Recombinant human fibronectin as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses recombinant human fibronectin as well as a preparation method and application thereof. The amino acid sequence of the recombinant human fibronectin disclosed by the invention is as shown in SEQ ID NO. 1. The recombinant human fibronectin comprises three fragments which are derived from natural fibronectin and have high hydrophilicity and multiple integrin binding capacity, the fragments are expressed in escherichia coli, and the recombinant human fibronectin with high soluble expression quantity is obtained; the recombinant human fibronectin has high activity of promoting proliferation and migration of human keratinocytes, and has potential application prospects in the aspects of skin repair and wound healing.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, and in particular relates to a recombinant human fibronectin, its preparation method and application. Background Technology

[0002] Fibronectin is a high-molecular-weight, multi-domain glycoprotein widely distributed in the extracellular matrix, connective tissue, and plasma. Based on its source and form, fibronectin can be divided into plasma fibronectin and cellular fibronectin. Plasma fibronectin is mainly a soluble dimer protein synthesized by hepatocytes. The normal human plasma fibronectin content is approximately 0.3 mg / mL, and its main functions include maintaining normal plasma function, promoting coagulation, and promoting wound healing. Cellular fibronectin is mainly secreted by fibroblasts and endothelial cells, assembling into insoluble multimers in the extracellular matrix to form a fibrous network structure. Cellular fibronectin is one of the important structural proteins, whose main functions are maintaining tissue structure, regulating cell activity, and mediating processes such as extracellular matrix assembly, adhesion, migration, proliferation, differentiation, and tissue repair.

[0003] Fibronectin's dimer structure resembles the letter V, consisting of two similar subunits of approximately 250 kDa linked by a C-terminal disulfide bond. The primary structure of fibronectin contains three types of repeating modules: 12 type I repeats (FNI), 2 type II repeats (FNII), and 15 type III repeats (FNIII). It also contains three alternative splicing regions: EDA, EDB, and IICS(V). These modules assemble into multiple functional domains, enabling the binding of integrins, heparin, collagen, fibrin, and other proteins, thus endowing fibronectin with rich biological functions. The FNIII module lacks disulfide bonds and contains multiple integrin binding sites. The RGD (Arg-Gly-Asp) on the 10th FNIII module (FNIII-10) is a key site mediating fibronectin adhesion to the extracellular matrix. The RGD specifically recognizes integrin α5β1 and enhances its affinity for integrins through the cooperating site PHSRN on FNIII-9, thereby regulating cell adhesion and migration. EDA is upregulated during the wound repair process. It has more specific binding sites for various types of integrins, such as α4β1 and α9β1, thereby enhancing cell adhesion and migration and playing an important role in the tissue damage repair process.

[0004] Initially, fibronectin was primarily used in the medical field. In recent years, with the development of the concept of functional skincare, its unique role in beauty and skincare has been discovered. However, the extraction of natural fibronectin is complex and yields extremely low rates, resulting in limited production and high costs, thus restricting its large-scale application. Recombinant expression has become an inevitable trend in the preparation of human functional proteins. Currently, several research institutions and companies have developed recombinant fibronectin and successfully applied it to skincare products, medical dressings, and other products. Due to the modular structure of fibronectin and the generally well-defined functions of each domain, recombinant expression typically involves splicing, repeating, or directly expressing modules with known functions. However, this approach suffers from problems such as low expression levels and the easy formation of inclusion bodies. Summary of the Invention

[0005] To address the aforementioned issues, this application designs a recombinant human fibronectin and provides its preparation method and applications.

[0006] Technical solution: The recombinant human fibronectin described in this application comprises three highly hydrophilic fragments derived from natural fibronectin: a fragment of 749-769aa from FNIII-2, which is added to the N-terminus to potentially promote the soluble expression of the recombinant protein; fragments of 1592-1805aa from FNIII-10, FNIII-11, and EDA, which provide abundant integrin-binding ability for the recombinant protein; and a fragment of 1716-1823aa from EDA, which doubles the EDA derivative to further enhance the integrin-binding ability of the recombinant protein. The amino acid sequence is shown in SEQ ID NO.1.

[0007] This application also provides a fusion protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] This application also provides the nucleic acid sequence of the fusion protein, as shown in SEQ ID NO.3.

[0009] This application also provides a recombinant expression vector containing the aforementioned nucleic acid. The vector is pET-32a, preferably a pET-32a (+) vector with the resistance gene replaced by kanamycin (KanR).

[0010] The engineered bacteria or cells described in this application contain the expression vector. The host cell is Escherichia coli BL21(DE3).

[0011] The preparation method of recombinant human fibronectin described in this application includes the following steps: after optimizing the codons of the amino acid sequence, the ligation is performed on a vector for fusion expression; the recombinant plasmid is transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain; after culturing the recombinant strain, it is centrifuged and purified to obtain recombinant human fibronectin.

[0012] This application also provides the application of the recombinant human fibronectin in the preparation of cosmetics, skin care products, biomaterials, and medical devices.

[0013] The recombinant human fibronectin can promote the proliferation of mouse embryonic fibroblasts NIH / 3T3 and the proliferation and migration of human keratinocytes HaCaT, and can be applied to skin repair and wound healing.

[0014] Beneficial effects: The recombinant human fibronectin of this application contains three highly hydrophilic fragments derived from natural fibronectin, each with multiple integrin-binding capabilities. These fragments were expressed in Escherichia coli, resulting in a recombinant human fibronectin with high soluble expression levels. The recombinant human fibronectin exhibits high activity in promoting the proliferation and migration of human keratinocytes, and has potential applications in skin repair and wound healing. Attached Figure Description

[0015] Figure 1 This is an SDS-PAGE gel image of the fermentation broth of a recombinant human fibronectin-expressing strain; Figure 2 It is the proliferative activity of recombinant human fibronectin on mouse embryonic fibroblast NIH / 3T3 cells; Figure 3 It is the proliferative activity of recombinant human fibronectin on human keratinocytes (HaCaT). Figure 4 It is the migration activity of recombinant human fibronectin on human keratinocytes (HaCaT). Detailed Implementation

[0016] The technical solution of this application will be described in detail below with reference to specific embodiments.

[0017] Example 1: Design and strain construction of recombinant human fibronectin Fragments derived from natural human fibronectin were selected for expression, specifically from FINC_HUMAN (UniprotP02751). The selected sequence fragments were derived from FINC_HUMAN fragments 749-769aa, 1592-1805aa, and 1716-1823aa. These three fragments were sequentially ligated to obtain the target sequence, as shown in SEQ ID NO.1. After codon optimization, this amino acid sequence was ligated into a pET-32a (+) vector with the resistance gene previously replaced with kanamycin resistance (KanR) for fusion expression. The amino acid sequence of the fusion protein is shown in SEQ ID NO.2, with an N-terminal TrxA tag, 6×His, and TEV protease cleavage sites. The nucleic acid sequence of the fusion protein is shown in SEQ ID NO.3. The recombinant plasmid was transformed into *E. coli* BL21(DE3), plated on kanamycin resistance selection plates, and clones were selected for PCR identification and gene sequencing verification. Positive clones were the obtained recombinant human fibronectin expression strain.

[0018] SEQ ID NO.1 GFRVEYELSEEGDEPQYLDLPSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSQMQVTDVQDNSISVKWLPSSSSPVTGYRVTTTPKNGPGPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKI AWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTAIPAPTDLKFTQ SEQ ID NO.2 MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGENLYFQGFRVEYELSEEGDEPQYLDLPSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVTTTPKNGPGPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTAIPAPTDLKFTQ SEQ ID NO.3 Example 2 Expression and purification of recombinant human fibronectin

[0019] Single positive colonies were picked from the plate and inoculated into 4 mL LB medium (yeast extract 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L) containing 50 μg / mL kanamycin. The culture was incubated at 37°C and 200 rpm for 12 h to obtain a seed culture. The seed culture was then transferred to a 1 L LB medium shake flask containing the same concentration of antibiotics and incubated at 37°C and 200 rpm until OD reached. 600 When the pH reached 0.6–0.8, IPTG at a final concentration of 0.2 mM was added for induction expression at 16℃ for 12 h. The seed culture medium for the recombinant fibronectin expression strain in the fermenter was LB medium, and the fermentation medium was TB medium (24.0 g / L yeast extract, 12.0 g / L peptone, 0.4% (v / v) glycerol, 17 mM potassium dihydrogen phosphate, 72 mM dipotassium hydrogen phosphate). The inoculum size was 10%, and the initial fermentation conditions were: temperature 37℃, aeration rate 0.8 vvm, rotation speed 150 rpm, pressure 0.03–0.04 MPa, pH 7.0. After adding the inducer, the fermentation temperature was 20℃, and the rotation speed, aeration rate, and pressure were adjusted to maintain dissolved oxygen at 20–40% for 12 h.

[0020] After high-speed centrifugation of the fermentation broth, the bacterial cells were collected and resuspended in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM imidazole, 10% (v / v) glycerol, 0.1% (v / v) Tween 20). The cells were then homogenized using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation for SDS-PAGE electrophoresis and purification. The purification process involved mixing the supernatant with Ni... 2+ The medium was mixed and added to the chromatography column, where it was allowed to elute naturally under gravity. The column was washed with 10 volumes of lysis buffer, followed by elution at 300–400 mM imidazole. The eluent was collected. To further improve sample purity and remove impurities such as pigments, ion-exchange chromatography was used for purification. The equilibration conditions were 50–100 mM NaCl, pH 5.0–6.5, and the elution conditions were 200–300 mM NaCl, pH 5.0–6.5. The purified target protein solution was stored at low temperature for testing cell viability and efficacy.

[0021] SDS-PAGE gel image of the fermentation broth of the recombinant human fibronectin expression strain is shown below. Figure 1As shown in the figure, M represents the protein marker. Lanes 1 and 2 represent the soluble and inclusion body components induced by the expression strain at 16°C in shake flasks, respectively, with a loading volume of 20 μL for each. Lanes 3 and 4 represent the soluble components induced by the expression strain at 20°C in a 10L fermenter, with loading volumes of 5 μL and 10 μL, respectively. Lane 5 represents the inclusion body component induced by the expression strain at 20°C in a 10L fermenter, with a loading volume of 10 μL. Lanes 6 and 7 represent bovine serum albumin as a reference. It can be seen that recombinant fibronectin is mainly expressed in a soluble form, and the expression level in the 10L fermenter is approximately 10 times that in the shake flasks, indicating that this protein has good amplification potential.

[0022] Example 3: Proliferative Activity of Recombinant Human Fibroin in Mouse Embryonic Fibroblasts The recombinant fusion protein obtained in Example 2 was digested with TEV protease to remove the tag portion, and the recombinant human fibronectin from SEQ ID NO.1 was purified and recovered. This recombinant human fibronectin was 100% derived from natural human fibronectin and contained no heterologous components. This example investigated the proliferative activity of recombinant human fibronectin against mouse embryonic fibroblasts. Mouse embryonic fibroblasts (NIH / 3T3) (CRL-1658, ATCC) were seeded at a density of approximately 50% in 96-well plates, with 100 μL of cell suspension added to each well, and cultured overnight at 37°C. On day 2, purified samples were added in serially diluted amounts of 500 μg / mL, 250 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, and 0.1 μg / mL to wells containing NIH / 3T3 cell culture. Bovine type I collagen (batch number 380008-202001, 20 mg / vial, China National Institutes for Food and Drug Control) was added as a control. Each concentration was added in triplicate. After incubation at 37°C for 48 h, cell proliferation was detected using a CCK8 assay kit (A311-01, Vazyme), 10 μL per well. The absorbance was measured at 450 nm using an ELISA reader, which indirectly reflects the number of viable cells. The average values ​​were used to calculate the Cell viability plateau value and EC50 value. Cell viability is defined as the cell proliferation survival rate (i.e., fold increase) compared to the untreated control. EC50 refers to the drug concentration required for cells to proliferate to half of the highest plateau value.

[0023] Proliferating activity of NIH / 3T3 cells, such as Figure 2 As shown, recombinant human fibronectin has a significant pro-proliferative effect on NIH / 3T3 cells. Figure 2The calculated Cell viability plateau value and EC50 value were 1.45 and 2.84 µg / mL, respectively, and the Cell viability plateau value was higher than that of bovine type I collagen under the same conditions (1.20).

[0024] Example 4: Human keratinocyte proliferation activity of recombinant human fibronectin This embodiment measures the proliferative effect of recombinant human fibronectin at different concentrations on human keratinocytes (HaCaT).

[0025] Cell culture and drug administration: Human keratinocytes (HaCaT) in logarithmic growth phase (BNCC339817, Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains) were cultured at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 1 / mL in 96-well plates. The plates were then incubated in a 5% CO2 incubator at 37°C. After 24 hours, the culture medium was aspirated, and the cells were washed with PBS. Serum-free medium containing the sample was then added to each well to create a blank control, experimental control, and zero control. The experimental control wells were filled with samples containing different concentration gradients of the active ingredient, corresponding to final concentrations of 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, respectively. Each control group was in triplicate and incubated for another 24 hours.

[0026] Cell viability assay: Add 10 μL of CCK8 to 100 μL of culture medium in each 96-well plate and incubate in a CO2 incubator for 1 hour in the dark. Follow the CCK8 kit instructions (A311-01, Nanjing Novizan Biotechnology Co., Ltd.) for experimental procedures, and measure absorbance at λ=450 nm using a microplate reader.

[0027] Formula for calculating cell viability:

[0028] Experimental results are as follows Figure 3 As shown, HaCaT cells were incubated with recombinant human fibronectin at concentrations ranging from 12.5 to 50 μg / mL. The results showed that the cell proliferation-promoting effect of recombinant human fibronectin was concentration-dependent. Cell viability was increased by 36% at 50 μg / mL compared to the blank control group, by 25% at 25 μg / mL, and by 17% at 12.5 μg / mL.

[0029] Example 5: Human keratinocyte migration activity of recombinant human fibronectin This embodiment evaluates the effect of recombinant human fibronectin on the migration of human keratinocytes to the site of injury and its role in promoting wound healing by detecting the promoting effect of recombinant human fibronectin samples on cell scratching of human keratinocytes.

[0030] Cell culture and drug administration: Human keratinocytes (HaCaT) in logarithmic growth phase (BNCC339817, Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains) were cultured at a concentration of 2 × 10⁻⁶ cells / cells. 5 The cells were seeded at a density of 1 mL per well in 12-well plates, divided into a negative control group (0 h), a negative control group (24 h), and a sample group, with three replicates per group. Cells were cultured in DMEM complete medium until 70-80% confluence. The cell growth surface was carefully washed three times with PBS. 300 μL of PBS was added to each well. Using a 20 μL pipette tip perpendicular to the plate surface, a scratch was made from one end of the well to the other, ensuring the pipette tip was vertical and not tilted. The scratches were clearly visible on the cell surface. After scratching, the PBS was removed, and the cell surface was washed three times with fresh PBS to remove the scratched cells, leaving clearly visible gaps. Microscopic images were taken of the 0 h group. For the negative control group, 1 mL of DMEM (serum-free) was added. For the sample groups, 1 mL of DMEM containing the test sample (serum-free) was added, resulting in final concentrations of 50 μg / mL and 25 μg / mL, respectively. Cells were cultured for another 24 h.

[0031] Observation, photography, and analysis of experimental results: Photographs were taken to record changes in cell scratches in 12-well plates after incubation of cells and samples for 0 h and 24 h.

[0032] ImageJ was used to calculate the cell scratch area. The formula for calculating cell migration rate is:

[0033] The results are as follows Figure 4 As shown, recombinant human fibronectin significantly promotes cell migration. Cell migration is a method for measuring cell migration and repair capabilities, and is closely related to skin repair and wound healing. When the amount of recombinant fibronectin added was 50 μg / mL, the migration rate of HaCaT cells was 61.32%, and when the amount added was 25 μg / mL, the migration rate of HaCaT cells was 51.57%, which were 38% and 28% higher than the negative control, respectively. This indicates that recombinant human fibronectin has a significant promoting effect on the migration activity of human keratinocytes, suggesting that recombinant fibronectin has a potential role in skin repair and wound healing.

Claims

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

1.

2. A fusion protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A nucleic acid encoding the fusion protein of claim 2, the sequence of which is shown in SEQ ID NO.

3.

4. A recombinant expression vector, characterized in that, It contains the nucleic acid described in claim 3.

5. The recombinant expression vector according to claim 4, characterized in that, The carrier is pET-32a.

6. An engineered bacterium or cell, characterized in that, It contains the expression vector as described in claim 5.

7. The method for preparing recombinant human fibronectin according to claim 1, characterized in that, The process includes the following steps: optimizing the codons of the amino acid sequence described in claim 1, ligating it into a vector for fusion expression, transforming the recombinant plasmid into Escherichia coli to obtain a recombinant strain, culturing the recombinant strain, centrifuging, and separating and purifying it to obtain recombinant human fibronectin.

8. The use of the recombinant human fibronectin according to claim 1 in the preparation of cosmetics, skin care products, biomaterials, and medical devices.

9. The application according to claim 8, characterized in that, The recombinant human fibronectin can promote the proliferation of mouse embryonic fibroblasts NIH / 3T3 and the proliferation and migration of human keratinocytes HaCaT, and can be used for skin repair and wound healing.

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