collagen specificity and its coading gene and application Download PDFInfo
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Abstract
A nerve growth factor with specific combination power to collagen features that a collagen combining structure domain is fused at the amino terminate of its beta subset. It can be used for treating the nerve injury. Its coding gene is also disclosed.
Description
Technical field
The present invention relates to nerve growth factor and encoding gene thereof and application, particularly relate to and a kind ofly having and the nerve growth factor of collagen specificity binding ability and the expression method of encoding gene and this nerve growth factor thereof.
Background technology
Nerve growth factor (Nerve Growtla Factor is a kind of by α NGF), the mixture that three kinds of different subunits of beta, gamma are formed, and molecular weight is about 140Kd.Studies show that, (β-NGF) is a kind of homodimer molecule to the β subunit of NGF, its strand contains 118 amino-acid residue (Greene, L.A.and E.M.Shooter, Thenerve growth factor:biochemistry, syn thesis, and mechanism of action.AnnuRev Neurosci, 1980.3:p.353-402.), it is the function subunit of NGF, with neuronic survival, migration, growth, differentiation and set up with other cell that functional all many-sides such as to get in touch closely related has the dual function of neurotrophy and short nervous process, is one of most important biologically active substance in the neural system, this is external to prevent the denervation amyotrophy aspect (Sofroniew that also plays an important role, M.V., C.L.Howe, and W.C.Mobley, Nerve growth factor signaling, neuroprotection, and neural repair.Annu Rev Neurosci, 2001.24:p.1217-81.).
Clinical application confirms that local application can make nerve directly obtain nutrition, helps neural axoplasm and flows, and promotes neurotization.Application method with neurotrophic factor treatment peripheral nerve injury mainly contains at present: (1) is in the local directly administration of damage; (2) local by the slow release administration of little osmotic pump; (3) to be transplanted to damage local for the cell that will express neurotrophic factor by transgenic technology.Mode by direct administration is applied to exogenous β-NGF in the body, affected factor is more, it is active keeps and action effect and use approach, mode, dosage etc. all have relation, disposable administration is difficult to guarantee the continuous action of medicine, the dose that successive administration needs is very big again, medical expense is very expensive, and owing to use NGF, the very worth worry of security in large quantities; Little osmotic pump has and absorbs difficult problem in rejection and the body; Vectors in Gene Therapy is selected and safety issue is difficult to resolve certainly, and therefore, above medication all is difficult at clinical application.
Collagen is one of main component of neuronal cell epimatrix, and it and other extracellular matrix have constituted the space frame of neure growth jointly.
Summary of the invention
The purpose of this invention is to provide a kind of nerve growth factor that has with the collagen specificity binding ability.
The nerve growth factor that has with the collagen specificity binding ability provided by the present invention, be that (aminoterminal (N end) of β-NGF) merges collagen binding domains (collagen binding domain, the fusion rotein that CBD) obtains at the nervegrowthfactor-subunit; The albumen that described collagen binding domains is made up of 7 amino-acid residues, its conserved sequence are the SEQ ID № in the sequence table: 1.
SEQ ID № in the sequence table: 1 is made up of 7 amino-acid residues.
In addition, for making things convenient for purifying, the described aminoterminal that has with the nerve growth factor of collagen specificity binding ability also can be connected with the Histidine affinity tag sequence of being made up of 6 Histidines.
Wherein, nerve growth factor in the N of β-NGF end having of merging that the collagen binding domains obtains and collagen specificity binding ability can have SEQ ID № in the sequence table: 2 amino acid residue sequence, SEQ ID № in the sequence table: 2 are made up of 159 amino-acid residues, are Histidine affinity tag sequence from aminoterminal 5-10 amino acids residue; From aminoterminal 22-28 amino acids residue is the conserved sequence of collagen binding domains, is β-NGF from aminoterminal 42-159 amino acids residue, is the connection peptides sequence from aminoterminal 29-41 amino acids residue.
The described gene that has with the nerve growth factor of collagen specificity binding ability of encoding can be the SEQID № in the sequence table: 3.
SEQ ID № in the sequence table: 3 by 480 based compositions, from 5 ' end 16-33 bit base encoding histidine affinity tag sequence, conserved sequence from 5 ' end 67-87 bit base coding collagen binding domains, from 5 ' end 127-480 bit base coding β-NGF, from 5 ' end 88-126 bit base coding connection peptides sequence.
Second purpose of the present invention provides a kind of above-mentioned expression method that has with the nerve growth factor of collagen specificity binding ability.
The expression method that has with the nerve growth factor of collagen specificity binding ability provided by the present invention, be to make up to contain the recombinant expression vector that has with the nerve growth factor gene of collagen specificity binding ability, the recombinant expression vector that makes up is imported host cell, cultivate host cell and make the nerve growth factor genetic expression that has with the collagen specificity binding ability.
The carrier that sets out that is used to make up described recombinant expression vector can be at expression in escherichia coli expression of exogenous gene carrier, as pET-28a, pET-28b, pET-28c, pET-21a (+) or pET-30a etc., is preferably pET-28a.
Be the carrier that sets out with pET-28a, the recombinant expression vector that the containing of structure has with the nerve growth factor gene of collagen specificity binding ability is pET-CBD-NGF.
Described host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Described intestinal bacteria can be E.coli BL21 (DE3), E.coli BL21 (DE3) plys, BLR (DE3) or B834 etc.
With E.coli BL21 (DE3) is starting strain, and the reorganization bacterium that pET-CBD-NGF is imported E.coli BL21 (DE3) acquisition is BL21 (DE3)-pET-CBD-NGF.
Above-mentioned recombinant expression vector and reorganization bacterium all can make up according to ordinary method.
Cultivation contains the present invention and has substratum and culture condition with the host cell of the nerve growth factor gene of collagen specificity binding ability, all can be substratum and the culture condition of cultivating the host that sets out.Wherein, need add inductor when cultivating described recombination bacillus coli host, as IPTG etc., add IPTG concentration be 0.8-1.2mmol/L, be preferably 1mmol/L, inducing temperature is 35-39 ℃, is preferably 37 ℃, induction time is 2-4 hour, is preferably 4 hours.
The invention provides a kind of nerve growth factor and encoding gene thereof that has with the collagen specificity binding ability.This nerve growth factor is based on being closely connected of neurone and collagen, consider from the angle of transforming nerve growth factor, by merging one section collagen land (collagen binding domain at sophisticated β-NGF molecule N end, CBD), to strengthen combining of nerve growth factor and collagen, between nerve growth factor and collagen, set up a kind of stronger physical-chemistry force, thereby β-NGF is anchored on the outer collagen of neurocyte, can guarantee to have identical when active like this, significantly reduce the consumption of β-NGF, avoided because of the excessive risk that causes of β-NGF consumption, the nerve growth factor of enrichment can obviously improve neuronic survival ability relatively simultaneously, further promote neuronic differentiation, thereby for the nerve injury reparation provides a new approach, potential applicability in clinical practice is wide.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is the SDS-PAGE detected result through the collagen binding domains with Histidine affinity tag and the β-NGF mature peptide fusion rotein of expression and purification
The morphological observation that Fig. 2 A is the PC12 cell after CBD-NGF and NAT-NGF induce
Fig. 2 B induces experiment for the PC12 cell axon after CBD-NGF and NAT-NGF induce
Fig. 2 C is the MTT experimental result of the PC12 cell after CBD-NGF and NAT-NGF induce
Fig. 3 A is the detected result of equivalent collagen to difference amount NAT-NGF and CBD-NGF binding capacity
Fig. 3 B is NAT-NGF and the CBD-NGF calculation result to the dissociation constant Kd of collagen
Fig. 4 is the external contractile studies of NAT-NGF and CBD-NGF
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
1, design primer
With three forward primer (CBDU of software Primer premier 5.05 aided design, NGFU1 and NGFU2, sequence sees Table 1) and two reverse primers (CBDD and NGFD), sequence sees Table 1, wherein, CBDU (introduce restriction enzyme Nde I recognition site) and CBDD (introducing restriction enzyme Hind III recognition site) be used to the to increase encoding sequence of collagen binding domains and connection peptides (LINKER), wherein, the conservative aminoacid sequence of collagen binding domains is that (the SEQ ID № in the sequence table: 1), the aminoacid sequence of connection peptides is GSAGSAAGSGGK to TKKTLRT; NGFU1 (introducing restriction enzyme Nde I recognition site) and NGFD (introducing terminator codon and the restriction enzyme Xho I recognition site) β that is used to increase-NGF encoding sequence; NGFU2 (introducing restriction enzyme Hind III recognition site) also is used to increase β-NGF encoding sequence with NGFD (introducing terminator codon and restriction enzyme Xho I recognition site).
Table 1 primer sequence
| CBDU：5’-ATC CATATGACTAAGAAAACCCTGCGTACTGGTAGCGCGGGCAGT-3 ' (band underscore base is a restriction enzyme Nde I recognition site) CBDD:5 '-ACT AAGCTTACCGCCAGAACCCGCAGCACTGCCCGCGCTACCAGT-3 ' (band underscore base is a restriction enzyme Hind III recognition site) NGFU1:5 '-CTA CATATGTCTTCGTCCCATCCCATCTTCCAC-3 ' (band underscore base is a restriction enzyme Nde I recognition site) NGFU2:5 '-GCT AAGCTTTCTTCGTCCCATCCCATCTTCCAC-3 ' (band underscore base is a restriction enzyme Hind III recognition site) NGFD:5 '-GAT CTCGAGTCATCTCACAGCCTTCCTGCTGAGCAC-3 ' (band underscore base is a restriction enzyme Xho I recognition site) |
2, has pcr amplification with the nerve growth factor encoding gene of collagen specificity binding ability
With CBDU and CBDD template each other, with the increase encoding sequence of collagen binding domains and connection peptides of the mode of bridging PCR, 50 μ l PCR reaction systems are: each 1pmol/ μ l of primer CBDU and CBDD, and dNTPs 200 μ mol/ μ l, Taq enzyme 1ul uses ddH
2O postreaction system to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min.After reaction finishes, pcr amplification product is carried out 1.5% agarose gel electrophoresis detect, the result obtains the band of an about 70bp of size.Behind recovery and this purpose band of purifying, with its with restriction enzyme Nde I be connected with prokaryotic expression carrier pET28a (Novagen company) after Hind III carries out double digestion through same enzyme double digestion, it is checked order, sequencing result shows and has obtained the correct collagen binding domains of sequence and the encoding sequence of connection peptides, will correctly be connected with the recombinant vectors called after pET-CBD of this sequence.
Full-length cDNA (GenBank number: NM 002506) with the human is a template, right at the primer of forming by NGFU1 and NGFD respectively, and pcr amplification human under the right guiding of the primer of forming by NGFU2 and NGFD, 50 μ l PCR reaction systems are: each 1pmol/ μ l of upstream and downstream primer, dNTPs 200 μ mol/ μ l, Taq enzyme 1ul uses ddH
2O postreaction system to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 45s then, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate 30 times; 72 ℃ are fully extended 10min.After reaction finishes, pcr amplification product is carried out 1.5% agarose gel electrophoresis detect, the result obtains the band of two about 350bp of size.Behind the band of recovery and purifying two clauses and subclauses, with the amplified band of NGFU1 and NGFD with restriction enzyme Nde I be connected with prokaryotic expression carrier pET28a after Xho I carries out double digestion through same enzyme double digestion, it is checked order, sequencing result shows the encoding sequence that has obtained the correct β with the Histidine affinity tag-NGF mature peptide of sequence, this sequence has the SEQ ID № in the sequence table: 5 nucleotide sequence, SEQID № in the sequence table: 5 by 420 based compositions, SEQ ID № in the code sequence tabulation: 4 amino acid residue sequence, SEQ ID № in the sequence table: 4 are made up of 139 amino-acid residues, will correctly be connected with the recombinant vectors called after pET-NAT-NGF of this sequence; Simultaneously with the amplified band of NGFU2 and NGFD with restriction enzyme HindIII be connected with prokaryotic expression carrier pET-CBD after XhoI carries out double digestion through same enzyme double digestion, it is checked order, sequencing result shows and has obtained the correct collagen binding domains with Histidine affinity tag of sequence and the encoding sequence of β-NGF mature peptide fusion rotein, this sequence has the SEQ ID № in the sequence table: 3 nucleotide sequence, SEQ ID № in the sequence table: 3 by 480 based compositions, from 5 ' end 16-33 bit base encoding histidine affinity tag sequence, conserved sequence from 5 ' end 67-87 bit base coding collagen binding domains, from 5 ' end 127-480 bit base coding β-NGF, from 5 ' end 88-126 bit base coding connection peptides sequence, SEQ ID № in the code sequence tabulation: 2 amino acid residue sequence, SEQ ID № in the sequence table: 2 are made up of 159 amino-acid residues, will correctly be connected with the recombinant vectors called after pET-CBD-NGF of this sequence.
3, have expression and purifying with the nerve growth factor of collagen specificity binding ability
The prokaryotic expression carrier pET-CBD-NGF of the prokaryotic expression carrier pET-NAT-NGF of the encoding sequence of the β with Histidine affinity tag-NGF mature peptide that step 1 is made up and collagen binding domains with Histidine affinity tag and the encoding sequence of β-NGF mature peptide fusion rotein distinguishes transformed into escherichia coli BL21 (DE3) competent cell, transformant is coated on the LB flat board, cultivate down at 37 ℃, selecting mono-clonal is forwarded in the LB liquid nutrient medium 37 ℃ of following cultivations 12-24 hour, be forwarded in the 100mL LB liquid nutrient medium with 2% ratio again, cultivate 3 hours down to OD at 37 ℃
600Value is about 0.8, and adding final concentration is the IPTG of 1mM, continues to cultivate 4 hours.After cultivating end, the centrifugal collection thalline of 8000rpm is with PBS (NaCl 8.5g, Na
2HPO
42.2g, NaH
2PO
40.4g be dissolved in 100ml distilled water, pH7.2) washing back recentrifuge is collected thalline, uses the resuspended thalline of 10mL PBS then, the ultrasonication thalline obtains the recombinant protein crude extract.Centrifugal collection inclusion body through washing, dissolving, utilizes immobilized metal (Ni
2+) part affinity column (Amersham biosciences) carries out purifying.With the renaturation of dialysing of two kinds of expression products behind the purifying, ultrafiltration and concentration is handled, and (citric acid 0.4g, Trisodium Citrate 2.38g are dissolved in 100mL distilled water, pH6.0) displacement, 4 ℃ of preservations with citrate buffer solution with the protein solvent system then.Expression and purified product to pET-CBD-NGF carry out the 15%SDS-PAGE detection, (swimming lane 1 is a protein molecular weight standard to detected result as shown in Figure 1, swimming lane 2 is the expression thalline after the ultrasonication, swimming lane 3 is the inclusion body (under the reductive condition) behind the purifying, expression has obtained the recombinant protein of the about 15.4Kd of molecular weight, conform to expected results, as can be seen from the figure target protein is mainly with the formal representation of inclusion body, obtained after purified purity higher have a nerve growth factor with the collagen specificity binding ability, the collagen binding domains and the β-NGF mature peptide fusion rotein that promptly have the Histidine affinity tag, with this albumen called after CBD-NGF, will express this proteic recombination bacillus coli called after BL21 (DE3)-pET-CBD-NGF.With the β with the Histidine affinity tag-NGF mature peptide called after NAT-NGF that expresses.
Rat adrenal pheochromocytoma PC12 can induce down at NGF, and differentiation generates aixs cylinder, in addition, NGF can promote neuronic survival, therefore, induce experiment and MTT experiment to detect the CBD-NGF of embodiment 2 acquisitions and the activity (Howe of NAT-NGF by PC12 (available from preclinical medicine institute of consonance medical university cell bank) aixs cylinder, C.L., Depolarization of PC12 cells induces neurite outgrowth and enhances nervegrowth factor-induced neurite outgrowth in rats.Neurosci Lett, 2003.351 (1): p.41-5.), method is: be CBD-NGF and the NAT-NGF stimulation PC12 cell of 0-64nM with concentration, with the PC12 cell without stimulation is contrast, after the effect as a result a day, the PC12 cell has aixs cylinder to induce generation (seeing Fig. 2 A); Calculate the prominent cell proportion of major axis, statistics is shown in Fig. 2 B, as can be seen along with the rising of CBD-NGF concentration, the prominent cell proportion of major axis is also along with rising, demonstrate dose-dependent effect, and through statistical study, there is significant difference (*: p＜0.05 in the β-NGF (CBD-NGF) with collagen land with the β-NGF (NAT-NGF) that does not have the collagen land; *: p＜0.01); In addition, carry out the MTT experiment after three days through β-NGF effect, the result is shown in Fig. 2 C, and is same, along with the rising of β-NGF concentration, OD
492Value illustrates that also along with rising the quantity of survivaling cell raises.Above-mentioned experimental result has all shown dose-dependent effect, and through statistical study, there is significant difference (*: p＜0.05 in two kinds of proteic experimental results; *: p＜0.01).The PROTEIN C BD-NGF of this presentation of results process dialysis renaturation and NAT-NGF be biologically active, and the activity of CBD-NGF is significantly higher than NAT-NGF.
The collagen binding ability of embodiment 4, CBD-NGF detects
Utilize genetic engineering technique and microbial fermentation to prepare CBD-NGF, purpose is to strengthen its binding ability to used in tissue engineering collagen three-dimensional stent material guaranteeing its active while, to reduce the effective level of β-NGF.Now detected NAT-NGF and CBD-NGF binding ability (Finnis respectively to collagem membrane with improved ELISA experiment, M.L.and M.A.Gibson, Microfibril-associated glycoprotein-1 (MAGP-1) bindsto the pepsin-resistant domain of the alpha3 (VI) chain of type VI collagen.J Biol Chem, 1997.272 (36): p.22817-23.), and the method (Matsushita that provides according to Matsushita, O., et al., A study of the collagen-binding domain of a 116-kDaClostridium histolyticum collagenase.J Biol Chem, 1998.273 (6): p.3643-8.) calculate the dissociation constant Kd of two kinds of albumen to collagen.On the collagem membrane (6.0mg) of the type i collagen preparation that NAT-NGF that dosage is increased gradually and CBD-NGF are carried in equivalent respectively, fully after the absorption, washing away through PBS can not bonded albumen, equivalent collagen (6.0mg) to the detected result of difference amount NAT-NGF and CBD-NGF binding capacity as shown in Figure 3A, under identical albumen heap(ed) capacity situation, the reservation amount of CBD-NGF on collagem membrane is apparently higher than NAT-NGF, and the joint efficiency that shows the former is apparently higher than the latter.In addition, the dissociation constant Kd of NAT-NGF and CBD-NGF is respectively 1.50 μ M and 0.51 μ M (seeing Fig. 3 B), and according to the definition to Kd, proteic Kd value is more little, and is strong more to the binding ability of collagen.Above-mentioned experimental result proves, compares with NAT-NGF, and CBD-NGF is significantly improved to the binding ability of collagen.
The external functional experiment of embodiment 5, CBD-NGF
At first NAT-NGF that concentration is improved gradually and CBD-NGF are carried in respectively with acid collagen (deriving from the mouse tail) bag by 48 orifice plates of (every hole 0.2mg collagen), adsorb after 1 hour, wash twice with PBS, then with the PC12 cell inoculation in orifice plate, the cell count of every hole inoculation is 5 * 10
3, cultivate after 3 days, detect survival cells for 37 ℃ with mtt assay, detected result is compared with NAT-NGF as shown in Figure 4, and CBD-NGF has higher activity on collagen, in the external survival and the growth that can better promote the PC12 cell, can be used for nerve injury reparation clinically.
Sequence table
<160>5
<210>1
<211>7
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Thr?Lys?Lys?Thr?Leu?Arg?Thr
1 5
<210>2
<211>159
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
1 5 10 15
Arg?Gly?Ser?His?Met?Thr?Lys?Lys?Thr?Leu?Arg?Thr?Gly?Ser?Ala?Gly
20 25 30
Ser?Ala?Ala?Gly?Ser?Gly?Gly?Lys?Leu?Ser?Ser?Ser?His?Pro?Ile?Phe
35 40 45
His?Arg?Gly?Glu?Phe?Ser?Val?Cys?Asp?Ser?Val?Ser?Val?Trp?Val?Gly
50 55 60
Asp?Lys?Thr?Thr?Ala?Thr?Asp?Ile?Lys?Gly?Lys?Glu?Val?Met?Val?Leu
65 70 75 80
Gly?Glu?Val?Asn?Ile?Asn?Asn?Ser?Val?Phe?Lys?Gln?Tyr?Phe?Phe?Glu
85 90 95
Thr?Lys?Cys?Arg?Asp?Pro?Asn?Pro?Val?Asp?Ser?Gly?Cys?Arg?Gly?Ile
100 105 110
Asp?Ser?Lys?His?Trp?Asn?Ser?Tyr?Cys?Thr?Thr?Thr?His?Thr?Phe?Val
115 120 125
Lys?Ala?Leu?Thr?Met?Asp?Gly?Lys?Gln?Ala?Ala?Trp?Arg?Phe?Ile?Arg
130 135 140
Ile?Asp?Thr?Ala?Cys?Val?Cys?Val?Leu?Ser?Arg?Lys?Ala?Val?Arg
145 150 155
<210>3
<211>480
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
atgggcagca?gccatcatca?tcatcatcac?agcagcggcc?tggtgccgcg?cggcagccat 60
atgactaaga?aaaccctgcg?tactggtagc?gcgggcagtg?ctgcgggttc?tggcggtaag 120
ctttcatcat?cccatcccat?cttccacagg?ggcgaattct?cggtgtgtga?cagtgtcagc 180
gtgtgggttg?gggataagac?caccgccaca?gacatcaagg?gcaaggaggt?gatggtgttg 240
ggagaggtga?acattaacaa?cagtgtattc?aaacagtact?tttttgagac?caagtgccgg 300
gacccaaatc?ccgttgacag?cgggtgccgg?ggcattgact?caaagcactg?gaactcatat 360
tgtaccacga?ctcacacctt?tgtcaaggcg?ctgaccatgg?atggcaagca?ggctgcctgg 420
cggtttatcc?ggatagatac?ggcctgtgtg?tgtgtgctca?gcaggaaggc?tgtgagatga 480
<210>4
<211>139
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>4
Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
1 5 10 15
Arg?Gly?Ser?His?Met?Ser?Ser?Ser?His?Pro?Ile?Phe?His?Arg?Gly?Glu
20 25 30
Phe?Ser?Val?Cys?Asp?Ser?Val?Ser?Val?Trp?Val?Gly?Asp?Lys?Thr?Thr
35 40 45
Ala?Thr?Asp?Ile?Lys?Gly?Lys?Glu?Val?Met?Val?Leu?Gly?Glu?Val?Asn
50 55 60
Ile?Asn?Asn?Ser?Val?Phe?Lys?Gln?Tyr?Phe?Phe?Glu?Thr?Lys?Cys?Arg
65 70 75 80
Asp?Pro?Asn?Pro?Val?Asp?Ser?Gly?Cys?Arg?Gly?Ile?Asp?Ser?Lys?His
85 90 95
Trp?Asn?Ser?Tyr?Cys?Thr?Thr?Thr?His?Thr?Phe?Val?Lys?Ala?Leu?Thr
100 105 110
Met?Asp?Gly?Lys?Gln?Ala?Ala?Trp?Arg?Phe?Ile?Arg?Ile?Asp?Thr?Ala
115 120 125
Cys?Val?Cys?Val?Leu?Ser?Arg?Lys?Ala?Val?Arg
130 135
<210>5
<211>420
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
atgggcagca?gccatcatca?tcatcatcac?agcagcggcc?tggtgccgcg?cggcagccat 60
atgtcatcat?cccatcccat?cttccacagg?ggcgaattct?cggtgtgtga?cagtgtcagc 120
gtgtgggttg?gggataagac?caccgccaca?gacatcaagg?gcaaggaggt?gatggtgttg 180
ggagaggtga?acattaacaa?cagtgtattc?aaacagtact?tttttgagac?caagtgccgg 240
gacccaaatc?ccgttgacag?cgggtgccgg?ggcattgact?caaagcactg?gaactcatat 300
tgtaccacga?ctcacacctt?tgtcaaggcg?ctgaccatgg?atggcaagca?ggctgcctgg 360
cggtttatcc?ggatagatac?ggcctgtgtg?tgtgtgctca?gcaggaaggc?tgtgagatga 420
Claims (10)
1, having nerve growth factor with the collagen specificity binding ability, is that the aminoterminal at the nervegrowthfactor-subunit merges the fusion rotein that the collagen binding domains obtains; The albumen that described collagen binding domains is made up of 7 amino-acid residues, its conserved sequence are the SEQ ID № in the sequence table: 1.
2, nerve growth factor according to claim 1 is characterized in that: the aminoterminal of described nerve growth factor is connected with the Histidine affinity tag sequence of being made up of 6 Histidines.
3, nerve growth factor according to claim 2 is characterized in that: described nerve growth factor has SEQ ID № in the sequence table: 2 amino acid residue sequence.
4, the gene of coding right 1 described nerve growth factor.
5, gene according to claim 4 is characterized in that: described gene has the SEQ ID № in the sequence table: 3 dna sequence dna.
6, the expression method of the described nerve growth factor of a kind of claim 1, be to make up the recombinant expression vector that contains the described gene of claim 4, the recombinant expression vector that makes up is imported host cell, cultivate host cell and make the nerve growth factor genetic expression that has with the collagen specificity binding ability.
7, expression method according to claim 6 is characterized in that: the carrier that sets out that is used to make up described recombinant expression vector is pET-28a, pET-28b, pET-28c, pET-21a (+) or pET-30a.
8, expression method according to claim 7 is characterized in that: described recombinant expression vector is pET-CBD-NGF.
9, according to claim 6 or 7 or 8 described expression methods, it is characterized in that: described host is E.coli BL21 (DE3), E.coli BL21 (DE3) plys, BLR (DE3) or B834; With E.coli BL21 (DE3) is that the reorganization bacterium that the host makes up is BL21 (DE3)-pET-CBD-NGF.
10, according to claim 6 or 7 or 8 described expression methods, it is characterized in that: need add the IPTG inductor when cultivating described recombination bacillus coli host, add IPTG concentration be 0.8-1.2mmol/L, inducing temperature is 35-39 ℃, induction time is 2-4 hour.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100815065A CN100400549C (en) | 2006-05-24 | 2006-05-24 | Nerve growth factor combined with collagen specificity and its coading gene and application |
| US12/159,369 US8802396B2 (en) | 2005-12-26 | 2006-12-01 | Activated collagen scaffold materials and their special fused active restoration factors |
| PCT/CN2006/003244 WO2007073661A1 (en) | 2005-12-26 | 2006-12-01 | Activated collagen scaffold materials and their special fused active restoration factors |
| EP06817938A EP1970382B1 (en) | 2005-12-26 | 2006-12-01 | Activated collagen scaffold materials and their special fused active restoration factors |
| AT06817938T ATE514712T1 (en) | 2005-12-26 | 2006-12-01 | ACTIVATED COLLAGEN SCROLLWORK MATERIALS AND THEIR SPECIAL CONDENSED ACTIVE RESTORATION FACTORS |
| US14/320,191 US9365617B2 (en) | 2005-12-26 | 2014-06-30 | Activated collagen scaffold materials and their special fused active restoration factors |
| US14/320,105 US9359403B2 (en) | 2005-12-26 | 2014-06-30 | Activated collagen scaffold materials and their special fused active restoration factors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100815065A CN100400549C (en) | 2006-05-24 | 2006-05-24 | Nerve growth factor combined with collagen specificity and its coading gene and application |
Publications (2)
| Publication Number | Publication Date |
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| CN1869072A true CN1869072A (en) | 2006-11-29 |
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| CN102115495A (en) * | 2010-12-24 | 2011-07-06 | 中国人民解放军第四军医大学 | Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar |
| CN101671396B (en) * | 2008-09-11 | 2012-07-18 | 烟台正海生物技术有限公司 | Vascular endothelial growth factor specifically combined with collagen and application thereof |
| CN101260398B (en) * | 2007-03-07 | 2013-06-05 | 舒泰神（北京）生物制药股份有限公司 | Nerve growth factor gene positioning reconstruction animal and its preparation method and application |
| CN101955543B (en) * | 2009-07-21 | 2013-08-14 | 中国科学院遗传与发育生物学研究所 | Nerve growth factor bound with laminin, and coding gene and application thereof |
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001007059A1 (en) * | 1999-07-21 | 2001-02-01 | University Of Southern California | Matrix-targeted fusion polypeptides for tissue regeneration and wound healing |
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| CN111494712A (en) * | 2020-05-12 | 2020-08-07 | 尧舜泽生物医药(南京)有限公司 | Preparation method of silk fibroin nerve graft fused with NT3 |
| CN111494712B (en) * | 2020-05-12 | 2021-04-09 | 尧舜泽生物医药(南京)有限公司 | Preparation method of silk fibroin nerve graft fused with NT3 |
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