CN101831432B - Biosynthesis method and application of bone morphogenetic protein-2 - Google Patents

Abstract

The invention discloses a biosynthesis method and application of a recombinative human bone morphogenetic protein-2 (rhBMP-2). The method comprises the following steps of: (1) obtaining a gene of the bone morphogenetic protein-2 (BMP-2) from a human osteosarcoma cell MG63 cell line; (2) constructing a prokaryotic expression carrier of the rhBMP-2; (3) constructing expression engineering bacteria; (4) carrying out induction expression of an inducer; and (5) purifying by affinity chromatography and renaturing by a gradient dialysis method to obtain the rhBMP-2. The invention has the following advantages that the selected material is the human osteosarcoma cell MG63 cell line, the rhBMP-2 gene is obtained through a reverse transcription PCR (Polymerase Chain Reaction) method, the material is convenient to take, the purity is high, the extraction process is simple, and the bone morphogenetic protein-2 has wide application potential in the fields of biological materials and bone tissue regeneration and repair.

Description

A kind of biosynthesis method and application of bone morphogenetic protein-2

technical field

The invention relates to the expression and purification of recombinant proteins in the field of genetic engineering, in particular to a biosynthesis method of bone morphogenetic protein-2.

Background technique

Bone morphogenetic protein-2 (bone morphogenetic proteins 2, BMP-2) belongs to the bone morphogenetic protein family. Bone morphogenetic proteins (BMPs) are a class of acidic glycoproteins discovered by Urist and colleagues in 1965 as an active ingredient in decalcified bone matrix, which induces bone formation, hence the name BMP protein. In 1988, Wozney and others isolated BMPs for the first time and cloned their cDNA. Studies have shown that, except for BMP-1 (belonging to procollagen c protease) in the BMPs family, they all belong to the members of the transforming growth factor (TGF-β) superfamily.

BMPs can regulate cell proliferation, differentiation and apoptosis. In 2009, Kato et al. used bone morphogenetic protein 2 to induce mesenchymal precursor cells ROB-C26 cell line to become mature osteoblasts and adipocytes. In addition, BMPs also participate in tissue remodeling and regeneration by regulating stem cells. At present, there are about 20 kinds of BMPs proteins known, some of which have completely different functions, and some have overlapping functions, which mainly depend on the receptors they act on and are expressed in different tissues. There is evidence that BMPs play an important role in regulating stem cell performance, but their roles vary among different stem cells.

BMPs affect the transcription of target genes through receptor-mediated intracellular signals. Two types of receptors are required for this process: type I receptors and type II receptors. At present, only one type II BMP receptor (BmprII) has been found, while there are three type I BMP receptors: Alk2, Alk3 and Alk6. Different combinations of receptors will lead to different results.

The BMP dimer binds to its specific type II receptor on the target cell membrane, and the type II receptor activates the type I receptor by phosphorylating the GS region of the type I receptor, and the activated type I receptor phosphorylates intracellular signal transduction Conductor proteins are Smads proteins, and the Smads family transduces signals from the cell membrane to the nucleus, and finally induces the transcription and protein expression of target genes of BMPs. Smad protein has been proved to be the only direct enzyme substrate that mediates the gene reaction process of TGF-β superfamily.

Smads signaling pathway is a well-researched one in BMPs signal transduction, and it has also been proved to be one of the most important pathways in BMPs signal transduction. BMPs also activate non-Smads intracellular signaling pathways. Ulsamer et al. found that BMPs can stimulate the expression of alkaline phosphatase and osteocalcin in osteoblasts by activating p38 in the MAPK family, and finally promote the osteogenesis of osteoblasts.

The most important function of BMPs is to induce the differentiation of mesenchymal cells into cartilage and bone, which can significantly promote the healing of refractory fractures and the repair of various bone defects, and has been applied clinically, among which BMP-2 is considered to be the The most active and only factor that can induce osteogenesis alone.

The full-length cDNA of BMP-2 is 1587bp, encoding 396 amino acids, including the N-terminal signal peptide, the intermediate propeptide and the C-terminal mature peptide. After cleavage of the BMP-2 precursor protein signal peptide, a dimer is formed. Specific proteolytic enzymes act on the RXXR site of the protein dimer to excise its propeptide, thereby forming a dimer mature protein with biological activity, that is, recombinant human bone morphogenetic protein-2 (recombinant human bone morphogenetic protein, Abbreviated as rhBMP-2).

The content of BMP-2 in natural bone tissue is low, the extraction process is complicated, the cost is high, and the purity of the product is limited, so it is difficult to meet the needs of scientific research and clinical application; the rhBMP-2 product prepared by eukaryotic expression system has relatively high activity, but the expression level is low, The production cost is high and the cycle is long; Escherichia coli is currently the most widely used host cell in genetic engineering, with the characteristics of simple operation, low cost, short cycle and large output.

Contents of the invention

The purpose of the present invention is to obtain rhBMP-2 gene from human osteosarcoma cell line MG63 by reverse transcription PCR method, and then induce and express rhBMP-2 gene in Escherichia coli, so as to obtain rhBMP-2 with good activity.

Technical scheme of the present invention is:

A biosynthetic method of recombinant human bone morphogenetic protein-2 (rhBMP-2), comprising the following steps in sequence:

(1) Obtain bone morphogenetic protein-2 (BMP-2) gene from human osteosarcoma cell line MG63 by reverse transcription PCR method;

(2) Apply DNA recombination technology to construct rhBMP-2 prokaryotic expression vector;

(3) rhBMP-2 prokaryotic expression vector transforms Escherichia coli host cells, and constructs expression engineering bacteria;

(4) fermenting and cultivating engineered bacteria, and carrying out inducer-induced expression;

(5) The expression product obtained in step (4) is purified by affinity chromatography and refolded by gradient dialysis to obtain rhBMP-2.

Step (1) is to use Trizol reagent (purchased from Invitrogen Company) to extract the total RNA of human osteosarcoma MG63 cells, and use oligo(dT) (purchased from Takara Company) as primers to obtain cDNA by reverse transcription PCR method; design full-length BMP -2 gene and rhBMP-2 gene PCR primers, wherein the upstream primer contains the EcoR V restriction site, and the downstream primer contains the Hind III restriction site; using cDNA as a template, the full-length BMP-2 gene is obtained by PCR method and cloned to the pMD-18T vector (purchased from Takara Company) to obtain the recombinant plasmid pMD-18T-BMP2; using the pMD-18T-BMP2 recombinant plasmid as a template, the rhBMP-2 gene was obtained by PCR.

Step (2) specifically clones the rhBMP-2 gene obtained in step (1) into the pMD-18T vector to obtain the recombinant plasmid pMD-18T-rhBMP2; ) and HindIII (purchased from Takara Company), the rhBMP2 gene was cloned into the expression plasmid pET30 (purchased from Takara Company) to construct the pET30-rhBMP2 prokaryotic expression vector.

Step (3) is specifically to transform the prokaryotic expression vector constructed in step (2) into the Escherichia coli host cell BL21 strain (purchased from Takara Company) after being double-digested with BamH I and HindIII to construct an expression engineering bacterium.

Step (4) specifically, the expression engineering bacteria constructed in step (3) were cultured overnight at 37° C. in 10 ml of LB medium containing 25 μg/ml Kan, and inoculated into 1 L containing In 25 μg/ml Kan LB medium (purchased from Shanghai Soleibao Biotechnology Co., Ltd.), culture at 37°C until _OD600 = 0.6-0.8, add IPTG (purchased from Takara Company) to a final concentration of 0.8mM, induce After expressing for 6h, the cells were collected.

Step (5) specifically is to collect the engineered bacteria collected after induced expression in step (4), centrifuge to collect the inclusion body precipitate after ultrasonic crushing, and purify rhBMP-2 by Ni ion affinity chromatography under denaturing conditions after the inclusion body dissolves; The purified rhBMP-2 was refolded by gradient dialysis to obtain soluble rhBMP-2.

The application of the recombinant human bone morphogenetic protein-2 of the present invention in biomaterials, regeneration and repair of bone tissue.

The benefits of the present invention are:

(1) The traditional rhBMP-2 gene extraction materials are human cartilage and other tissues. These tissues have few sources and are difficult to obtain, and the relative content of rhBMP-2 gene is low. The material selected in the present invention is human osteosarcoma cell MG63 cell line, and the rhBMP-2 gene is obtained by reverse transcription PCR method, which has the advantages of convenient material collection, high purity and simple extraction process.

(2) The present invention has high expression efficiency, simple separation and purification, easy operation, easy amplification, good stability, and is suitable for large-scale industrial production. Therefore, the present invention is of great value for the development of rhBMP-2 protein with good biological activity, and has broad application prospects in the fields of biomaterials, bone tissue regeneration and repair.

Description of drawings

Fig. 1 is the electrophoresis pattern of the total RNA extracted from the MG63 cell line in the embodiment of the present invention.

Fig. 2 is the identification electrophoretic pattern of recombinant expression plasmid pET30a-rhBMP2 in the embodiment of the present invention;

Among them: Swimming lane M is DNA standard molecular weight maker; Swimming lane 1 is the product of plasmid pET30a-rhBMP2 after EcoR V and Hind III double enzyme digestion; Swimming lane 2 is the band obtained by using plasmid pET30a-rhBMP2 as template and using rhBMP2 upstream and downstream primers PCR , Lane 3 is the negative control in the PCR reaction.

Fig. 3 is the SDS-PAGE electrophoresis result of the total protein expressed by rhBMP-2 in Escherichia coli in an embodiment of the present invention;

Among them: lane M is the protein standard molecular weight maker; lanes 1, 2, 3, and 4 are the total bacterial protein after IPTG induction; lane con is the control group, that is, the total bacterial protein without IPTG induction.

Fig. 4 is the result after purification of rhBMP-2 by Ni ion affinity chromatography in the embodiment of the present invention;

Among them: Lane M is the protein standard molecular weight maker; Lane 1 is the result after purification of rhBMP2.

Fig. 5 is the result after refolding of rhBMP2 gradient dialysis in the embodiment of the present invention;

Among them: Lane M is the protein standard molecular weight maker; Lane 1 is the sample after rhBMP-2 refolding.

Fig. 6 shows the effect of rhBMP-2 on alkaline phosphatase activity in MG63 cells in an embodiment of the present invention.

Detailed ways

The technical scheme of the present invention is described below in conjunction with embodiment:

Example 1:

1. Acquisition of rhBMP-2 gene

The total RNA of the MG63 cell line (purchased from the Cell Resource Center, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences) was extracted using Trizol reagent (purchased from Invitrogen). The extraction results are shown in Figure 1, and 28S and 18S bands can be clearly seen. It shows that the extraction result of total RNA is better. Using oligo(dT) as a primer, cDNA was obtained by reverse transcription PCR. Using cDNA as a template, the full-length BMP2 of the target gene was amplified by PCR. The PCR primers are as follows (the site of the endonuclease is underlined, and the name of the endonuclease is marked in brackets):

Sense (EcoR V): 5′-TAGGATATCATGGTGGCCGGGACCCG-3′

Antisense (Hind III): 5′-TGCAAGCTTCTAGCGACACCCACAACCCTCC-3′

The PCR reaction system is:

ddH ₂ O 13 μl

10×PCR buffer 2.5μl

25mM MgCl2 _2μl

2.5mM dNTP (purchased from 2μl

Invitrogen)

Primer 1 1 μl

Primer 2 1μl

cDNA 3 μl

tag enzyme (purchased from 0.5 μl

Invitrogen)

Total 25μl

The reaction procedure of PCR is:

Pre-denaturation at 94°C for 5 minutes

Extend at 72°C for 10min

The amplified product was identified by agarose gel electrophoresis, and a specific amplification band of about 1.2 kb was seen, which was consistent with the expected result. The amplified product was recovered and ligated into the pMD-18T vector to obtain the recombinant plasmid pMD-18T-BMP2. The recombinant plasmid was identified by enzyme digestion and sequencing, and its sequence was the same as the expected result.

Using the recombinant plasmid pMD-18T-BMP2 as a template, the target fragment rhBMP-2 gene was amplified by PCR method. The PCR primers are as follows (the site of the endonuclease is underlined, and the name of the endonuclease is marked in brackets):

Sense (EcoR V): 5′-TAGGATATCCAAGCCAAACACAAACAG-3′

Antisense (HindIII): 5′-TGCAAGCTTCTAGCGACACCCACAACCCTCC-3′

The amplified product was recovered and ligated into the pMD-18T vector to obtain the recombinant plasmid pMD-18T-rhBMP2. The recombinant plasmid was identified by enzyme digestion and sequencing, which was consistent with the expected result.

2. Construction of rhBMP-2 prokaryotic expression vector

After the recombinant plasmid pMD-18T-rhBMP2 was digested with EcoR V and Hind III, the rhBMP-2 gene fragment (about 350bp in size) was recovered and ligated with the pET30a expression plasmid digested with EcoR V and Hind III to obtain The recombinant expression plasmid pET30a-rhBMP2 was established. The recombinant plasmid was identified by EcoR V and Hind III double enzyme digestion and PCR method (Figure 2). A band of about 350bp can be seen in both enzyme digestion and PCR products, indicating that the recombinant expression vector pET30a-rhBMP2 was successfully constructed. Sequencing results also indicated that the plasmid was constructed successfully.

3. Construction of expression engineering bacteria and expression of rhBMP-2

The recombinant expression vector pET30a-rhBMP2 was transformed into the E. coli host cell BL21 strain, and the expression engineered bacteria were constructed. The engineered bacteria were inoculated into 10ml LB medium (containing 25 μg/ml Kan) at a ratio of 1:1000, and cultivated overnight at 37°C; the next day Inoculate it into 1 L of LB medium (containing 25 μg/ml Kan) at a ratio of 1:100 (v/v), culture at 37°C until OD ₆₀₀ = 0.6-0.8, add IPTG to a final concentration of 0.8 mM, and induce expression 6h, take 1ml of bacterial liquid from each bottle, collect the bacterial cells, use uninduced bacterial cells as a control, and detect the total protein expression products of the bacterial cells by SDS-PAGE electrophoresis; centrifuge at 8000rpm for 5min, and collect the bacterial cells.

4. Purification and renaturation of rhBMP-2

Resuspend the bacteria in PBS, sonicate for 30 minutes, centrifuge for 5 minutes, collect the supernatant and precipitate separately, and take some samples for SDS-PAGE electrophoresis to detect the expression product; precipitate with inclusion body solution (8mol/L urea; 0.1mol/L Na _{2 HPO} ; 0.01mol/L Tris; pH=8.0) was fully dissolved, centrifuged at 14000rpm for 30min, and the supernatant was purified by Ni ion affinity chromatography to purify rhBMP-2. Use the inclusion body washing solution (8mol/L urea; 0.1mol/L Na ₂ HPO ₄ ; 0.01mol/L Tris; 0.05mol/L imidazole; pH=6.3) to wash the foreign protein, and use the inclusion body eluent (8mol/L urea; 0.1 mol/L Na ₂ HPO ₄ ; 0.01 mol/L Tris; 0.25 mol/L imidazole; pH=5.9) to elute the recombinant protein from the Ni ion affinity chromatography column. Purified samples were detected by SDS-PAGE electrophoresis.

Figure 3 is the electrophoresis diagram of the total bacterial protein. Compared with the control group, the IPTG-induced group has an obvious band at about 20kD, indicating that rhBMP-2 can be successfully expressed in E. coli. After sonication, the result of SDS-PAGE electrophoresis of supernatant obtained by centrifugation and precipitation showed that rhBMP-2 expressed in Escherichia coli existed in the form of inclusion body. Fig. 4 is rhBMP-2 purified by Ni ion affinity chromatography. It can be seen from the figure that rhBMP-2 with higher purity and higher concentration can be obtained by Ni ion affinity chromatography.

The present invention refolds rhBMP-2 by gradient dialysis, and the specific scheme is as follows:

(1) Put the rhBMP-2 protein solution purified by the column in dialysate I (6mol/L urea; 0.1mol/L Nacl; 0.02mol/L Tris; 5mmol/L EDTA; pH=7.4) at 4°C Under dialysis for 8h;

(2) The rhBMP-2 protein solution was dialyzed at 4°C for 8 hours in dialysate II (4mol/L urea; 0.1mol/L Nacl; 0.02mol/L Tris; 5mmol/L EDTA; pH=7.4);

(3) The rhBMP-2 protein solution was dialyzed at 4°C for 8 hours in dialysate III (2mol/L urea; 0.1mol/L Nacl; 0.02mol/L Tris; 5mmol/L EDTA; pH=7.4);

(4) RhBMP-2 protein solution was dialyzed in PBS solution (137mmol/L Nacl; 2.7mmol/L Kcl; 10mmol/L Na ₂ HPO ₄ ; 2mmol/L KH ₂ PO ₄ ; pH=7.4) at 4°C 24h, during which the PBS solution was replaced once;

(5) Centrifuge the dialyzed rhBMP-2 protein solution at 12,000 rpm for 30 min at 4°C, and collect the supernatant;

(6) The protein concentration was measured by BCA method, and the refolding results were detected by SDS-PAGE electrophoresis.

The results of rhBMP-2 renaturation are shown in Figure 5, indicating that rhBMP-2 can be successfully renatured by gradient dialysis. The BCA method was used to measure the protein concentration and calculate the recovery rate. It was found that the recovery rate was 34.5% after gradient dialysis of the dialysate with pH=7.4 under the condition of 4°C. The recovery rate can reach 93% at pH=9, but the cell test shows that its activity is very low, so the present invention adopts the dialysate with pH=7.4 to refold rhBMP-2.

Example 2

Activity detection of rhBMP-2

The invention detects the rhBMP-2 activity through the alkaline phosphatase activity in the in vitro experiment.

(1) Inoculate human MG63 cells in a 12-well plate according to 1×10 ⁵ cells per well, add DMEM medium containing 10% fetal bovine serum, and culture at 37°C for 24 hours;

(2) Aspirate the original medium, wash with PBS three times, add DMEM medium containing 0.5% fetal bovine serum, add rhBMP-2, the final concentration is 1 μg/ml and 10 μg/ml respectively, and the group without recombinant protein is used as the control ;

(3) Alkaline phosphatase activity of 1d, 3d and 5d was detected by alkaline phosphatase detection kit (purchased from Nanjing Jiancheng).

The results are shown in Figure 6, indicating that the addition of rhBMP-2 can significantly increase the alkaline phosphatase activity of MG63, and with the increase of rhBMP-2 concentration, the alkaline phosphatase activity increases significantly. It shows that rhBMP-2 with good activity can be successfully obtained by gradient dialysis.

Finally, it should also be noted that the above examples are only specific implementation examples of the present invention. Apparently, the present invention is not limited to the above examples, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

sequence listing

SEQ ID NO 1:

27 taggatatca tggtggccg ggacccg

SEQ ID NO 2:

31 tgcaagcttc tagcgacacc cacaaccctc c

SEQ ID NO 3:

27 taggatatcc aagccaaaca caaacag

SEQ ID NO 4:

31 tgcaagcttc tagcgacacc cacaaccctc c

Claims (5)

1. the biosynthetic means of a recombinant human bone morphogenesis protein-2 is characterized in that may further comprise the steps successively:

(1) from people's human osteosarcoma cell MG63 clone, obtain the rhBMP-2 gene through the reverse transcription PCR method:

Extracting people's osteogenic sarcoma MG63 cell total rna with Trizol reagent, is primer with oligo (dT), obtains cDNA through the reverse transcription PCR method; Design total length BMP-2 gene primer SEQ ID NO:1 and SEQ ID NO:2 and rhBMP-2 gene PCR primer SEQ ID NO:3 and SEQ ID NO:4, wherein upstream primer contains EcoR V restriction enzyme site, and downstream primer contains Hind III restriction enzyme site; With cDNA is template, and PCR method obtains total length BMP-2 gene, is cloned into the pMD-18T carrier, obtains recombinant plasmid pMD-18T-BMP2; With the pMD-18T-BMP2 recombinant plasmid is template, and PCR method obtains the rhBMP-2 gene;

(2) Application of DNA recombinant technology makes up the rhBMP-2 prokaryotic expression carrier;

(3) rhBMP-2 prokaryotic expression carrier transformed into escherichia coli host cell, the construction expression engineering bacteria;

(4) fermentation culture engineering bacteria carries out the inductor abduction delivering;

(5) step (4) gained expression product is obtained rhBMP-2 after affinitive layer purification, gradient dialysis method renaturation.

2. the biosynthetic means of a kind of recombinant human bone morphogenesis protein-2 according to claim 1 is characterized in that: step (2) specifically be rhBMP-2 gene clone that step (1) is obtained to the pMD-18T carrier, obtain recombinant plasmid pMD-18T-rhBMP2; PMD-18T-rhBMP2 in expression plasmid pET30, makes up the pET30-rhBMP2 prokaryotic expression carrier with the rhBMP2 gene clone behind restriction enzyme EcoR V and Hind III double digestion.

3. the biosynthetic means of a kind of recombinant human bone morphogenesis protein-2 according to claim 1; It is characterized in that: step (3) specifically is a prokaryotic expression carrier that step (2) is made up after BamH I and the affirmation of HindIII double digestion; Be transformed into e. coli host cell BL21 bacterial strain, the construction expression engineering bacteria.

4. the biosynthetic means of a kind of recombinant human bone morphogenesis protein-2 according to claim 1; It is characterized in that: step (4) specifically is that the expression engineering bacteria that step (3) makes up is contained in the LB substratum of 25 μ g/ml Kan at 10ml; 37 ℃ of overnight cultures; Be seeded to it in LB substratum that 1L contains 25 μ g/ml Kan at 1: 100 by volume next day, and 37 ℃ are cultured to OD ₆₀₀=0.6～0.8 o'clock, adding IPTG was 0.8mM to final concentration, and abduction delivering 6h collects thalline.

5. the biosynthetic means of a kind of recombinant human bone morphogenesis protein-2 according to claim 1; It is characterized in that: step (5) specifically is the engineering bacteria of collecting behind step (4) abduction delivering; Centrifugal collection inclusion body deposition after ultrasonication is passed through Ni ion affinity chromatography purifying rhBMP-2 under the sex change condition behind the solubilization of inclusion bodies; RhBMP-2 behind the purifying through gradient dialysis method renaturation, is obtained the rhBMP-2 of solubility.

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