Recombinant human fibronectin as well as expression method and application thereof

Abstract

The invention belongs to the technical field of recombinant protein, and particularly relates to recombinant human fibronectin as well as an expression method and application thereof. The recombinant human fibronectin provided by the invention selects a fibronectin core functional domain III9-10 / III12-14, the core functional domain III9-10 / III12-14 is modified and transformed to obtain an encoding gene, and the encoding gene is transferred into escherichia coli and expressed to obtain the recombinant human fibronectin. The obtained recombinant human fibronectin has good stability, proliferation promoting activity and cell migration promoting capacity, can be applied to skin care products or cosmetics, and has important use value and practical significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of recombinant proteins, and in particular, relates to a recombinant human fibronectin and an expression method and application thereof. Background Art

[0002] Fibronectin (FN) is a high molecular weight extracellular matrix glycoprotein. FN mediates a variety of interactions between cells and the extracellular matrix (ECM) and plays an important role in cell adhesion, migration, growth and differentiation. FN can promote the phagocytosis of pathogens by macrophages, the deposition and orderly arrangement of collagen to accelerate wound healing. FN is composed of two similar subunits connected by disulfide bonds, and each subunit is composed of type I, type II and type III homologous repeating units. FN contains functional domains that can bind to collagen, heparin and cell surface receptors. The sequence Arg-Gly-Arp (RGD) in FN III10 can bind to a variety of integrins, thereby affecting cell adhesion, migration and proliferation. The PHSRN sequence in FN III9 can promote the binding of RGD to integrins. As a highly promiscuous growth factor binding domain, FN III12-14 can bind to a variety of growth factors and enhance their functions.

[0003] Currently, natural fibronectin extracted from human or animal blood and tissues has problems such as high cost and low safety. The production of recombinant fibronectin (rh FN) using genetic engineering technology has become a research hotspot. However, natural fibronectin has a large molecular weight and is difficult to express recombinantly. Therefore, how to select the structure of fibronectin to maximize its biological activity becomes the key.

[0004] Therefore, the main technical problem solved by the present invention is to provide a recombinant fibronectin that can be fully expressed and has high biological activity, good proliferation promoting effect and good cell migration promoting ability and an expression method thereof. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant human fibronectin and an expression method and application thereof.

[0006] To solve the above problems, the technical solution adopted by the present invention is: Technical topic 1: A recombinant human fibronectin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Technical theme 2: A method for expressing recombinant human fibronectin, comprising the following steps: S1. Construction of a recombinant expression vector: Select the core functional domain FNIII9-10 / III12-14 of fibronectin to obtain the amino acid sequence shown in SEQ ID NO.1, modify and transform to obtain the coding gene sequence of recombinant human fibronectin, double-enzyme-cut the coding gene of recombinant human fibronectin and the expression vector, and then perform enzyme ligation to obtain a recombinant expression vector; S2. Construction of engineering bacteria and fermentation expression of recombinant human fibronectin: transferring the recombinant expression vector into Escherichia coli to obtain engineering bacteria of recombinant human fibronectin; performing fermentation expression of the engineering bacteria to obtain engineering bacteria containing recombinant human fibronectin; S3. Purification of recombinant human fibronectin: The engineered bacteria were crushed, and the supernatant was collected by centrifugation to obtain a total protein extract, which was purified using Ni column affinity chromatography and anion column.

[0008] As a further embodiment of the present invention, the restriction endonucleases used in the double digestion are NdeI and XhoI enzymes; and the enzyme used in the enzyme ligation is T4 DNA ligase.

[0009] As a further embodiment of the present invention, the expression vector is pET30a or pET28a.

[0010] As a further embodiment of the present invention, the Escherichia coli is E. coli BL21 (DE3).

[0011] As a further embodiment of the present invention, the fermentation expression is: at a temperature of 30-40°C, pH=6.5-7.5, and 150-205rpm, ferment to an OD600 value of 8-10, cool to 20-40°C, add an IPTG inducer with a final concentration of 0.1mM-1mM, and use non-feedback variable speed flow feeding at the same time. After the feeding is completed, the expression is induced for 8-12h.

[0012] As a further embodiment of the present invention, the non-feedback variable speed flow feeding is: feeding at a rate of 0.2-0.4 mL / min for 2-4 h, then feeding at a rate of 0.4-0.8 mL / min for 2-4 h, and finally feeding at a rate of 0.8-1.2 mL / min for 2-4 h; the total volume of the feed is 0.5-1.5 L; the ingredients of the feed liquid are: peptone 10 g / L-20 g / L, yeast powder 15 g / L-30 g / L, potassium dihydrogen phosphate 1.0 g / L-3 g / L and dipotassium hydrogen phosphate 10 g / L-18 g / L.

[0013] Technical Theme 3: A recombinant human fibronectin engineering bacterium constructed in the preparation method described in Technical Topic 2.

[0014] Technical Theme 4: A use of the recombinant human fibronectin as described in Technical Topic 1 in the field of cosmetics or skin care products.

[0015] The beneficial effects of adopting the above technical solution are: 1. The present invention selects the core functional domain sequence of human fibronectin and designs a new recombinant human fibronectin sequence. The obtained recombinant human fibronectin is successfully expressed in the Escherichia coli expression system, and its soluble expression level can reach 30%. The purity of the obtained recombinant human fibronectin can reach more than 95%, overcoming the limitation that natural human fibronectin is difficult to recombinantly express due to its large molecular weight.

[0016] 2. The present invention selects FN III9-10 / FN III12-14 as the main structure, and the sequence arrangement is close to human fibronectin, which provides a guarantee for the safety of the obtained recombinant human fibronectin. Experiments have shown that the recombinant human fibronectin obtained by the present invention has no cytotoxicity within a certain concentration range.

[0017] 3. The sequence disclosed in the present invention selects the integrin binding site, integrin synergistic site and growth factor domain of human fibronectin, and the obtained recombinant human fibronectin has good ability to promote cell proliferation and cell migration.

[0018] 4. The recombinant human fibronectin provided by the present invention has high safety and good performance, and can effectively promote the proliferation and migration activity of skin fibroblasts and human immortalized keratinocytes. It can be made into freeze-dried preparations or hydrogels by adding auxiliary materials, and can be used in the fields of cosmetics or cosmeceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of the structure of the recombinant expression plasmid pET30a-rh FN obtained in Example 1 of the present invention; Figure 2 is the fermentation condition control curve of rh FN 3L fermenter in Example 2 of the present invention; Figure 3 This is the result of rh FN nickel column purification in Example 3 of the present invention; wherein, lane M is a marker, lane 1 is a supernatant of engineering bacteria crushing and centrifugation, lane 2 is a precipitate of engineering bacteria crushing and centrifugation, lane 3 is a flow-through, lane 4 is a wash buffer-1, lane 5 is a wash buffer-2, lane 6 is a wash buffer-3, lane 7 is an eluent of Elution Buffer-1, lane 8 is an eluent of Elution Buffer-2, lane 9 is a re-concentrated waste liquid, and lane 10 is a desalted liquid; Figure 4This is the result of rh FN anion column purification in Example 3 of the present invention; lane M is a marker, lane 1 is a reconcentrated solution, lane 2 is a flow-through solution, lane 3 is a Wash Buffer-4 washing solution, lane 4 is a Wash Buffer-5 washing solution, lane 5 is an Elution Buffer-3 eluent, lane 6 is a reconcentrated waste solution, and lane 7 is a desalted solution; Figure 5 This is the experimental result of the cytotoxicity of rh FN obtained in Example 3 of the present invention on mouse embryonic fibroblasts (NIH-3T3); Figure 6 This is a graph showing the experimental results of promoting the proliferation of mouse embryonic fibroblasts (NIH-3T3) using rh FN cells obtained in Example 3 of the present invention; Figure 7 This is a graph showing the experimental results of promoting the proliferation of human immortalized keratinocytes (HaCaT) using rh FN cells obtained in Example 3 of the present invention; Figure 8 This is a graph showing the migration promotion test of mouse embryonic fibroblasts (NIH-3T3) by rh FN obtained in Example 3 of the present invention; Fig. 9 This is a graph showing the results of a migration promotion test of mouse embryonic fibroblasts (NIH-3T3) by rh FN obtained in Example 3 of the present invention; Fig.10 This is a graph showing the migration promotion test of rh FN obtained in Example 3 of the present invention on human immortalized keratinocytes (HaCaT); Fig.11 This is a graph showing the results of a migration promotion test of rh FN obtained in Example 3 of the present invention on human immortalized keratinocytes (HaCaT). DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0021] Example 1 Obtaining recombinant human fibronectin expression vector 1. Sequence design: The present invention intercepts the core functional domain of fibronectin FNIII9-10 / III12-14 to obtain the amino acid sequence of recombinant human fibronectin, and its amino acid sequence is shown in SEQ ID NO.1. A His-Tag purification tag is added to the C-terminus of the recombinant human fibronectin amino acid sequence to obtain the amino acid sequence shown in SEQ ID NO.2.

[0022] 2. Sequence optimization and synthesis: The amino acid sequence shown in SEQ ID NO.2 was converted into a DNA sequence, and restriction sites and the termination codon TAA were added. Then, the DNA sequence was optimized according to the codon preference of Escherichia coli to obtain the recombinant human fibronectin (rh FN) encoding gene sequence. The obtained target gene sequence was commissioned to GenScript Biotech Co., Ltd. for gene synthesis.

[0023] 3. Construction of recombinant expression vector: The obtained rh FN gene and vector plasmid pET30a were double-digested with restriction endonucleases to obtain linearized plasmid pET30a and rh FN gene fragments; the linearized plasmid pET30a and rh FN gene fragments were connected with T4 DNA ligase to obtain the recombinant expression vector pET30a-rh FN, the structure of which is as follows: Figure 1 shown.

[0024] Example 2 Construction of engineered bacteria and expression of recombinant human fibronectin 1. Transformation: Take 100 μL of E. coli BL21 (DE3) competent cells from -80℃, melt and place on ice for 5-10 minutes; add 1 μL of recombinant expression vector pET30a-rh FN to E. coli BL21 (DE3) competent cells, mix and place on ice for 30 minutes; shake the obtained bacterial solution and heat shock it in a 42℃ water bath for 90 seconds, then immediately place on ice for 5 minutes; add 1mL of LB liquid medium without resistance to the bacterial solution obtained after heat shock, and resuscitate and culture at 37℃ and 150rpm for 60 minutes; after the resuscitation culture, take 150 μL of the transformation mixture and inoculate it into LB solid medium containing ampicillin (50 mg / mL), and spread it evenly with a disposable coating rod; first place it in a clean bench until the bacterial liquid on the surface of the culture medium is completely penetrated, then invert it and place it in a 37℃ constant temperature incubator for overnight culture to obtain recombinant human fibronectin engineering bacteria.

[0025] LB liquid medium: Add 4 g of LB beef extract medium powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to a pH between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes. LB solid medium: add 4 g LB beef extract medium powder and 4 g agar powder to 100 mL purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes; LB solid culture medium containing ampicillin (0.05 mg / mL): add 4 g of LB beef extract culture medium powder and 4 g of agar powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize it by high pressure at 121°C for 30 minutes; after sterilization, wait until the temperature drops to 60°C, and add 0.1 mL of 50 mg / mL ampicillin solution filtered through a 0.22µm filter membrane in the clean bench.

[0026] 2. Fermentation expression: (1) TB medium: peptone 15 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1.5 g / L, and potassium dihydrogen phosphate 16 g / L.

[0027] (2) The transformed E. coli was inoculated into 2 L of TB medium at an inoculum size of 4%. The fermentation program was set as follows: temperature 37 °C, pH = 7.0, 200 rpm. When the OD600 value reached 8-10, the temperature was lowered to 30 °C, 0.5 mM IPTG inducer was added, and non-feedback variable rate flow feeding was adopted. TB medium was fed at a rate of 0.3 mL / min for 3 h, then at a rate of 0.6 mL / min for 3 h, and finally at a rate of 1 mL / min for 3 h. The total volume of TB medium added was 1 L. After the feeding was completed, the expression was induced for 10 h, and the fermentation was terminated. The bacterial liquid was centrifuged and the bacterial sludge was collected.

[0028] Example 3 Purification of recombinant human fibronectin 1. Obtain protein extract: Each 1 g of the engineered bacterial sludge obtained in Example 1 was resuspended in 10 mL of Lysis Buffer-1, crushed 2-3 times under a pressure of 800 Pa, centrifuged at 4° C. and 12000 rpm for 30 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain a total protein extract.

[0029] 2. Nickel column purification: (1) Buffer preparation: Lysis Buffer-1: 50 mM Tris-HCl, 150 mM NaCl and 50 mM imidazole, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; Wash Buffer-1: 50 mM Tris-HCl, 150 mM NaCl, and 50 mM imidazole, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; Wash Buffer-2: 50 mM Tris-HCl, 150 mM NaCl, and 100 mM imidazole, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; Wash Buffer-3: 50 mM Tris-HCl, 150 mM NaCl, and 150 mM imidazole, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; Elution Buffer-1: 50 mM Tris-HCl, 150 mM NaCl, and 200 mM imidazole, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; Elution Buffer-2: 50 mM Tris-HCl, 150 mM NaCl, and 500 mM imidazole, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; (2) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-1; (3) Loading the total protein extract obtained in step 1 onto a nickel column and collecting the flow-through; (4) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-1; (5) Add 5 column volumes of Wash Buffer-1, 5 column volumes of Wash Buffer-2, 5 column volumes of Wash Buffer-3, 5 column volumes of Elution Buffer-1, and 5 column volumes of Elution Buffer-2 in sequence, and collect the wash buffer and eluent in sections; (6) The collected eluates are combined and concentrated, desalted to obtain a desalted solution, re-concentrated to obtain a re-concentrated solution, and the re-concentrated waste liquid is collected; SDS-PAGE electrophoresis analysis was performed, and the results were as follows Figure 3 As shown, its purity is >90%.

[0030] 3. Anion column purification: (1) Buffer preparation: Lysis Buffer-2: 50 mM Tris-HCl, 50 mM NaCl, pH adjusted to 8.0 with NaOH solution, sterilized by filtration through a 0.22 μm filter membrane; Wash Buffer-3: 50 mM Tris-HCl and 50 mM NaCl, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; Wash Buffer-4: 50 mM Tris-HCl and 200 mM NaCl, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; Elution Buffer-3: 50 mM Tris-HCl and 500 mM NaCl, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter membrane; (2) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-2; (3) loading the reconcentrated solution obtained in step 1 onto a nickel column and collecting the flow-through 2; (4) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-2; (5) Add 5 column volumes of Wash Buffer-3, 5 column volumes of Wash Buffer-4, and 5 column volumes of Elution Buffer-3 in sequence, and collect the wash buffer and eluent in sections; (6) The eluates are combined and concentrated, and desalted to obtain a desalted solution. The desalted solution is reconcentrated to obtain recombinant human fibronectin, and the reconcentrated waste liquid is collected; SDS-PAGE electrophoresis analysis was performed, and the results were as follows Figure 4 As shown, its purity is >95%.

[0031] Effect Example 1 Cytotoxicity Test 1. Digestion: Take out the T25 flask from the CO2 constant temperature incubator and observe the density of NIH-3T3 cells (mouse embryonic fibroblasts) under an inverted microscope. When the cell growth density reaches 80%~90%, add 1mL of 0.25% trypsin (containing EDTA) to the T25 flask for digestion. Under the microscope, it is observed that the cells shrink and become round, and the gaps increase. When most of the cells float under gentle shaking, add 3mL of complete culture medium to terminate the digestion, and gently shake the culture flask to detach the remaining cells from the wall.

[0032] 2. Dilute the digested cells to 5×10 3 The cell resuspension was inoculated into a 96-well plate, with 100 μL in each well; the culture plate was transferred to a 37°C cell culture incubator containing 5% carbon dioxide and cultured overnight to allow it to adhere to the wall.

[0033] 3. To the experimental groups 1-5, 100 μL of serum-free DMEM medium containing 125, 250, 500, 1000, and 2000 mg / mL of the recombinant human fibronectin rh FN obtained in Example 3 was added, respectively; to the control group, 100 μL of serum-free DMEM medium not containing the recombinant human fibronectin rh FN obtained in Example 3 was added, with 3 replicate wells in each group; 15 h after administration, the cell culture plate was taken out, 10 μL of CCK-8 solution was added to each well, and after incubation at 37°C for 2 h, the absorbance of each well was detected at 450 nm to obtain the OD value.

[0034] The results are as follows Figure 5 As shown, the experimental results showed that within the concentration gradient range of 125-2000 μM, the recombinant protein rh FN had no cytotoxicity to NIH-3T3 cells.

[0035] Effect Example 2 Cell Proliferation Experiment 1. Digestion: Take out the T25 flask from the CO2 constant temperature incubator and observe the density of NIH-3T3 cells (mouse embryonic fibroblasts) under an inverted microscope. When the cell growth density reaches 80%~90%, add 1mL of 0.25% trypsin (containing EDTA) to the T25 flask for digestion. Under the microscope, it is observed that the cells shrink and become round, and the gaps increase. When most of the cells float under gentle shaking, add 3mL of complete culture medium to terminate digestion, and gently shake the culture flask to allow the remaining cells to detach from the wall.

[0036] 2. Dilute the cells to 4×10 3 The resuspension was inoculated into a 96-well plate, 100 μL per well, and the culture plate was transferred to a 37°C cell culture incubator with 5% CO2 to adhere overnight.

[0037] 3. Take the culture plate out of the cell culture incubator; (1) NIH-3T3 cell proliferation assay: The experimental group was added with 100 μL of serum-free DMEM medium containing 31.25, 62.5, 125, 250, and 500 mg / mL of the recombinant human fibronectin rh FN obtained in Example 3; The control group was added with 100 μL of serum-free DMEM medium without the recombinant human fibronectin rh FN obtained in Example 3; (2) HaCaT cell proliferation experiment: The experimental group was added with 100 μL of serum-free DMEM medium containing 125 μM recombinant human fibronectin rh FN obtained in Example 3; The control group was added with 100 μL of serum-free DMEM medium without the recombinant human fibronectin rh FN obtained in Example 3; There were 3 replicate wells in each group. 24 hours after administration, the cell culture plate was taken out and 10 μL of CCK-8 solution was added to each well. After incubation at 37°C for 1-4 hours, the absorbance of each well was detected at 450 nm.

[0038] 4. The results are as follows Figure 6 and Figure 7 As shown, the experimental results show that within the concentration gradient range of 31.25-500 μM, the recombinant protein rh FN has a proliferation-promoting effect on HaCaT cells; within the concentration gradient range of 31.25-250 μM, the recombinant protein rhFN has a proliferation-promoting effect on NIH-3T3 cells.

[0039] Effect Example 3: Cell Migration Promotion Experiment 1. Take out the T25 flask from the CO2 constant temperature incubator and observe the density of NIH-3T3 cells (mouse embryonic fibroblasts) under an inverted microscope. When the cell growth density reaches 80%~90%, add 1mL of 0.25% trypsin (containing EDTA) to the T25 flask for digestion. Under the microscope, it is observed that the cells shrink and become round, and the gaps increase. When most of the cells float after gentle shaking, add 3mL of complete culture medium to terminate digestion, and gently shake the culture flask to detach the remaining cells from the wall.

[0040] 2. Dilute NIH-3T3 cells and HaCaT cells to 1×10 cells / mL with 10 mL of DMEM medium containing 10% fetal bovine serum. 6 The cell resuspension was inoculated into different 6-well plates at 2 mL per well, and the culture plates were transferred to a 37°C cell culture incubator containing 5% CO2 and cultured for 24 h to allow the cells to grow confluently.

[0041] 3. Use a 200 μL pipette tip to draw a cross in each well, and wash with sterile PBS three times to remove the crossed cells; (1) NIH-3T3 cell migration assay: The experimental group was added with 2 mL of serum-free DMEM medium containing 125 μM rh FN; The control group was added with 2 mL of serum-free DMEM medium; (2) HaCaT cell migration experiment: The experimental group was added with 2 mL of serum-free DMEM medium containing 125 μM rh FN; The control group was added with 2 mL of serum-free DMEM medium.

[0042] 4. Observe and take photos under an inverted microscope to record the wound healing status at 0, 24, 48, and 72 hours. The results of the cell migration experiment are as follows: Figure 8-11 The experimental results showed that within the concentration gradient range of 31.25-500 μM, the recombinant protein rh FN had a significant effect on promoting the migration of NIH-3T3 cells and HaCaT cells.

[0043] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human fibronectin, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for expressing recombinant human fibronectin, characterized in that: Here are the steps: S1. Construction of a recombinant expression vector: selecting the core functional domain FNIII9-10 / III12-14 of fibronectin to obtain the amino acid sequence shown in SEQ ID NO.1 in claim 1, and obtaining the coding gene sequence of recombinant human fibronectin by modification and transformation, and then double-digesting the coding gene of recombinant human fibronectin and the expression vector and performing enzyme ligation to obtain a recombinant expression vector; S2. Construction of engineering bacteria and fermentation expression of recombinant human fibronectin: transferring the recombinant expression vector into Escherichia coli to obtain engineering bacteria of recombinant human fibronectin; performing fermentation expression of the engineering bacteria to obtain engineering bacteria containing recombinant human fibronectin; S3. Purification of recombinant human fibronectin: The engineered bacteria were crushed, and the supernatant was collected by centrifugation to obtain a total protein extract, which was purified using Ni column affinity chromatography and anion column.

3. The method for expressing recombinant human fibronectin according to claim 2, characterized in that: The restriction endonucleases used in the double digestion are NdeI and XhoI enzymes; the enzyme used in the enzyme ligation is T4 DNA ligase.

4. The method for expressing recombinant human fibronectin according to claim 2, characterized in that: The expression vector is pET30a or pET28a.

5. The method for expressing recombinant human fibronectin according to claim 2, characterized in that: The Escherichia coli is E. coli BL21 (DE3).

6. The method for expressing recombinant human fibronectin according to claim 2, characterized in that: The fermentation was performed at a temperature of 30-40°C, a pH of 6.5-7.5, and a speed of 150-205 rpm until OD 600 The value was 8-10, the temperature was lowered to 20-40℃, IPTG inducer was added with a final concentration of 0.1mM-1mM, and feeding was performed by non-feedback variable speed flow. After the feeding was completed, the expression was induced for 8-12h.

7. The method for expressing recombinant human fibronectin according to claim 6, characterized in that: The non-feedback variable speed flow feeding is as follows: feeding at a rate of 0.2-0.4 mL / min for 2-4 hours, then feeding at a rate of 0.4-0.8 mL / min for 2-4 hours, and finally feeding at a rate of 0.8-1.2 mL / min for 2-4 hours; the total feeding volume is 0.5-1.5 L; the ingredients of the feeding liquid are: peptone 10 g / L-20 g / L, yeast powder 15 g / L-30 g / L, potassium dihydrogen phosphate 1.0 g / L-3 g / L and dipotassium hydrogen phosphate 10 g / L-18 g / L.

8. A recombinant human fibronectin engineering bacterium constructed by the expression method according to claims 2-7.

9. Use of the recombinant human fibronectin as claimed in claim 1 in the field of cosmetics or skin care products.

Images