CN100400549C - Nerve growth factor specifically combined with collagen and its coding gene and application - Google Patents

Abstract

The invention discloses a nerve growth factor and its coding gene and application. The nerve growth factor with the ability to specifically bind to collagen is a fusion protein obtained by fusing the collagen binding domain at the amino terminal of the nerve growth factor β subunit; the collagen binding domain is a protein composed of 7 amino acid residues, Its conserved sequence is SEQ ID No. 1 in the sequence listing. The nerve growth factor of the present invention can greatly reduce the dosage of β-NGF while ensuring the same activity, avoiding the risk caused by excessive dosage of β-NGF, and at the same time, the relatively enriched nerve growth factor can significantly improve the survival ability of neurons , to further promote the differentiation of neurons, thereby providing a new way for nerve injury repair, and has broad prospects for clinical application.

Description

Nerve growth factor specifically combined with collagen and its coding gene and application

technical field

The present invention relates to nerve growth factor and its encoding gene and application, in particular to a nerve growth factor capable of specifically combining with collagen, its encoding gene and the expression method of the nerve growth factor.

Background technique

Nerve Growth Factor (NGF) is a complex composed of three different subunits of α, β, and γ, with a molecular weight of about 140Kd. Studies have shown that the β subunit of NGF (β-NGF) is a homodimeric molecule, and its single chain contains 118 amino acid residues (Greene, L.A. and E.M. Shooter, Thenerve growth factor: biochemistry, synthesis, and mechanism of action.AnnuRev Neurosci, 1980.3: p.353-402.), it is a functional subunit of NGF, which is closely related to many aspects such as the survival, migration, growth, differentiation and establishment of functional connections with other cells of neurons. It is one of the most important biologically active substances in the nervous system, and it also plays an important role in preventing denervated muscle atrophy (Sofroniew, 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 has proved that local administration can directly nourish the nerves, facilitate the axoplasmic flow of the nerves, and promote nerve regeneration. Currently, neurotrophic factors are used in the treatment of peripheral nerve injury mainly in the following ways: (1) direct administration at the injured part; (2) local slow release of drug through micro-osmotic pumps; The cells are transplanted into the injured area. The application of exogenous β-NGF into the body through direct administration is affected by many factors. The maintenance of its activity and effect are related to the route, method, and dosage of the drug. The continuous effect of continuous administration requires a large amount of medicine, the cost of treatment is very expensive, and because of the large amount of NGF used, the safety is very worrying; the micro-osmotic pump has the problems of rejection and difficult absorption in the body; gene therapy Carrier selection and safety issues are difficult to solve, therefore, the above drug delivery methods are difficult to apply clinically.

Collagen is one of the main components of the extracellular matrix of neurons, and together with other extracellular matrices, it constitutes the spatial scaffold for neuron growth.

Contents of the invention

The purpose of the present invention is to provide a nerve growth factor with specific binding ability to collagen.

The nerve growth factor with the ability to specifically bind to collagen provided by the present invention is obtained by fusing a collagen binding domain (CBD) to the amino terminal (N-terminal) of the nerve growth factor β subunit (β-NGF) Fusion protein; the collagen-binding domain is a protein consisting of 7 amino acid residues, and its conserved sequence is SEQ ID №1 in the sequence listing.

SEQ ID No. 1 in the sequence listing consists of 7 amino acid residues.

In addition, for the convenience of purification, a histidine affinity tag sequence consisting of 6 histidines may also be connected to the amino terminal of the nerve growth factor having the ability to specifically bind to collagen.

Wherein, the nerve growth factor obtained by fusing the collagen-binding domain at the N-terminal of β-NGF with the ability to specifically bind to collagen may have the amino acid residue sequence of SEQ ID №: 2 in the sequence listing, and the SEQ ID № in the sequence listing: 2 consists of 159 amino acid residues, the 5th-10th amino acid residue from the amino terminal is a histidine affinity tag sequence; the 22nd-28th amino acid residue from the amino terminal is the conserved sequence of the collagen binding domain. Amino acid residues 42-159 of the amino terminal are β-NGF, and amino acid residues 29-41 of the amino terminal are connecting peptide sequences.

The gene encoding the nerve growth factor with specific binding ability to collagen may be SEQID №3 in the sequence listing.

SEQ ID №3 in the sequence listing consists of 480 bases, the 16-33 bases from the 5' end encode the histidine affinity tag sequence, and the 67-87 bases from the 5' end encode collagen binding The conserved sequence of the structural domain, the 127th-480th base from the 5' end encodes β-NGF, and the 88th-126th base from the 5' end encodes the connecting peptide sequence.

The second object of the present invention is to provide a method for expressing the aforementioned nerve growth factor with the ability to specifically bind to collagen.

The method for expressing nerve growth factor with specific binding ability to collagen provided by the present invention is to construct a recombinant expression vector containing the gene of nerve growth factor with specific binding ability to collagen, introduce the constructed recombinant expression vector into host cells, and cultivate the host The cells express the gene for nerve growth factor, which has the ability to specifically bind collagen.

The starting vector for constructing the recombinant expression vector can be an expression vector for expressing foreign genes in Escherichia coli, such as pET-28a, pET-28b, pET-28c, pET-21a(+) or pET-30a, etc., Preferred is pET-28a.

Using pET-28a as the starting vector, the constructed recombinant expression vector containing the nerve growth factor gene with the ability to specifically bind to collagen is pET-CBD-NGF.

The host can be Escherichia coli, yeast, mammalian cells, insect cells or Bacillus subtilis, etc., preferably Escherichia coli.

The escherichia coli can be E.coli BL21(DE3), E.coli BL21(DE3)plys, BLR(DE3) or B834 etc.

Using E.coli BL21(DE3) as the starting strain, the recombinant strain obtained by introducing pET-CBD-NGF into E.coli BL21(DE3) is BL21(DE3)-pET-CBD-NGF.

The above-mentioned recombinant expression vectors and recombinant bacteria can be constructed according to conventional methods.

The culture medium and culture conditions for culturing the host cells containing the nerve growth factor gene with specific binding ability to collagen of the present invention can be the culture medium and culture conditions for culturing the starting host. Wherein, when cultivating the recombinant Escherichia coli host, it is necessary to add an inducer, such as IPTG, etc., the concentration of the added IPTG is 0.8-1.2mmol/L, preferably 1mmol/L, and the induction temperature is 35-39°C, preferably 37°C , the induction time is 2-4 hours, preferably 4 hours.

The invention provides a nerve growth factor capable of specifically combining with collagen and its coding gene. The nerve growth factor is based on the tight connection between neurons and collagen. From the perspective of transforming the nerve growth factor, a collagen binding domain (CBD) is fused to the N-terminus of the mature β-NGF molecule to enhance nerve growth. The combination of factor and collagen establishes a strong physical and chemical force between nerve growth factor and collagen, so that β-NGF is anchored on the collagen outside the nerve cells, which can greatly reduce β-NGF while having the same activity. -The dosage of NGF avoids the risk caused by excessive dosage of β-NGF. At the same time, the relatively enriched nerve growth factor can significantly improve the survival ability of neurons and further promote the differentiation of neurons, thereby providing a way for nerve damage repair. A new approach with broad prospects for clinical application.

The present invention will be described in further detail below in conjunction with specific embodiments.

Description of drawings

Figure 1 is the SDS-PAGE detection result of the expressed and purified collagen binding domain with histidine affinity tag and β-NGF mature peptide fusion protein

Figure 2A is the morphological observation of PC12 cells induced by CBD-NGF and NAT-NGF

Figure 2B is the axon induction experiment of PC12 cells induced by CBD-NGF and NAT-NGF

Figure 2C is the MTT experiment results of PC12 cells induced by CBD-NGF and NAT-NGF

Figure 3A is the detection results of the same amount of collagen on the binding amount of different amounts of NAT-NGF and CBD-NGF

Figure 3B is the calculation result of the dissociation constant Kd of NAT-NGF and CBD-NGF on collagen

Figure 4 shows the results of in vitro functional experiments of NAT-NGF and CBD-NGF

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1. Cloning, Expression and Purification of Nerve Growth Factor Encoding Gene with Collagen-Specific Binding Ability

1. Design primers

Three forward primers (CBDU, NGFU1 and NGFU2, whose sequences are shown in Table 1) and two reverse primers (CBDD and NGFD), whose sequences are shown in Table 1, were designed with the help of the software Primer premier 5.05, wherein, CBDU (introduced restriction endonuclease Enzyme Nde I recognition site) and CBDD (introduction of restriction endonuclease Hind III recognition site) are used to amplify the coding sequence of collagen binding domain and connecting peptide (LINKER), wherein, the amino acid sequence of collagen binding domain conservation For TKKTLRT (SEQ ID №: 1 in the sequence table), the amino acid sequence of the linking peptide is GSAGSAAGSGGK; NGFU1 (introduction of restriction endonuclease Nde I recognition site) and NGFD (introduction of stop codon and restriction endonuclease Xho I recognition site) is used to amplify the β-NGF coding sequence; NGFU2 (introduction of restriction endonuclease Hind III recognition site) and NGFD (introduction of stop codon and restriction endonuclease Xho I recognition site) are also used for the amplification of the β-NGF coding sequence.

Table 1 Primer Sequence

CBDU: 5'-ATC<u>CATATG</u>ACTAAGAAAAACCCTGCGTACTGGTAGCGCGGGCAGT-3' (the underlined base is the restriction endonuclease Nde I recognition site) CBDD: 5'-ACT<u>AAGCTT</u>ACCGCCAGAACCCGCAGCACTGCCCGCGCTACCAGT -3' (underlined base is restriction endonuclease Hind III recognition site) NGFU1: 5'-CTA<u>CATATG</u>TCTTCGTCCCATCCCCATCTTCCAC-3' (underlined base is restriction endonuclease Nde I recognition site) NGFU2: 5'-GCT<u>AAGCTT</u>TCTTCGTCCCATCCCATCTTCCAC-3' (the underlined base is the restriction endonuclease Hind III recognition site)NGFD: 5'-GAT<u>CTCGAG </u>TCATCTCACAGCCTTCCTGCTGAGCAC-3' (the underlined base is the restriction endonuclease Xho I recognition site)

2. PCR amplification of nerve growth factor coding gene with specific binding ability to collagen

Using CBDU and CBDD as templates, amplify the coding sequence of the collagen binding domain and the connecting peptide by bridging PCR. The 50 μl PCR reaction system is: primers CBDU and CBDD 1 pmol/μl each, dNTPs 200 μmol/μl, Taq enzyme 1ul, Supplement the reaction to 50 μl with ddH ₂ O. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; then denaturation at 94°C for 30 s, annealing at 55°C for 1 min, extension at 72°C for 1 min, and 30 cycles; finally, extension at 72°C for 10 min. After the reaction, the PCR amplification product was detected by 1.5% agarose gel electrophoresis, and a band with a size of about 70 bp was obtained as a result. After recovering and purifying the target band, it was double-digested with restriction endonucleases Nde I and Hind III and then connected to the prokaryotic expression vector pET28a (Novagen Company) that was double-digested with the same enzyme, and sequenced. Sequencing results showed that the coding sequence of the correct collagen binding domain and connecting peptide was obtained, and the recombinant vector with the correct sequence was named pET-CBD.

Using the full-length cDNA of the human β-NGF gene (GenBank number: NM 002506) as a template, PCR amplifies human β-NGF under the guidance of the primer pair consisting of NGFU1 and NGFD, and the primer pair consisting of NGFU2 and NGFD, respectively. Gene, 50 μl PCR reaction system: 1 pmol/μl of upstream and downstream primers, 200 μmol/μl of dNTPs, 1 ul of Taq enzyme, and supplement the reaction system with ddH ₂ O to 50 μl. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes; then denaturation at 94°C for 45 seconds, annealing at 55°C for 1 minute, extension at 72°C for 1 minute, and 30 cycles; full extension at 72°C for 10 minutes. After the reaction, the PCR amplification product was detected by 1.5% agarose gel electrophoresis, and two bands with a size of about 350 bp were obtained. After recovering and purifying the two target bands, the amplified bands of NGFU1 and NGFD were double-digested with restriction endonucleases Nde I and Xho I, and then connected to the prokaryotic expression vector pET28a that had been double-digested with the same enzymes. It was sequenced, and the sequencing results showed that the coding sequence of the β-NGF mature peptide with the correct sequence of histidine affinity tag was obtained, and the sequence had the nucleotide sequence of SEQ ID No. 5 in the sequence listing, and the SEQID №: 5 consists of 420 bases, encodes the amino acid residue sequence of SEQ ID №: 4 in the sequence listing, and SEQ ID № in the sequence listing: 4 consists of 139 amino acid residues. The recombinant vector was named pET-NAT-NGF; at the same time, the amplified bands of NGFU2 and NGFD were double-digested with restriction endonucleases HindIII and XhoI, and then connected to the prokaryotic expression vector pET-CBD that had been double-digested with the same enzymes. It was sequenced, and the sequencing results showed that the coding sequence of the fusion protein of the collagen-binding domain with the histidine affinity tag and the mature peptide of β-NGF was obtained, and the sequence has SEQ ID No. 3 in the sequence listing Nucleic acid sequence, SEQ ID No. 3 in the sequence listing consists of 480 bases, the 16th-33rd base from the 5' end encodes a histidine affinity tag sequence, and the 67th-87th base from the 5' end The base codes for the conserved sequence of the collagen-binding domain, the 127th-480th base from the 5' end encodes β-NGF, and the 88th-126th base from the 5' end encodes the connecting peptide sequence, and the SEQ ID No. in the coding sequence list: The amino acid residue sequence of 2, SEQ ID No. 2 in the sequence table consists of 159 amino acid residues, and the recombinant vector correctly connected with this sequence is named pET-CBD-NGF.

3. Expression and purification of nerve growth factor with specific binding ability to collagen

The prokaryotic expression vector pET-NAT-NGF constructed in step 1 with the coding sequence of the β-NGF mature peptide with histidine affinity tag and the collagen binding domain with histidine affinity tag were fused with the β-NGF mature peptide The prokaryotic expression vector pET-CBD-NGF of the protein coding sequence was transformed into Escherichia coli BL21 (DE3) competent cells, and the transformed cells were spread on LB plates, cultured at 37°C, and single clones were selected and transferred to LB liquid culture Culture medium at 37°C for 12-24 hours, then transfer to 100mL LB liquid medium at a ratio of 2%, culture at 37°C for 3 hours until the OD ₆₀₀ value is about 0.8, add IPTG at a final concentration of 1mM , continue to incubate for 4 hours. After the cultivation, the cells were collected by centrifugation at 8000rpm, washed with PBS (NaCl 8.5g, Na ₂ HPO ₄ 2.2g, NaH ₂ PO ₄ 0.4g dissolved in 100ml distilled water, pH 7.2) and then centrifuged again to collect the cells, and then washed with 10mL The bacteria were resuspended in PBS, and the bacteria were disrupted by ultrasonic to obtain the crude extract of the recombinant protein. The inclusion bodies were collected by centrifugation, washed, dissolved, and purified using an immobilized metal ion (Ni ²⁺ ) ligand affinity chromatography column (Amersham biosciences). The purified two expression products were refolded by dialysis, concentrated by ultrafiltration, and then the protein solvent system was replaced with citric acid buffer (citric acid 0.4g, sodium citrate 2.38g, dissolved in 100mL distilled water, pH6.0) , stored at 4°C. The expression and purification products of pET-CBD-NGF were detected by 15% SDS-PAGE, and the detection results are shown in Figure 1 (lane 1 is the protein molecular weight standard, lane 2 is the expressed bacteria after ultrasonic disruption, and lane 3 is the purified Inclusion bodies (under reducing conditions), the recombinant protein with a molecular weight of about 15.4Kd was expressed, which was consistent with the expected results. It can be seen from the figure that the target protein is mainly expressed in the form of inclusion bodies. After purification, a relatively high-purity protein The nerve growth factor with the ability to specifically bind to collagen, that is, the fusion protein of the collagen binding domain with histidine affinity tag and the mature peptide of β-NGF, named the protein as CBD-NGF, and the recombinant Escherichia coli expressing the protein Named BL21(DE3)-pET-CBD-NGF. The expressed β-NGF mature peptide with histidine affinity tag was named NAT-NGF.

Example 3, Activity Detection of Nerve Growth Factor with Collagen-Specific Binding Ability

Rat adrenal chromaffin cells PC12 can differentiate and generate axons under the induction of NGF. In addition, NGF can promote the survival of neurons. Therefore, through PC12 (purchased from the cell bank of the Basic Medical College of Peking Union Medical College) axon induction experiments and MTT experiments Detect the activity of CBD-NGF and NAT-NGF obtained in Example 2 (Howe, CLDepolarization of PC12 cells induces neurite outgrowth and enhances nerve growth factor-induced neurite outgrowth in rats. Neurosci Lett, 2003.351 (1): p.41-5. ), the method is: stimulate PC12 cells with CBD-NGF and NAT-NGF at a concentration of 0-64 nM, and use unstimulated PC12 cells as a control. As a result, after one day of action, PC12 cells have axon induction (see Figure 2A ); Calculate the proportion of long axon cells, the statistical results are shown in Figure 2B, it can be seen that with the increase of CBD-NGF concentration, the proportion of long axon cells also increases, showing a dose-dependent effect, and after statistics Analysis showed that there was a significant difference between β-NGF with collagen binding region (CBD-NGF) and β-NGF without collagen binding region (NAT-NGF) (*: p<0.05; **: p<0.01); In addition, the MTT experiment was carried out three days after being treated with β-NGF, and the results are shown in Figure 2C. Similarly, as the concentration of β-NGF increased, the OD ₄₉₂ value also increased, indicating that the number of surviving cells increased. The above experimental results all showed a dose-dependent effect, and after statistical analysis, there was a significant difference between the experimental results of the two proteins (*: p<0.05; **: p<0.01). The results indicated that both CBD-NGF and NAT-NGF had biological activity, and the activity of CBD-NGF was significantly higher than that of NAT-NGF.

Example 4, Detection of Collagen Binding Ability of CBD-NGF

Using genetic engineering technology and microbial fermentation to prepare CBD-NGF, the purpose is to enhance its binding ability to collagen three-dimensional scaffold materials for tissue engineering while ensuring its activity, so as to reduce the effective dosage of β-NGF. The improved ELISA experiments were used to detect the binding ability of NAT-NGF and CBD-NGF to collagen membranes (Finnis, M.L. and M.A.Gibson, Microfibril-associated glycoprotein-1 (MAGP-1) binds to the pepsin-resistant domain of the alpha3 (VI)chain of type VI collagen.J Biol Chem, 1997.272(36):p.22817-23.), and according to the method provided by Matsushita (Matsushita, O.et al.A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. J Biol Chem, 1998.273 (6): p.3643-8.) Calculate the dissociation constant Kd of the two proteins for collagen. Load gradually increasing doses of NAT-NGF and CBD-NGF on the collagen membrane (6.0 mg) prepared by the same amount of type I collagen. mg) The detection results of different amounts of NAT-NGF and CBD-NGF binding amount are shown in Figure 3A. Under the same protein loading, the retention of CBD-NGF on the collagen membrane was significantly higher than that of NAT-NGF, indicating that The binding efficiency of the former is significantly higher than that of the latter. In addition, the dissociation constants Kd of NAT-NGF and CBD-NGF were 1.50 μM and 0.51 μM, respectively (see Figure 3B). According to the definition of Kd, the smaller the Kd value of a protein, the stronger its binding ability to collagen. The above experimental results prove that, compared with NAT-NGF, the binding ability of CBD-NGF to collagen is significantly improved.

Example 5. In vitro functional experiment of CBD-NGF

Firstly, NAT-NGF and CBD-NGF with gradually increasing concentrations were respectively loaded on 48-well plates coated with acidic collagen (derived from rat tail) (0.2 mg collagen per well). After adsorption for 1 hour, they were washed twice with PBS. Then PC12 cells were inoculated in the well plate, the number of cells inoculated in each well was 5×10 ³ , and after culturing at 37°C for 3 days, the number of surviving cells was detected by MTT method. Compared with CBD-NGF, it has higher activity on collagen, can better promote the survival and growth of PC12 cells in vitro, and can be used for clinical nerve injury repair.

sequence listing

<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 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 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 (9)

1. having the 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; Described collagen binding domains is made up of 7 amino-acid residues shown in the SEQ ID NO: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: the amino acid residue sequence of described nerve growth factor is shown in SEQ ID NO:2.

4. the gene of the described nerve growth factor of claim 1 of encoding.

5. gene according to claim 4 is characterized in that: the base sequence of described gene is shown in SEQ ID NO:3.

6. the expression method of the described nerve growth factor 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. according to claim 6 or 7 described expression methods, it is characterized in that: described host is E.coli BL21 (DE3), E.coli BL21 (DE3) plys, BLR (DE3) or B834.

9. expression method according to claim 8 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.

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