CN114480269A - Recombinant protein hydrogel culture scaffold and preparation method thereof - Google Patents

Recombinant protein hydrogel culture scaffold and preparation method thereof Download PDF

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CN114480269A
CN114480269A CN202210113285.4A CN202210113285A CN114480269A CN 114480269 A CN114480269 A CN 114480269A CN 202210113285 A CN202210113285 A CN 202210113285A CN 114480269 A CN114480269 A CN 114480269A
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夏小霞
田凯凯
黄盛晨
钱志刚
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Shanghai Jiaotong University
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Abstract

A recombinant protein hydrogel-based culture scaffold and a preparation method thereof are disclosed, wherein a recombinant protein expression vector of a dendroid silk fused with a protamine-glycerol-aspartic tripeptide (RGD) sequence is constructed, introduced into an expression host cell, subjected to recombinant expression, separation and purification and temperature stimulation treatment, mixed with sodium alginate, subjected to enzyme catalytic crosslinking and cyclic treatment of calcium chloride and sodium citrate, and then a dynamic fiber mesh hydrogel scaffold for three-dimensional culture of stem cells is obtained. According to the invention, the recombinant protein hydrogel matrix suitable for three-dimensional culture is prepared by constructing the recombinant protein of the analogous limb silk fused with the RGD sequence and utilizing the self-assembly behavior of temperature-sensitive fibers, and the life processes of adhesion, proliferation, extension, differentiation and the like of mesenchymal stem cells (BMSCs) can be effectively regulated and controlled.

Description

Recombinant protein hydrogel culture scaffold and preparation method thereof
Technical Field
The invention relates to a technology in the field of biomedical materials, in particular to a recombinant protein hydrogel culture scaffold for promoting the growth and differentiation of mesenchymal stem cells and a preparation method thereof.
Background
The efficient repair of tissue or organ damage caused by trauma or infection and other factors is still a problem to be solved urgently in the biomedical field. Because of the problems of insufficient source, low cell activity and mobility and the like of autologous or allogeneic transplantation, the biological material bionic by transplantation is the most powerful means for repairing damaged tissues at present. The ideal repair material not only can be used as an inert support to provide a place for cell survival, but also can simulate the physicochemical properties of extracellular matrix to promote life activities such as cell adhesion and proliferation. The extracellular matrix is used as a gel medium consisting of fiber bundles, and the dynamic circulating degradation and regeneration process of the extracellular matrix plays an extremely important role in regulating and controlling the cell development process. At present, most of the existing hydrogel systems cannot realize a dynamic circulation process similar to that of a natural extracellular matrix. Therefore, constructing a dynamic biomimetic fiber mesh hydrogel can have a profound impact on the biomedical and tissue engineering fields.
The existing protein hydrogel preparation technology cannot finely regulate and control the mechanical property of hydrogel so as to regulate and control the life processes of stem cell growth, differentiation and the like; the fiber network structure and the dynamic regulation process of the natural extracellular matrix do not exist, and the cycle process of degradation and regeneration of the extracellular matrix in the cell development process cannot be simulated.
Inventive procedure
Aiming at the defects in the prior art, the invention provides a culture scaffold based on recombinant protein hydrogel and a preparation method thereof, and the bionic matrix suitable for three-dimensional culture of mesenchymal stem cells is prepared by mixing the nodular silk recombinant protein fused with RGD sequence with sodium alginate, so that the life processes of adhesion, proliferation, extension, differentiation and the like of the mesenchymal stem cells (BMSCs) can be effectively regulated and controlled.
The invention is realized by the following technical scheme:
the invention relates to a preparation method of a culture scaffold based on recombinant protein hydrogel, which comprises the steps of constructing an ampullaria-like silk recombinant protein expression vector fused with a spermine-gan-aspartyl tripeptide (RGD) sequence, introducing the ampullaria-like silk recombinant protein expression vector into an expression host cell, carrying out recombinant expression, separation and purification and temperature stimulation treatment, mixing with sodium alginate, carrying out enzyme catalytic crosslinking and cyclic treatment of calcium chloride and sodium citrate, and thus obtaining the dynamic fiber mesh hydrogel.
The construction refers to: integration of the Gene expressing the Arg-Gly-ASP (RGD) sequence into recombinant protein (R) from Arthropoda silk Using an inverse PCR site-directed mutagenesis kit (purchased from Toyo Boseki Co., Ltd.)4S8)5The expression vector pET-19b3-R4S8-5 is used for constructing the expression vector pET-19b3-RGD-R4S8-5 of the recombinant protein of the joint-like silk fused with the RGD sequence. .
The recombinant protein of the joint-like silk is a fusion protein formed by an elastic protein block (R) of the joint-like silk and a fibroin-like block (S), and the amino acid sequence of the recombinant protein is [ (GGRPSDSYGAPGGGN)4-(GAGAGS)8]5。
The forward primer RGD-F adopted by the reverse PCR site-directed mutagenesis is shown as Seq ID No.1, and specifically comprises the following components: 5'-CGTGGTGATATCGACGACGACGACAAG-3', the reverse primer RGD-R is shown as Seq ID No.2, and specifically comprises: 5'-ATGGCCGCTGCTGTGATGATG-3' are provided.
The recombinant Protein expression vector pET-19b3-R4S8-5 is described in controlled fibrosis reinforced Genetically Engineered rubber-like Protein hydrogel (Huang et al. controllable fibrous tissue engineering Protein hydrogel, Biomacromolecules 2021,22, 961-970).
The recombinant expression refers to: transferring the recombinant protein expression vector pET-19b3-RGD-R4S8-5 of the nodular-like silkworm silk fused with the RGD sequence into an expression host escherichia coli BL21(DE3) (purchased from Tiangen Biochemical technology Co., Ltd.), culturing recombinant bacteria, and inducing the expression of the nodular-like silkworm silk fused with the RGD sequence by using isopropyl thiogalactoside (IPTG).
The separation and purification refers to: and (3) separating and purifying the obtained nodular limb silk protein fused with the RGD sequence by using a nickel ion metal chelate (Ni-NTA) affinity chromatographic column.
The temperature stimulation refers to the following steps: the purified different concentrations of the RGD sequence fused arthropod fibroin were incubated at 37 ℃ for 30 minutes.
The cyclic treatment is as follows: adding 0.2% (w/v) sodium alginate solution, horseradish peroxidase and hydrogen peroxide into 5% (w/v) of the 5% (w/v) ampullar silk protein solution fused with the RGD sequence after temperature stimulation, fully mixing the mixture, reacting the mixture for 10 minutes at 37 ℃, soaking the mixture in 25mM calcium chloride solution for 30 minutes to obtain initial hydrogel, soaking the initial hydrogel in an alpha-MEM culture medium for 12 hours, respectively soaking the initial hydrogel in 25mM sodium citrate solution, 125mM sodium citrate solution and 250mM sodium citrate solution for 30 minutes, then soaking the initial hydrogel in the alpha-MEM culture medium for 12 hours, and then sequentially circulating the soaking systems of the calcium chloride, the culture medium and the sodium citrate.
The enzyme activity of the horseradish peroxidase is 600U/mL, and the concentration of hydrogen peroxide is 0.03% (w/v).
The invention relates to a dynamic fiber net hydrogel prepared by the method, which has the fiber morphology similar to a natural extracellular matrix and the compressive modulus is circularly regulated and controlled at 2-22 kPa.
The circulation regulation and control specifically comprises the following steps: after 30 minutes of calcium chloride soaking, 11.5 hours of culture medium soaking is carried out once within 24 hours, then 30 minutes of sodium citrate soaking is carried out once, and 11.5 hours of culture medium soaking is carried out again.
The invention relates to an application of the dynamic fiber mesh hydrogel, which is used for three-dimensional culture of mesenchymal stem cells, and specifically comprises the following steps: and dripping the digested and suspended BMSCs into the mixed solution for preparing the dynamic fiber mesh hydrogel scaffold, and circularly replacing the soaking system of sodium alginate, calcium chloride and the culture medium after the BMSCs form hydrogel, so that the BMSCs can be used for three-dimensional culture.
Technical effects
By means of the self-assembly behavior of the fibers of the recombinant protein and the complex reaction of the calcium chloride and the sodium citrate, the invention realizes the cooperative and dynamic regulation of the mechanical property, the fiber morphology and the internal space of the cell culture matrix and effectively promotes the proliferation and the extension of the mesenchymal stem cells in the three-dimensional culture.
Drawings
FIG. 1 is RGD-(R4S8)5The matrix-assisted laser desorption ionization time-of-flight mass spectrogram;
in the figure: the position pointed by the arrow is the molecular weight of the target protein;
FIG. 2 shows RGD- (R)4S8)5After the solution was incubated at 37 ℃ for 30 minutes, the resulting topography was photographed by atomic force microscopy.
FIGS. 3 to 10 are diagrams illustrating effects of the embodiment;
FIG. 3 shows the compressive modulus of 5% (w/v) RGD- (R4S8)5 solution after incubation at 37 deg.C for 30 min, mixed with 0.2% (w/v) sodium alginate solution, and soaked in calcium chloride solution to form initial hydrogel after enzyme-catalyzed cross-linking and then soaked in sodium citrate solution of different concentrations.
FIG. 4 shows RGD- (R) at 5% (w/v)4S8)5After the solution is incubated for 30 minutes at 37 ℃, the solution is fully mixed with 0.2 percent (w/v) sodium alginate solution, initial hydrogel is formed by enzymatic crosslinking and calcium chloride solution soaking, and the compressive modulus of the hydrogel is obtained after repeated cyclic soaking by sodium citrate solution with different concentrations and calcium chloride solution with certain concentration.
FIG. 5 shows RGD- (R) at 5% (w/v)4S8)5After the solution is incubated for 30 minutes at 37 ℃, the solution is fully mixed with 0.2% (w/v) sodium alginate solution, initial hydrogel is formed by enzyme-catalyzed crosslinking and calcium chloride solution soaking, and the appearance image of the hydrogel after being soaked by sodium citrate solution with different concentrations is an electron scanning microscope.
FIG. 6 shows RGD- (R) at 5% (w/v)4S8)5After the solution is incubated for 30 minutes at 37 ℃, the solution is fully mixed with 0.2% (w/v) sodium alginate solution, initial hydrogel is formed by enzymatic catalysis crosslinking and calcium chloride solution soaking, and the hydrogel is subjected to electron scanning microscope topography after ten times of circulating soaking by sodium citrate with different concentrations and calcium chloride solution with certain concentration.
FIG. 7 shows RGD- (R) at 5% (w/v)4S8)5Incubating the solution at 37 deg.C for 30 min, mixing with 0.2% (w/v) sodium alginate solution and BMSCs suspension, enzyme-catalyzed crosslinking, and soaking in calcium chloride solutionAfter initial hydrogel wrapping BMSCs is formed, the BMSCs are soaked in ammonium citrate solutions with different concentrations and calcium chloride solutions with certain concentrations in a circulating mode, and BMSCs live-dead-dye images are obtained after three days of culture.
FIG. 8 shows RGD- (R) at 5% (w/v)4S8)5After the solution is incubated for 30 minutes at 37 ℃, the solution is fully mixed with 0.2% (w/v) sodium alginate solution and BMSCs suspension, initial hydrogel wrapping the BMSCs is formed by enzyme catalytic crosslinking and calcium chloride solution soaking, and then the initial hydrogel is circularly soaked by sodium citrate solutions with different concentrations and calcium chloride solutions with certain concentrations, and the proliferation activity of the BMSCs is cultured for 1, 3, 5 and 7 days.
FIG. 9 shows RGD- (R) at 5% (w/v)4S8)5After the solution is incubated for 30 minutes at 37 ℃, the solution is fully mixed with 0.2% (w/v) sodium alginate solution and BMSCs suspension, initial hydrogel wrapping the BMSCs is formed by enzyme-catalyzed crosslinking and calcium chloride solution soaking, and then the BMSCs are cultured for three days after the initial hydrogel is circularly soaked by sodium citrate solutions with different concentrations and calcium chloride solutions with certain concentrations.
FIG. 10 is an immunofluorescence of CD31 after incubation of 5% (w/v) RGD- (R4S8)5 solution at 37 ℃ for 30 minutes, mixing with 0.2% (w/v) sodium alginate solution and BMSCs suspension, enzyme-catalyzed cross-linking, soaking in calcium chloride solution to form initial hydrogel wrapping BMSCs, circularly soaking in sodium alginate-sodium citrate solution with different concentrations and calcium chloride solution with certain concentration, and inducing differentiation for seven days.
Detailed Description
Example 1
In this example, the vector was constructed by the following specific procedures: in this embodiment, expression production is achieved by constructing an expression vector of the fusion protein and introducing the expression vector into escherichia coli, and the following specific operation processes are provided: the gene segments of the RGD-encoding polypeptide, namely 5'-CGTGGTGATATCGACGACGACGACAAG-3' and 5'-ATGGCCGCTGCTGTGATGATG-3', are introduced into a plasmid for encoding the arthropod-like fibroin by a point mutation kit to construct an expression vector pET-19b3-RGD-R4S8-5 of the arthropod-like fibroin fused with the RGD sequence. Then the expression vector is transferred into an expression host cell, the cell is cultured in 4mL LB culture medium containing ampicillin (0.1mg/mL) at 37 ℃ until OD600 reaches 1.8-2.0, then the cell is transferred into 100mL LB culture medium containing ampicillin at 37 ℃ until OD600 reaches 3.0-4.0, all the cells are transferred into 800mL TB culture medium containing ampicillin until OD600 reaches 6.0-8.0, and finally 1mM IPTG is added to induce the cells at 16 ℃ for 12-16 hours and then the cells are centrifugally harvested.
The strains, plasmids, antibiotics and culture media involved in the above examples were: cloning host cell Escherichia coli DH5 alpha, expression host cell Escherichia coli BL21(DE 3); expression plasmid pET-19b 3; ampicillin; LB medium, TB medium.
The LB culture medium comprises the following components: 10g/L tryptone, 5g/L yeast powder and 10g/L sodium chloride.
The TB culture medium comprises the following components: 12g/L tryptone, 24g/L yeast powder, 5g/L glycerol and 10% (v/v) TB salt solution.
The above TB salt solution comprises the following components: 23.1g/L potassium dihydrogen phosphate and 164.3g/L dipotassium hydrogen phosphate trihydrate.
Example 2
In this example, the separation and purification of the target protein is further performed based on example 1, and the specific implementation process is as follows: in this example, the cells collected in example 1 were resuspended in a ratio of 1g of wet cells to 10mL of equilibration buffer; after high-pressure homogenate and crushing, centrifugally collecting supernatant at 12000rpm, filtering by a 0.45-micron filter membrane, and loading the supernatant into a Ni-Sepharose affinity chromatography column after being balanced by a balance buffer solution; washing away the non-specifically bound hybrid protein by using a buffer solution for removing impurities, and eluting the target protein by using an elution buffer solution. Carrying out pure water dialysis for 36 hours by using a dialysis bag with the interception amount of 3.5kDa, and replacing pure water dialysate every 6 hours; concentrating the dialyzed target protein to 20-50mg/mL by using a concentration tube with the cut-off of 3kDa, freezing the concentrated target protein in a refrigerator at-80 ℃ for 12 hours, and then freeze-drying the target protein in a freeze dryer for 72 hours.
And (3) determining the molecular weight of the dialyzed target protein by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, wherein the position indicated by an arrow is the peak position of the target protein.
The equilibration buffer comprises the following components: 20mM Tris.HCl, 150mM NaCl and 5mM imidazole.
The impurity removal buffer comprises the following components: 20mM Tris.HCl, 150mM NaCl and 65mM imidazole.
The elution buffer comprises the following components: 20mM Tris.HCl, 150mM NaCl and 250mM imidazole.
Example 3
In this embodiment, a temperature stimulation is further used to prepare an RGD sequence-fused nodular fibroin fiber solution based on embodiment 2, and the specific implementation process is as follows: the target protein obtained in example 2 was dissolved in D-PBS buffer solution to prepare a protein solution with a concentration of 100ng/mL, incubated at 37 ℃ for 30 minutes, dropped on the surface of mica sheets, adsorbed for 10 minutes, washed with deionized water, dried, and photographed with an atomic force microscope.
As a result, the protein after temperature stimulation can be self-assembled to form a more uniform fiber morphology, as shown in FIG. 2.
Example 4
In this example, a dynamic fiber mesh hydrogel is further prepared based on example 3, and the specific implementation process is as follows: dissolving the nodular fibroin fused with the RGD sequence obtained in the example 2 in a D-PBS solution, incubating for 30 minutes at 37 ℃, adding sodium alginate, peroxidase and hydrogen peroxide solution, reacting for 10 minutes at 37 ℃, then adding calcium chloride solution, soaking for 30 minutes to obtain initial hydrogel, then soaking in an alpha-MEM culture medium for 12 hours, adding sodium citrate with different concentrations, soaking for 30 minutes, soaking in the alpha-MEM culture medium again for 12 hours, and then circularly soaking in the calcium chloride solution, the culture medium and the sodium citrate solution according to the steps to obtain the nodular fibroin/sodium alginate dynamic fiber net hydrogel fused with the RGD sequence.
The concentration of each component in the mixed solution is as follows: a 5% (w/v) protein solution; 0.2% (w/v) of sodium alginate; 600U/mL horseradish peroxidase solution; 0.03% (w/v) hydrogen peroxide solution.
The concentration of the calcium chloride solution is as follows: 25 mM.
The concentrations of the sodium citrate solution are respectively as follows: 25 mM; 125 mM; 250 mM.
In this example, the compressive modulus of the hydrogel was measured by using a universal tensile tester, and as shown in fig. 3, the compressive modulus of the hydrogel was reduced to different degrees after primary treatment with sodium citrate of different concentrations; as shown in FIG. 4, after the calcium chloride solution and the sodium citrate solutions with different concentrations are subjected to multiple circulating treatments, the compressive modulus of the hydrogel treated by the sodium citrate solutions with different concentrations is restored to different degrees.
In the embodiment, the appearance of the hydrogel is photographed by using an electron scanning microscope, as shown in fig. 5, the composite hydrogel has a clearly visible fiber structure inside, and the hydrogel treated by the sodium citrate solutions with different concentrations has different degrees of damage inside; as shown in fig. 6, after ten times of circulating treatment of the calcium chloride solution and the ammonium alginate solution, the inside of the composite hydrogel still maintains a clearly visible fiber structure, and the internal space is restored to different degrees.
Example 5
In this embodiment, a dynamic fiber mesh hydrogel is used to regulate the three-dimensional culture behavior of BMSCs, and the specific implementation process is as follows: the nodular fibroin fused with the RGD sequence obtained in example 2 is dissolved in a culture medium, filtered by a 0.22-micron filter head, incubated for 30 minutes at 37 ℃, sequentially added with sodium alginate filtered by the 0.22-micron filter head, peroxidase, hydrogen peroxide solution and suspension containing 100000 BMSCs, the mixed solution is dripped into a 48-hole cell culture plate, reacted for 10 minutes at 37 ℃, and then added with 500-mu L of calcium chloride solution to be soaked for 30 minutes to obtain initial hydrogel. Then soaking the initial hydrogel in 200 mu L of culture medium for 12 hours, soaking the hydrogel in 500 mu L of sodium citrate solution with different concentrations for 30 minutes, then soaking the hydrogel in 200 mu L of culture medium for 12 hours, and then circularly soaking the hydrogel in calcium chloride solution, culture medium and sodium citrate solution according to the steps to carry out three-dimensional culture of BMSCs. The viability, proliferation, morphology and endothelial differentiation of BMSCs cultured in three dimensions were determined.
The concentration of each component of the mixed solution is as follows: a 5% (w/v) protein solution; 0.2% (w/v) of sodium alginate; 600U/mL horseradish peroxidase solution; 0.03% (w/v) hydrogen peroxide solution.
The concentration of the calcium chloride solution is as follows: 25 mM.
The concentration of the sodium citrate solution is respectively as follows: 25 mM; 125 mM; 250 mM.
The culture medium is as follows: the culture medium for measuring the cell viability, proliferation and morphology is alpha-MEM, and the culture medium for endothelial differentiation is alpha-MEM containing 50 ng/mL.
The cell viability refers to that: BMSCs cultured in hydrogel for three days are stained with a live-dead staining solution at normal temperature for 1 hour, and photographed with a living cell ultrahigh-resolution multiphoton laser confocal microscope.
The cell proliferation refers to: BMSCs cultured in dynamic fiber mesh hydrogel at 1, 3, 5, and 7 were immersed in a mixture containing 200. mu.L of fresh alpha-MEM medium and 20. mu.L of CCK-8 solution, reacted at 37 ℃ for 4 hours, and then the mixture was taken out and placed in a 98-well plate, and the absorbance at 450nm was measured using a multifunctional microplate reader.
The cell expansion refers to: BMSCs cultured in dynamic fiber net hydrogel for 3 days are fixed by 4% paraformaldehyde at room temperature for 15 minutes, penetrated by 0.1% Triton X-100 at room temperature for 15 minutes, sealed in a quick sealing solution for 30 minutes, stained by FITC-phalloidin at 4 ℃ overnight, counterstained by DAPI staining solution at room temperature, and photographed by a living cell ultrahigh-resolution multi-photon laser confocal microscope.
The endothelial differentiation refers to: BMSCs seven days after induced differentiation in dynamic web hydrogels were assayed for CD31 expression using immunofluorescence.
The immunofluorescence is as follows: after BMSCs induced to differentiate are fixed by 4% paraformaldehyde at room temperature for 15 minutes, the BMSCs are penetrated by 0.1% Triton X-100 at room temperature for 15 minutes, then the BMSCs are sealed in a rapid sealing solution for 30 minutes, the BMSCs are soaked in a primary antibody of anti-CD 31 diluted by the rapid sealing solution according to a ratio of 1:100, after the BMSCs are soaked for 12 hours at 4 ℃, a secondary antibody labeled by Fluorescein Isothiocyanate (FITC) diluted by a ratio of 1:10 is added, after the BMSCs are soaked for 1 hour at room temperature, cell nuclei are counterstained by 4', 6-diamidino-2-phenylindole (DAPI), and shooting is carried out by a living cell ultrahigh-resolution multiphoton laser confocal microscope.
As shown in fig. 7, the recombinant protein/sodium alginate dynamic fiber network hydrogel fused with RGD sequence can promote the adhesion of stem cells and exhibit good biocompatibility.
As shown in fig. 8, the prepared dynamic fiber mesh hydrogel can promote three-dimensional proliferation of BMSCs, and the proliferation efficiency of BMSCs can be effectively adjusted through the synergistic regulation and control of the mechanical properties and the internal pore size of the dynamic fiber mesh hydrogel.
As shown in fig. 9, by dynamically regulating the composite fiber mesh hydrogel, the extension of BMSCs in the three-dimensional culture process can be effectively regulated.
As shown in FIG. 10, the prepared dynamic fiber mesh hydrogel was effective in regulating endothelial differentiation of BMSCs, and the dynamic fiber mesh hydrogel treated with 25mM sodium citrate and 25mM calcium chloride in cycles had the best effect of inducing endothelial differentiation.
The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
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Claims (10)

1. A preparation method of a culture scaffold based on recombinant protein hydrogel is characterized in that a dynamic fiber mesh hydrogel is obtained by constructing a recombinant protein expression vector of a dendroid silk fused with a protamine-glycerol-aspartyl tripeptide sequence, introducing the recombinant protein expression vector into an expression host cell, carrying out recombinant expression, separation and purification and temperature stimulation treatment, mixing with sodium alginate, carrying out enzyme catalytic crosslinking and cyclic treatment of calcium chloride and sodium citrate.
2. The method for preparing a culture scaffold based on a recombinant protein hydrogel as claimed in claim 1, wherein the constructing comprises: integrating the gene expressing the Arg-Gly-ASP (RGD) sequence into the recombinant protein (R) of the silkworm cocoon joint-like silk by using an inverse PCR site-directed mutagenesis kit4S8)5The expression vector pET-19b3-R4S8-5 is used for constructing the recombinant protein (R) of the nodular-like silkworm silk fused with RGD sequence4S8)5The expression vector pET-19b3-RGD-R4S 8-5.
3. The method for preparing a scaffold based on recombinant protein hydrogel of claim 2, wherein said recombinant protein is a fusion protein consisting of an elastin-like block (R) and a fibroin-like block (S), and the amino acid sequence thereof is [ (GGRPSDSYGAPGGGN)4-(GAGAGS)8]5；
The forward primer RGD-F adopted by the reverse PCR site-directed mutagenesis is shown as Seq ID No.1, and specifically comprises the following components: 5'-CGTGGTGATATCGACGACGACGACAAG-3', the reverse primer RGD-R is shown as Seq ID No.2, and specifically comprises: 5'-ATGGCCGCTGCTGTGATGATG-3' are provided.
4. The method for preparing a culture scaffold based on a recombinant protein hydrogel as claimed in claim 1, wherein the recombinant expression is: transferring the recombinant protein expression vector pET-19b3-RGD-R4S8-5 of the nodular-like silkworm silk fused with the RGD sequence into an expression host escherichia coli BL21(DE3), culturing recombinant bacteria, and inducing the expression of the nodular-like silkworm silk fused with the RGD sequence by using isopropyl thiogalactoside.
5. The method for preparing a culture scaffold based on a recombinant protein hydrogel as claimed in claim 1, wherein the separation and purification comprises: and (3) separating and purifying the expressed nodular fibroin by using a nickel ion metal chelate (Ni-NTA) affinity chromatography column.
6. The method for preparing a culture scaffold based on a recombinant protein hydrogel as claimed in claim 1, wherein the temperature stimulation is: the purified different concentrations of the RGD sequence fused arthropod fibroin were incubated at 37 ℃ for 30 minutes.
7. The method for preparing a recombinant protein hydrogel-based culture scaffold according to claim 1, wherein the cyclic treatment comprises: adding 0.2% (w/v) sodium alginate solution, horseradish peroxidase and hydrogen peroxide into 5% (w/v) of the 5% (w/v) ampullar silk protein solution fused with the RGD sequence after temperature stimulation, fully mixing the mixture, reacting the mixture for 10 minutes at 37 ℃, soaking the mixture in 25mM calcium chloride solution for 30 minutes to obtain initial hydrogel, soaking the initial hydrogel in an alpha-MEM culture medium for 12 hours, respectively soaking the initial hydrogel in 25mM sodium citrate solution, 125mM sodium citrate solution and 250mM sodium citrate solution for 30 minutes, then soaking the initial hydrogel in the alpha-MEM culture medium for 12 hours, and then sequentially circulating the soaking systems of the calcium chloride, the culture medium and the sodium citrate.
8. The method for preparing a culture scaffold based on a recombinant protein hydrogel of claim 7, wherein the enzyme activity of horseradish peroxidase is 600U/mL, and the concentration of hydrogen peroxide is 0.03% (w/v).
9. A dynamic fiber mesh hydrogel prepared according to any one of claims 1 to 8, having a fiber morphology similar to a natural extracellular matrix and a compressive modulus that is cyclically controlled at 2-22 kPa;
the circulation regulation and control specifically comprises the following steps: after 30 minutes of calcium chloride soaking once every 24 hours, culture medium soaking is carried out for 11.5 hours, then 30 minutes of sodium citrate soaking is carried out once, and culture medium soaking is carried out again for 11.5 hours.
10. Use of a dynamic fiber mesh hydrogel prepared according to any one of claims 1 to 8 or according to claim 9 for three-dimensional culture of mesenchymal stem cells, in particular: and dripping the digested and suspended BMSCs into the mixed solution for preparing the dynamic fiber mesh hydrogel scaffold, and circularly replacing the soaking system of sodium alginate, calcium chloride and the culture medium after the BMSCs form hydrogel, so that the BMSCs can be used for three-dimensional culture.
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