Recombinant human fibronectin and its expression method and application

Abstract

The present invention belongs to the field of recombinant protein technology, specifically, to a recombinant human fibronectin and its expression method and application. The recombinant human fibronectin provided by the present invention selects the fibronectin core functional domain III9-10 / III12-14, is modified and transformed to obtain a coding gene, which is transferred into Escherichia coli to express and obtain recombinant human fibronectin. The recombinant human fibronectin obtained by the present invention has good stability, pro-proliferation activity and the ability to promote cell migration, 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 multiple interactions between cells and the extracellular matrix (ECM) and plays an important role in cell adhesion, migration, growth, and differentiation. FN promotes phagocytosis of pathogens by macrophages and the deposition and orderly arrangement of collagen, accelerating wound healing. FN is composed of two similar subunits linked by a disulfide bond, each composed of type I, type II, and type III homologous repeats. FN contains functional domains that bind to collagen, heparin, and cell surface receptors. The sequence Arg-Gly-Arp (RGD) in FN III10 binds to various integrins, thereby affecting cell adhesion, migration, and proliferation. The PHSRN sequence in FN III9 promotes the binding of RGD to integrins. FN III12-14, as highly promiscuous growth factor-binding domains, can bind to a variety of growth factors and enhance their function.

[0003] Currently, natural fibronectin extracted from human or animal blood and tissues has problems such as high cost and low safety. The use of genetic engineering technology to produce recombinant fibronectin (rhFN) 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] In order to solve the above problems, the technical solution adopted by the present invention is:

[0007] Technical Topic 1:

[0008] A recombinant human fibronectin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Technical Topic 2:

[0010] A method for expressing recombinant human fibronectin, comprising the following steps:

[0011] S1. Construction of a recombinant expression vector: Select the core functional domain of fibronectin, FNIII9-10 / III12-14, 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-digest the recombinant human fibronectin coding gene and the expression vector, and then enzymatically ligate them to obtain a recombinant expression vector;

[0012] S2. Construction of engineered bacteria and fermentation expression of recombinant human fibronectin: Transform the recombinant expression vector into Escherichia coli to obtain recombinant human fibronectin engineered bacteria; perform fermentation expression of the engineered bacteria to obtain engineered bacteria containing recombinant human fibronectin;

[0013] 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 then purified using Ni column affinity chromatography and anion column.

[0014] As a further embodiment of the present invention, the restriction endonucleases used in the double enzyme digestion are NdeI and XhoI enzymes; and the enzyme used in the enzyme ligation is T4 DNA ligase.

[0015] As a further embodiment of the present invention, the expression vector is pET30a or pET28a.

[0016] As a further embodiment of the present invention, the Escherichia coli is E. coli BL21 (DE3).

[0017] As a further embodiment of the present invention, the fermentation expression is as follows: at a temperature of 30-40°C, pH = 6.5-7.5, and 150-205rpm, fermentation is carried out to an OD600 value of 8-10, the temperature is lowered to 20-40°C, an IPTG inducer is added with a final concentration of 0.1mM-1mM, and non-feedback variable speed flow feeding is adopted at the same time. After the feeding is completed, the expression is induced for 8-12h.

[0018] 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 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 feeding liquid components 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.

[0019] Technical Theme 3:

[0020] A recombinant human fibronectin engineering bacterium constructed in the preparation method described in Technical Topic 2.

[0021] Technical Topic 4:

[0022] A use of the recombinant human fibronectin as described in Technical Topic 1 in the field of cosmetics or skin care products.

[0023] The beneficial effects of adopting the above technical solution are:

[0024] 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 an 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 of natural human fibronectin that is difficult to recombinantly express due to its large molecular weight.

[0025] 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 resulting recombinant human fibronectin. Experiments have also shown that the recombinant human fibronectin obtained in the present invention is non-cytotoxic within a certain concentration range.

[0026] 3. The sequence disclosed in the present invention selects the integrin binding site, integrin cooperative site and growth factor domain of human fibronectin, and the resulting recombinant human fibronectin has good ability to promote cell proliferation and cell migration.

[0027] 4. The recombinant human fibronectin provided by the present invention has high safety and good performance. It can effectively promote the proliferation and migration activity of skin fibroblasts and human immortalized keratinocytes. It can be made into lyophilized preparations or hydrogels by adding excipients, and can be used in the fields of cosmetics or cosmeceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the structure of the recombinant expression plasmid pET30a-rh FN obtained in Example 1 of the present invention;

[0029] Figure 2 This is the fermentation condition control curve of rh FN 3L fermenter in Example 2 of the present invention;

[0030] Figure 3This is the rhFN nickel column purification result of Example 3 of the present invention; wherein, lane M is a marker, lane 1 is the supernatant of the engineered bacteria crushing and centrifugation, lane 2 is the precipitate of the engineered bacteria crushing and centrifugation, lane 3 is the flow-through, lane 4 is the wash buffer-1, lane 5 is the wash buffer-2, lane 6 is the wash buffer-3, lane 7 is the eluate of Elution Buffer-1, lane 8 is the eluate of Elution Buffer-2, lane 9 is the reconcentration waste liquid, and lane 10 is the desalted liquid;

[0031] Figure 4 This is the result of rhFN anion column purification in Example 3 of the present invention; lane M is the marker, lane 1 is the reconcentrate, lane 2 is the flow-through, lane 3 is the Wash Buffer-4 wash solution, lane 4 is the Wash Buffer-5 wash solution, lane 5 is the Elution Buffer-3 eluate, lane 6 is the reconcentrate waste solution, and lane 7 is the desalted solution;

[0032] 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);

[0033] Figure 6 This is a graph showing the experimental results of promoting the proliferation of mouse embryonic fibroblasts (NIH-3T3) using rhFN cells obtained in Example 3 of the present invention;

[0034] Figure 7 This is a graph showing the experimental results of promoting the proliferation of human immortalized keratinocytes (HaCaT) using rhFN cells obtained in Example 3 of the present invention;

[0035] 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;

[0036] Figure 9 This is a graph showing the results of a migration promotion test on mouse embryonic fibroblasts (NIH-3T3) by rh FN obtained in Example 3 of the present invention;

[0037] Figure 10 This is a graph showing the migration promotion test of human immortalized keratinocytes (HaCaT) by rh FN obtained in Example 3 of the present invention;

[0038] Figure 11 This is a graph showing the results of a migration promotion test on human immortalized keratinocytes (HaCaT) by rh FN obtained in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0040] Example 1 Obtaining a recombinant human fibronectin expression vector

[0041] 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, whose 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.

[0042] 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. The DNA sequence was then optimized based on the codon preference of E. coli to obtain the recombinant human fibronectin (rh FN) encoding gene sequence. The resulting target gene sequence was commissioned to GenScript Biotech Co., Ltd. for gene synthesis.

[0043] 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 fragment; the linearized plasmid pET30a and rh FN gene fragment were ligated with T4 DNA ligase to obtain the recombinant expression vector pET30a-rh FN, the structure of which is shown in the following figure: Figure 1 shown.

[0044] Example 2 Construction of engineered bacteria and expression of recombinant human fibronectin

[0045] 1. Transformation: Take 100 μL of E. coli BL21 (DE3) competent cells from -80℃, thaw and place on ice for 5-10 minutes; add 1 μL of recombinant expression vector pET30a-rh FN to the E. coli BL21 (DE3) competent cells, mix well and place on ice for 30 minutes; shake the resulting bacterial solution and heat shock it in a 42℃ water bath for 90 seconds, then immediately place on ice for 5 minutes; add 1 mL of non-resistant LB liquid medium to the heat-shocked bacterial solution and recover and culture at 37℃ and 150 rpm for 60 minutes; after the recovery culture is completed, 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 completely penetrates the surface of the medium, then invert it and place it in a 37℃ constant temperature incubator for overnight culture to obtain recombinant human fibronectin engineered bacteria.

[0046] LB liquid medium: Add 4 g of LB beef extract medium powder to 100 mL of purified water. Adjust the pH of the solution to 6.8-7.3 with 5%-20% sodium hydroxide solution. Sterilize by autoclaving at 121°C for 30 minutes.

[0047] LB solid medium: Add 4 g of LB beef extract powder and 4 g of agar powder to 100 mL of purified water. Adjust the pH of the solution to 6.8-7.3 with 5%-20% sodium hydroxide solution. Autoclave at 121°C for 30 minutes.

[0048] LB solid medium containing ampicillin (0.05 mg / mL): Add 4 g of LB beef extract medium powder and 4 g of agar powder to 100 mL of purified water. Adjust the pH of the prepared solution to between 6.8 and 7.3 with 5%-20% sodium hydroxide solution. Autoclave at 121°C for 30 minutes. After sterilization, cool the temperature to 60°C and add 0.1 mL of 50 mg / mL ampicillin solution filtered through a 0.22 µm filter in a clean bench.

[0049] 2. Fermentation expression:

[0050] (1) TB culture medium: peptone 15 g / L, yeast powder 20 g / L, potassium dihydrogen phosphate 1.5 g / L, and potassium dihydrogen phosphate 16 g / L.

[0051] (2) The transformed E. coli was inoculated into 2 L of TB culture 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 speed flow feeding was used at the same time. TB culture medium was added 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 culture 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.

[0052] Example 3 Purification of recombinant human fibronectin

[0053] 1. Obtain protein extract:

[0054] 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 800 Pa pressure, 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.

[0055] 2. Nickel column purification:

[0056] (1) Buffer preparation:

[0057] 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;

[0058] 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;

[0059] 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;

[0060] 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;

[0061] 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;

[0062] 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.

[0063] (2) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-1;

[0064] (3) Load the total protein extract obtained in step 1 onto a nickel column and collect the flow-through;

[0065] (4) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-1;

[0066] (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 solution and eluate in sections;

[0067] (6) The collected eluates are combined and concentrated, desalted to obtain a desalted solution, and re-concentrated to obtain a re-concentrated solution, and the re-concentrated waste liquid is collected;

[0068] SDS-PAGE electrophoresis analysis was performed, and the results were as follows Figure 3 As shown, its purity is >90%.

[0069] 3. Anion column purification:

[0070] (1) Buffer preparation:

[0071] Lysis Buffer-2: 50 mM Tris-HCl, 50 mM NaCl, adjusted to pH 8.0 with NaOH solution, and sterilized by filtration through a 0.22 μm filter.

[0072] 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;

[0073] 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;

[0074] 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.

[0075] (2) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-2;

[0076] (3) Loading the reconcentrated solution obtained in step 1 onto a nickel column and collecting the flow-through 2;

[0077] (4) Equilibrate the nickel column with 10 column volumes of Lysis Buffer-2;

[0078] (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 eluate in sections;

[0079] (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;

[0080] SDS-PAGE electrophoresis analysis was performed, and the results were as follows Figure 4 As shown, its purity is >95%.

[0081] Effect Example 1 Cytotoxicity Test

[0082] 1. Digestion: Remove 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 1 mL of 0.25% trypsin (containing EDTA) to the T25 flask for digestion. Under the microscope, the cells will shrink and become round, and the gaps between them will increase. When most of the cells float when gently shaken, add 3 mL of complete culture medium to terminate the digestion. Gently shake the culture flask to detach the remaining cells from the wall.

[0083] 2. Dilute the digested cells to 5×10 3 The cells were resuspended and inoculated into a 96-well plate with 100 μL per well. The culture plate was transferred to a 37°C cell culture incubator containing 5% carbon dioxide and cultured overnight to allow the cells to adhere to the wall.

[0084] 3. Experimental groups 1-5 were added with 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, respectively. The control group was added with 100 μL of serum-free DMEM medium without the recombinant human fibronectin rh FN obtained in Example 3, with three replicates per group. 15 h after administration, the cell culture plate was removed, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance of each well was measured at 450 nm to obtain the OD value.

[0085]

[0086] The results are as follows Figure 5 As shown, the experimental results showed that the recombinant protein rh FN had no cytotoxicity to NIH-3T3 cells within the concentration gradient range of 125-2000 μM.

[0087] Effect Example 2 Cell Proliferation Experiment

[0088] 1. Digestion: Remove 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 1 mL of 0.25% trypsin (containing EDTA) to the T25 flask for digestion. Under the microscope, observe that the cells shrink and become round, and the gaps between them increase. When most of the cells float under gentle shaking, add 3 mL of complete culture medium to terminate digestion. Gently shake the culture flask to detach the remaining cells from the wall.

[0089] 2. Dilute the cells to 4×103 The resuspension was inoculated into a 96-well plate with 100 μL per well, and the culture plate was transferred to a 37°C cell culture incubator with 5% CO2 to adhere overnight.

[0090] 3. Take the culture plate out of the cell culture incubator;

[0091] (1) NIH-3T3 cell proliferation assay:

[0092] 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;

[0093] The control group was added with 100 μL of serum-free DMEM medium without the recombinant human fibronectin rh FN obtained in Example 3;

[0094] (2) HaCaT cell proliferation experiment:

[0095] 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;

[0096] The control group was supplemented with 100 μL of serum-free DMEM medium without the recombinant human fibronectin rh FN obtained in Example 3;

[0097] Each group had three replicate wells. 24 hours after administration, the cell culture plate was removed 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.

[0098]

[0099] 4. The results are as follows Figure 6 and Figure 7 As shown, the experimental results showed that within the concentration gradient range of 31.25-500 μM, the recombinant protein rh FN had a proliferation-promoting effect on HaCaT cells; within the concentration gradient range of 31.25-250 μM, the recombinant protein rhFN had a proliferation-promoting effect on NIH-3T3 cells.

[0100] Effect Example 3: Cell Migration Promotion Experiment

[0101] 1. Remove 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 1 mL of 0.25% trypsin (containing EDTA) to the T25 flask for digestion. Under the microscope, observe that the cells shrink and become round, and the gaps between them increase. When most of the cells float after gentle shaking, add 3 mL of complete culture medium to terminate the digestion. Gently shake the culture flask to detach the remaining cells from the wall.

[0102] 2. Dilute NIH-3T3 cells and HaCaT cells to 1×10 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.

[0103] 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.

[0104] (1) NIH-3T3 cell migration assay:

[0105] The experimental group was added with 2 mL of serum-free DMEM medium containing 125 μM rh FN;

[0106] The control group was added with 2 mL of serum-free DMEM medium;

[0107] (2) HaCaT cell migration assay:

[0108] The experimental group was added with 2 mL of serum-free DMEM medium containing 125 μM rh FN;

[0109] The control group was added with 2 mL of serum-free DMEM medium.

[0110] 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.

[0111] 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 replacements for some of the technical features therein; and these modifications or replacements 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: Select the core functional domain of fibronectin, FNIII9-10 / III12-14, to obtain the amino acid sequence as shown in SEQ ID NO. 1 in claim 1, and modify and transform the sequence to obtain the coding gene sequence of recombinant human fibronectin. The recombinant human fibronectin coding gene and the expression vector are double-enzyme-digested and then ligated by enzyme to obtain a recombinant expression vector; S2. Construction of engineered bacteria and fermentation expression of recombinant human fibronectin: Transform the recombinant expression vector into Escherichia coli to obtain recombinant human fibronectin engineered bacteria; perform fermentation expression of the engineered bacteria to obtain engineered 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 then purified using Ni column affinity chromatography and anion column.

3. The method for expressing recombinant human fibronectin according to claim 2, wherein: The restriction endonucleases used in the double enzyme 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, wherein: The expression vector is pET30a or pET28a.

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

6. The method for expressing recombinant human fibronectin according to claim 2, wherein: 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 the 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, wherein: The non-feedback variable-speed flow feeding method includes: 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; and the feeding liquid components include: 10 g / L-20 g / L of peptone, 15 g / L-30 g / L of yeast powder, 1.0 g / L-3 g / L of potassium dihydrogen phosphate, and 10 g / L-18 g / L of dipotassium hydrogen phosphate.

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

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