Preparation and Application of Recombinant Human Fibronectin

Abstract

The present invention discloses a method for preparing recombinant human fibronectin, belonging to the technical field of genetic engineering. The recombinant human fibronectin prepared by the present invention reasonably combines the core fragments of fibronectin by adjusting the composition of the sequence, removes unnecessary or some domains that have a greater impact on the structure, and through reasonable optimization, obtains recombinant human fibronectin with higher purity and better yield. The recombinantly expressed human fibronectin still retains high biological activity in cell proliferation, adhesion, and differentiation, and has good application prospects in cosmetics, medical devices, and other aspects.

Description

Technical Field

[0001] The present invention relates to the preparation and application of recombinant human fibronectin, and belongs to the technical field of genetic engineering. Background Art

[0002] Fibronectin (FN) is a high-molecular-weight glycoprotein with a carbohydrate content of 4.5%-9.5%. It is a multifunctional glycoprotein abundant in plasma and widely distributed in the extracellular matrix. Its primary function is to mediate cell adhesion. Fibronectin (FN) is a large glycoprotein with a molecular weight of approximately 440,000 daltons. It is a dimer composed of two subunits with a molecular mass of 220,000 daltons connected by disulfide bonds. The entire molecule is composed of two similar A and B chains, forming a V-shape. Each FN subunit contains multiple binding domains closely related to its function: two heparin-binding domains; three fibrin-binding domains; and one collagen-binding domain. The collagen-binding domains on FN bind to collagen in the cell matrix, thereby adsorbing cells to the extracellular matrix. One or two eukaryotic cell-binding domains are located on Fn III 10, and the second is located on Fn III 9. Fibronectin recognizes and binds to integrin heterodimers via the arginine-glycine-aspartate (RGD) sequence on Fn III 10 and Fn III 9, thereby influencing cell adhesion and migration. Purified fibronectin can enhance cell-cell adhesion and cell-matrix adhesion. Cell adhesion and migration are the result of specific recognition, binding, and interaction between cells and the extracellular matrix. The interaction between extracellular matrix protein molecules and their corresponding cell membrane receptors, integrins, is a key mechanism determining cell adhesion and migration. The interaction between the ligand molecule fibronectin and the corresponding integrin is the central link in the regulation of cell adhesion and migration. FN can connect cells to the extracellular matrix.

[0003] Fibronectin has multiple adhesion functions, such as intercellular adhesion, and is also important in wound healing. Soluble fibronectin deposited on damaged collagen and fibrin enhances platelet adhesion, phagocyte and fibroblast migration, and cell proliferation. In addition to adhesion, fibronectin regulates cell shape and cytoskeletal organization through cell signaling pathways, promoting cell spreading. During embryogenesis, fibronectin is essential for the migration and differentiation of many cell types. Fibronectin is also important in wound repair. During blood clot formation, fibronectin promotes platelet adhesion to damaged blood vessels. Therefore, fibronectin has broad application prospects in medicine, beauty, and skincare.

[0004] Although animal tissues and blood contain fibronectin, its content is extremely limited, and the protein extracted from animals is a full-length protein with poor skin permeability. This results in tissue-derived fibronectin with low production, high cost, low product purity, and low skin penetration. Naturally extracted fibronectin has a large molecular weight and is not uniform, resulting in limited amounts of pure extracted protein and difficulty in large-scale production. Products extracted from animals also have limitations such as low product purity, the risk of viral transmission, the risk of immunogenicity, and high cost. Therefore, it is necessary to express and prepare highly purified humanized fibronectin in prokaryotic or eukaryotic microorganisms through genetic engineering techniques.

[0005] The existing fibronectin source sequences are not completely human, and in application scenarios, there may be problems with immunogenicity, which limits its application scenarios. In terms of production technology, there are problems such as low fermentation yield and easy plasmid loss in fermentation strains. There are also problems such as cumbersome purification processes, low purity, and low purification yield, which are not conducive to large-scale production and purification applications. At present, the development of recombinant human fibronectin has been carried out, but there are a series of problems, such as low yield, which cannot form large-scale production; low purity, and certain safety risks in large-scale production; low activity, and high cost of large-scale production; poor stability, high cost of large-scale production, and it is not conducive to product use, and the application scenarios are simple. In addition, the molecular weight of the recombinant fibronectin produced is relatively large, and its hydrophilicity is strong, which makes it difficult to penetrate the skin and difficult to perform its function. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides a preparation and application of recombinant fibronectin, aiming to solve the problem that some fibronectin source sequences are not completely human, and there may be immunogenicity problems in application scenarios, which limits its application scenarios; in terms of molecular weight, there are problems such as difficulty in penetration; in terms of production process, there are problems such as low fermentation process yield and easy plasmid loss in fermentation strains; there are problems such as complicated purification process, low purity, low purification yield, and low stability, which are not conducive to large-scale production and purification and other technical problems.

[0007] The first technical solution provided by the present invention is a recombinant human fibronectin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The second technical solution provided by the present invention is a gene encoding the protein described in the first technical solution.

[0009] In certain embodiments, the nucleotide sequence of the gene is shown in any one of SEQ ID NOs. 2 to 4.

[0010] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0011] In certain embodiments, pET32a(+) or pET28a(+) is used as the expression vector.

[0012] The fourth technical solution provided by the present invention is a genetically engineered bacterium, which is a host cell expressing the protein described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the plasmid described in the third technical solution.

[0013] In certain embodiments, the host cell comprises a prokaryotic or eukaryotic microorganism.

[0014] In certain embodiments, the host cell is based on Escherichia coli, preferably, E. coli BL21(DE3) was used as the expression host.

[0015] The fifth technical solution provided by the present invention is a method for preparing recombinant fibronectin, which comprises culturing the genetically engineered bacteria described in the fourth technical solution, inducing the bacteria to express recombinant fibronectin, and disrupting the bacteria to obtain a crude recombinant fibronectin product.

[0016] In certain embodiments, the genetically engineered bacteria are inoculated in TB liquid culture medium, cultured at 37°C, 220 rpm for 4-6 hours, 0.1-1 M IPTG is added, induced at 25-37°C, 220 rpm for 6-8 hours, centrifuged to remove the supernatant, ultrasonically disrupted the bacteria, and centrifuged to collect the supernatant to obtain a crude product.

[0017] In certain embodiments, the crude recombinant fibronectin is purified to obtain pure recombinant fibronectin.

[0018] Furthermore, the purification process includes the following steps: the crude recombinant fibronectin is separated by nickel column affinity chromatography to obtain a pure recombinant fibronectin product.

[0019] The sixth technical solution provided by the present invention is the use of the protein described in the first technical solution, or the gene described in the second technical solution, or the plasmid described in the third technical solution, or the genetically engineered bacteria described in the fourth technical solution, or the method described in the fifth technical solution in the preparation of a product containing fibronectin.

[0020] The seventh technical solution provided by the present invention is a method for detecting fibronectin activity, wherein the method comprises culturing mouse fibroblasts NIH 3T3 in 1640 medium supplemented with a fibronectin sample to be detected, and after 60 hours, replacing the medium with 1640 medium containing 10% CCK8 for 2 hours, and detecting the absorbance at 450 nm;

[0021] Alternatively, the method comprises culturing human keratinocytes HaCaT in a DMEM medium supplemented with a fibronectin sample to be tested, replacing the medium with a DMEM medium containing 0.5 mg / mL MTT after 24 hours, culturing for 4 hours, discarding the supernatant DMSO, and detecting the absorbance at 570 nm.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The human fibronectin sequence selected by the present invention is combined and optimized to obtain the original amino acid sequence. The original amino acid sequence is codon-optimized to enable heterologous expression in microorganisms. At the same time, some tags are selected as auxiliary proteins of the recombinant fibronectin to construct a fusion protein. The fusion protein is constructed into a protein expression vector and transformed into a microorganism. The expression of the fermentation strain is optimized to obtain a microbial strain with high fibronectin production. The strain is crushed and subjected to column chromatography, enzyme digestion, and other methods to obtain recombinant human fibronectin. The activity of its active protein is tested by a pharmacopoeia-like method and has the characteristic of high activity at low concentrations. The present invention obtains recombinant human fibronectin with high purity and good yield. The recombinantly expressed human fibronectin still retains efficient biological activity in cell proliferation, adhesion, and differentiation, and has good application prospects in cosmetics, medical devices, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Structure prediction for proteins under different tags.

[0025] Figure 2 Schematic diagram of the Escherichia coli recombinant plasmid containing recombinant human fibronectin.

[0026] Figure 3 This is the nucleic acid electrophoresis diagram of the recombinant plasmid, where lane 1: pET32a(+) + original recombinant human fibronectin gene fragment; lane 2: 10 kb DNA marker.

[0027] Figure 4 The SDS-PAGE images are of the wild type Escherichia coli high-copy genetically engineered strain expressing recombinant human fibronectin and the supernatant of the broken cell wall of the high-copy genetically engineered Escherichia coli expressing recombinant human fibronectin (induced for 5 hours).

[0028] Figure 5 This is a comparison of the purity of recombinant human fibronectin from different sources (HPLC). The left figure shows the results of fibronectin from a commercial company, and the right figure shows the fibronectin shown in the present invention.

[0029] Figure 6 These are the results of the thermal stability experiment of recombinant human fibronectin.

[0030] Figure 7 These are the results of the freeze-thaw stability experiment of recombinant human fibronectin.

[0031] Figure 8 The results are for detecting the proliferation activity of mouse fibroblast NIH 3T3 cells.

[0032] Figure 9 This figure shows the test results of the human keratinocyte cell proliferation activity test method.

[0033] Figure 10 This is the SDS-PAGE image of the fermentation optimization of recombinant human fibronectin. Lane 1 is the original sequence without codon optimization, lane 2 is the untagged sequence after codon optimization, lane 3 is the conventional fermentation result of TrxA tag; lane 4 is the optimized fermentation result of TrxA tag; lane 5 is the uninduced protein band.

[0034] Figure 11 This is a comparison of the purity of recombinant human fibronectin from different sources (SDS-PAGE). Lane 1 is the fibronectin obtained by other purification methods, lane 2 is the fibronectin of the present invention, and lane 3 is the fibronectin method of a commercial company. DETAILED DESCRIPTION

[0035] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0036] Test method:

[0037] 1. HPLC detection of fibronectin: The present invention refers to the Chinese Pharmacopoeia General Chapter 0512 High Performance Liquid Chromatography, the Guiding Principles of 0532 Critical Point Chromatography, and the industry standard YY / T 1805.3-2022 method, and selects appropriate SEC molecular sieves for detection; the mobile phase is 20 mM PBS aqueous solution; the flow rate is 0.5 ml / min.

[0038] 2. Protein Solubility Measurement: The protein solution content was measured using the Kjeldahl nitrogen method (Method 1, Method 1) as described in General Chapter 0731 of the Chinese Pharmacopoeia for Protein Content Measurement. The total amount of protein was calculated. The elastin solution was lyophilized using a freeze-drying apparatus, the solution was converted into a powder, and the protein powder was gradually dissolved in a small amount of water until it was completely dissolved. The ratio of the total protein content of the protein solution to the volume of dissolved solids obtained can be used to roughly determine the protein solubility.

[0039] Materials used in the examples:

[0040] 1. The plasmids and cells involved in the following examples are as follows:

[0041] strain E coliBL21(DE3) and plasmid pET32a(+) are commercial strains and plasmids.

[0042] 2. NIH 3T3 cells were from Science Compass, and HaCaT cells were from Lexus.

[0043] 3. The formula of TB medium is: 11.8 g tryptone, 23.6 g yeast extract, 9.4 g K2HPO4, 2.2 g KH2PO4, 4 ml glycerol, 996 ml water.

[0044] 4. 1640 culture medium is Sepunov RPMI-1640 culture medium.

[0045] 5. DMEM culture medium is Sepu DMEM culture medium.

[0046] Example 1 Sequence of recombinant human fibronectin

[0047] This embodiment provides a recombinant human fibronectin, whose amino acid sequence is shown in SEQ ID NO.1, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. The nucleotide sequence shown in SEQ ID NO.2 is subjected to genetic engineering codon optimization and optimized to a nucleotide sequence shown in SEQ ID NO.3, so that the amino acid sequence shown in SEQ NO.1 can be better expressed in the host bacteria. Different tag sequences are added to both sides of the nucleotide sequence shown in SEQ ID NO.3. The improvement of amino acid expression by adding different tag sequences is shown in Table 1. The different tagged proteins are predicted and analyzed by Rosetta. After comprehensive comparison, the preferred sequence is TrxA (containing a HIS tag sequence, as shown in SEQ ID NO.4).

[0048] Table 1

[0049]

[0050] Figure 1 The structure predictions for proteins under different tags are from the Rosetta website. The left figure shows the experimental results without tags, and the right figure shows the results with tags added. The results show that the proteins with tags have a more compact structure, which promotes protein stability. The TrxA tag cannot predict protein results, so there is no specific protein structure diagram. It is speculated that using this tag will produce very different results compared to traditional protein expression. The GST domain promotes the linearity of protein results, making the protein structure more independent and three-dimensional, increasing the possibility of expression. The protein structure prediction results are also consistent with the predictions in Table 1.

[0051] Example 2 Construction and screening of recombinant human fibronectin strains

[0052] The specific process is as follows:

[0053] (1) Plasmid synthesis: The gene encoding human fibronectin (as shown in SEQ ID NO.4) was subcloned into the NcoⅠ and KpnⅠ restriction sites of pET32a(+) to obtain the recombinant plasmid pET32a-FN. Figure 2 The recombinant plasmid shown has a full set of operational elements for gene expression and can express the target heterologous protein through the Lac operon. The protein after codon optimization has good compatibility with the host and can be expressed efficiently in the host cell. The electrophoresis diagram of the recombinant plasmid pET32a-FN is shown in Figure 3 shown.

[0054] (2) Host cell transformation: Transform the recombinant plasmid pET32a-FN and the successfully constructed expression plasmid into E. coli competent cells BL21 (DE3). The specific process is as follows:

[0055] Remove the EP tube containing competent E. coli BL21(DE3) cells from the ultra-low temperature freezer and place it on ice. When the cells are half-thawed, add 2 μl of the plasmid to be transformed to the competent E. coli BL21(DE3) cells and manually mix by inverting the EP tube 2-6 times. Place the mixture of recombinant plasmid pET32a-FN and BL21(DE3) competent cells on ice for 25-35 minutes, then heat shock in a 42°C water bath for 60-90 seconds. Remove the mixture and immediately place it on ice for 1.5-2.5 minutes. Transfer the transformed BL21(DE3) cells to a clean bench, add 700 μl of liquid SOB medium, and incubate in a constant temperature shaking incubator at 37°C, 220 rpm for 40-120 minutes. Spread 100-200 μl of the culture evenly on five SOB plates containing 50 μg / ml ampicillin sodium. The coated plate was placed upside down in a 37°C constant temperature incubator and cultured for 16-24 hours until monoclonal colonies clearly visible to the naked eye grew.

[0056] (3) Strain screening: 5-10 monoclonal strains were picked from the above plates and inoculated into TB medium shake flasks containing 100 μg / ml ampicillin sodium. The culture was incubated in a constant temperature shaker at 220 rpm and 37°C for 4-6 hours. The shake flask was then cooled to 25-28°C and IPTG was added to induce expression for 6-8 hours. The bacterial solution was divided into centrifuge bottles and centrifuged at 8500 rpm and 4°C for 10-15 minutes. The cells were collected and their weights were recorded. Samples were taken (2 ml of bacterial solution was taken every hour before and after induction and centrifuged at 8000 rpm for 2 minutes to obtain cells) for electrophoresis detection. The collected cells were resuspended in PB buffer (20 mMPB, pH 7.4) and disrupted by ultrasound. After completion, the disrupted bacterial solution was collected and centrifuged at 8000 rpm and 8°C for 45 minutes. The supernatant and precipitate were collected for electrophoresis detection. The electrophoresis results were scanned and analyzed, and a monoclonal strain with a higher expression level of recombinant human fibronectin was selected for strain preservation to obtain a genetically engineered strain expressing human recombinant fibronectin.

[0057] Example 3 Fermentation and optimization of recombinant human fibronectin strain

[0058] The fermentation and optimization of the strain were performed in a shake flask: 600 μl of ampicillin and 6000 μl of glycerol seed solution (genetically engineered bacteria obtained in Example 2) were pipetted into 600 ml of sterile TB medium, the bottle mouth was bandaged, and the mixture was placed in a shaker as a basic configuration; before induction, the mixture was cultured at 37°C until a specific OD 600 value.

[0059] The fermentation conditions were optimized as shown in Table 2, and the optimal expression conditions were screened using the single factor analysis method.

[0060] Table 2

[0061]

[0062] After optimization, the optimal expression conditions are as follows: culture at 37°C, 150 rpm for 6-8 h, OD 600 After reaching 2-3, IPTG was added to reach a final concentration of 0.5 mM / l, and induction was continued at 37 degrees for 5 hours; this was the end of induction. The expression level of fibronectin after optimization was tested, and the results were as follows Figure 4 As shown, from Figure 4 Comparison of lane 1 with the other lanes shows that IPTG-induced fibronectin expression gradually increases over time. Optimized expression yields a relatively high yield, with wet cell counts reaching 95 g / l.

[0063] Example 4 Purification of recombinant human fibronectin protein

[0064] The induced bacterial solution obtained in Example 3 was balanced and placed in a centrifuge, centrifuged at 6500 rpm for 35 min at a temperature of 8°C, and the bacterial precipitate was collected; the bacteria were resuspended in 50 mM PB (pH 8.0) and ultrasonically disrupted for 45 min, 3 s at intervals of 2 s, at a power of 455 watts, to release the recombinant protein from the bacteria. The disrupted bacteria were centrifuged to separate the protein supernatant and the precipitate, and the centrifugation conditions were 6500 rpm for 35 min at a temperature of 8°C.

[0065] Because the 6*HIS-histidine moiety of the TrxA tag on the vector binds strongly to a nickel column under appropriate conditions, affinity chromatography is used to separate impurities. Prior to chromatography, the supernatant should be filtered through a 0.45 μm or 0.22 μm filter. The chromatography buffer used for equilibration was 20 mM imidazole, 50 mM PB (pH 8.0), and the elution buffer was 500 mM imidazole, 50 mM PB (pH 8.0). Linear elution was performed, and peaks of appropriate molecular weight were selected by SDS-PAGE analysis for subsequent purification. After desalting the eluted peak, 0.01-1% EK enzyme was added to the system, and the reaction was incubated at 4°C overnight. The enzymatically digested protein solution was subjected to affinity chromatography again as described above, and the flow-through peak was collected. The flow-through peak was concentrated by ultrafiltration and stored in 50 mM PBS (pH 8.0).

[0066] The purity of commercially available fibronectin was tested by HPLC. The results were as follows: Figure 5 As can be seen from the figure, the fibronectin purified by the purification method of the present invention has a higher purity than the commercially available fibronectin.

[0067] The solubility and stability of the recombinant fibronectin obtained in this example were tested. Natural fibronectin is a water-insoluble protein. The concentration of commercially available recombinant human fibronectin is mostly at the level of 1000 ppm. The recombinant human fibronectin prepared in the present invention currently has a concentration greater than 8 mg / ml.

[0068] The fibronectin of this example has strong stability. The fibronectin of the fibronectin can maintain temperature stability for at least 6 hours in an 80-degree water bath environment. For detailed results, see Figure 6 In addition, the fibronectin of the present invention is freeze-thaw stable. With the addition of an appropriate amount of salt, it exhibits a characteristic opposite to the traditional fibronectin that is cold insoluble. It also maintains the stability of the aqueous solution at low temperatures. After repeated freezing and thawing, no bands with obvious molecular weight reduction appear within three times. The recombinant human fibronectin has freeze-thaw stability. For detailed results, see Figure 7 .

[0069] Example 5 Cellular Activity Detection of Recombinant Human Fibronectin Protein

[0070] NIH 3T3 cell proliferation assay:

[0071] Control group: commercially available product (sale-Fn), Aladdin full-length recombinant (rfull-Fn), catalog number: rp155944-200μg

[0072] Test sample: recombinant protein (rFn) obtained in Example 4

[0073] The four groups of samples were sample 1 (rFn), control 1 (sale-Fn), and control 2 (rfull-Fn);

[0074] Control 1 served as a negative control, and Control 2 served as a positive control. Appropriate amounts of each sample were weighed and dissolved in sterile PBS. Filtered through a 0.22 μM filter, the sample was serially diluted to 100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, 0.16 μg / ml, 0.032 μg / ml, and 0.0064 μg / ml. After mixing, the sample was added to a 96-well plate at 100 μl / well and coated at 37°C for 1 hour. Each well was coated in triplicate.

[0075] Pour out the liquid in the coated 96-well plate, add 200 μl sterile PBS to each well, wash twice, and set aside. Take NIH 3T3 cells in the logarithmic growth phase, count the cells, and adjust the cell concentration to 6×10 3 Inoculate 100 μL of culture medium / well into each treated 96-well plate and incubate in a 5% CO2, 37°C incubator for 60 hours. Remove the RMPI 1640 medium. Wash each well three times with PBS, then add 100 μL / well of culture medium containing 10% CCK-8. Incubate in a 5% CO2, 37°C incubator for 2 hours. Measure absorbance at 450 nm using a microplate reader.

[0076] The results of cell activity were as follows Figure 8 As shown in the figure, the cell proliferation activity results under the method of the present invention are compared with fibronectin from different sources. The results show that the fibronectin shown in the present invention has similar activity results to the recombinant full-length fibronectin (rice expression), the commercial fibronectin has no cell proliferation activity, and the control recombinant human fibronectin obtained from other sources also has no cell proliferation activity.

[0077] HaCaT cell proliferation activity detection;

[0078] Samples were diluted to appropriate concentrations (0.0008 μg / ml, 0.0016 μg / ml, 0.0032 μg / ml, 0.0064 μg / ml, 0.0125 μg / ml, 0.025 μg / ml, 0.05 μg / ml, and 0.1 μg / ml) in serum-free DMEM. Cells were seeded in 24-well plates and incubated overnight in an incubator (37°C, 5% CO₂, 95% RH). The experiment was divided into 10 groups: a blank control group received serum-free medium, a positive control group received serum-free medium containing DMSO, and an experimental group received serum at the aforementioned concentrations. When the cell plating rate in the 24-well plate reached approximately 80%, the drug was administered to each group, with triplicate wells per group. Cultures were incubated in an incubator (37°C, 5% CO₂, 95% RH) for 24 hours. The cells were washed three times with PBS, 200 μl of 0.5 mg / ml MTT was added, and the cells were incubated at 37°C for 4 h. The supernatant was discarded, and 200 μl of DMSO was added to measure the absorbance at 570 nm.

[0079] like Figure 9 As shown, the proliferation activity of HaCaT cells under the method of the present invention gradually increases with the increase of the concentration of the sample used.

[0080] Comparative Example 1

[0081] According to the method of Example 2, a genetically engineered bacterium containing a gene fragment having a nucleotide sequence such as SEQ ID NO. 2-3 was constructed, and protein expression was performed according to the optimal fermentation expression conditions of Example 3, and protein expression was detected by SDS-PAGE. Figure 10 As can be seen from the figure, the sequence without codon optimization has no expression or very low expression in the host, the expression level of the untagged protein in the body is low, and the fermentation yield of the optimized strain with tag is higher than that of the conventional fermentation with tag.

[0082] Comparative Example 2

[0083] The crude product obtained under the optimal fermentation expression conditions in Example 3 was purified using conventional purification methods, specifically as follows:

[0084] Traditional purification methods involve purification through a saturated ammonium sulfate gradient followed by molecular sieves or anion-cation exchange. Specifically, the saturated ammonium sulfate concentration is 33%, and the anion-cation exchange is strong anion exchange. The purified fibronectin obtained above, the purified fibronectin obtained in Example 4, and other commercially available fibronectin products were analyzed by SDS-PAGE.

[0085] like Figure 11As shown, the commercially available fibronectin has a higher purity after the purification method of the present invention than that obtained by other traditional purification methods.

[0086] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

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

1.

2. A gene encoding the protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria are host cells containing the gene described in claim 2 or transformed with the plasmid described in claim 3.

5. The genetically engineered bacterium according to claim 4, characterized in that: The host cell is Escherichia coli BL21 (DE3).

6. A method for preparing recombinant human fibronectin, characterized in that: The method comprises culturing the genetically engineered bacteria according to claim 4, inducing the bacteria to express recombinant fibronectin, disrupting the bacteria, and obtaining a crude recombinant fibronectin product.

7. The method according to claim 6, characterized in that The genetically engineered bacteria were inoculated in a liquid culture medium, cultured at 37°C and 220 rpm for 4-6 hours, 0.1-1 M IPTG was added, induced at 25-37°C and 220 rpm for 6-8 hours, centrifuged to remove the supernatant, ultrasonically disrupted the bacteria, and centrifuged to collect the supernatant to obtain a crude product.

8. The method according to claim 6, characterized in that The crude recombinant fibronectin product is purified to obtain a pure recombinant fibronectin product.

9. Use of the protein according to claim 1, or the gene according to claim 2, or the plasmid according to claim 3, or the genetically engineered bacteria according to claim 4 or 5, or the method according to any one of claims 6 to 8 in the preparation of a product containing fibronectin.

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