Recombinant human fibronectin having complex function of soothing, preparation method therefor, and use thereof

Abstract

Provided are a recombinant human fibronectin (FN) having a complex function of soothing, a preparation method therefor, and the use thereof. Said recombinant human fibronectin has an amino acid sequence shown as SEQ ID NO.1. By adding a hyaluronic acid interaction segment of human fibronectin while retaining main segments of human fibronectin cell adhesion and integrin receptor interaction, and linking the two domains by means of a flexible "linker" amino acid sequence GGS, the recombinant human FN protein having the complex function is constructed. While possessing core characteristics of traditional recombinant fibronectins, the present recombinant human fibronectin also exhibits an effect of soothing cell irritations. Therefore, the prepared recombinant human fibronectin is expected to exhibit a good effect in soothing skin irritation in the field of functional active skincare products.

Description

A recombinant human fibronectin with soothing complex function, its preparation method, and its applications. Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a recombinant human fibronectin with a soothing complex function, its preparation method, and its application. Background Technology

[0002] Fibronectin (FN) is widely distributed in the animal kingdom (from freshwater sponges to humans). It is a large extracellular membrane protein on the cell surface and a major non-collagenous glycoprotein in the extracellular matrix and basement membrane. Its subunit molecular weight is 220-250 kDa, composed of approximately 2500 amino acid residues. It plays a central role in cell adhesion and regulates cell polarity, differentiation, and growth. It is widely distributed in the body: it exists in soluble form in plasma and various body fluids; and in soluble form in the extracellular matrix (including some basement membranes) and on the cell surface. The former is called plasma FN, and the latter is called cellular FN. Limited proteolytic cleavage can break it down into several domains, which can bind to plasma fibrin, heparin, collagen, and cell surface receptors. The N-terminal domain of FN protein contains a region that can bind to hyaluronic acid (HA), and this region also plays an important role in the binding of fibrin and heparin.

[0003] Natural fibronectin has a large molecular weight, composed of more than 2,000 amino acids, making it difficult for the skin to absorb. Therefore, genetic engineering techniques are used to select and express different functional domains of fibronectin (FN) to obtain recombinant fibronectin with different functions. Currently, recombinant fibronectin has excellent skin care effects and has broad application prospects in the medical, cosmetic, and medical fields. Technical issues

[0004] However, most of the current recombinant fibronectin sequences focus on the recombinant expression of the C-terminal cell adhesion-related domain of the protein. There are no recombinant fibronectins that have been successfully fused, expressed and purified with the N-terminal functional domain. Therefore, the functions of the currently synthesized recombinant proteins are limited, and there are few reported cases of enhancing the skin cell irritation relief effect of recombinant fibronectin through the function of the N-terminal domain. Technical solutions

[0005] To address the problems existing in the prior art, this invention utilizes techniques such as codon optimization and macromolecular protein solubility optimization to overcome the shortcomings of poor solubility in prokaryotic expression of macromolecular proteins and recombinant protein molecules containing the N-terminal functional domain of human fibronectin, and provides macromolecular recombinant human fibronectin with soothing complex function, its preparation method, and its applications.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] Firstly, a recombinant human fibronectin with a soothing complex function, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The recombinant human fibronectin has a molecular weight of approximately 95 kDa and includes the promoter corresponding amino acid, the human fibronectin fibrin and hyaluronic acid interaction region as shown in SEQ ID NO.2, and the human fibronectin cell adhesion-related and integrin receptor binding region as shown in SEQ ID NO.3. The protein domains at both ends of SEQ ID NO.2 and SEQ ID NO.3 are connected by the flexible protein "linker" amino acid motif GGS. The recombinant protein has a 6×His protein tag at its C-terminus for protein purification.

[0009] The recombinant fibronectin of this invention has a hyaluronic acid binding region and a segment related to the interaction between the extracellular matrix and cell integrin receptors. The finally purified recombinant human fibronectin contains 780 amino acids, has a molecular size of about 95 kDa, and a purity of more than 95%. This recombinant fibronectin has a significant promoting effect on the adhesion and proliferation of human keratinocyte HaCat cells, and at the same time has a significant soothing effect on SDS stimulation of keratinocytes.

[0010] Secondly, the coding sequence of a recombinant human fibronectin with a soothing complex function, after codon optimization, is shown in SEQ ID NO.4.

[0011] The coding sequence is a nucleotide sequence optimized by codons in a prokaryotic expression system. It contains the promoter ATG and is inserted into the pET28a prokaryotic expression vector at both ends of the sequence via restriction enzyme sites “NdeI” (CATATG) and “XhoI” (CTCGAG). The recombinant protein contains the nucleotide coding sequence “CACCACCACCACCACCAC” corresponding to the 6×His protein tag at the C-terminus, and the corresponding stop codon is TGA.

[0012] Thirdly, a method for preparing recombinant human fibronectin with a soothing complex function includes the following steps:

[0013] S1. Based on the amino acid sequence SEQ ID NO.1, codons for the E. coli expression system were optimized to generate the corresponding nucleotide sequence, and a protein expression vector was constructed.

[0014] S2. The expression vector was transferred into the Escherichia coli expression strain, and positive monoclonal colonies with high expression levels were screened, amplified, and a library was constructed. Positive monoclonal colonies were picked and inoculated into liquid culture medium for culture. After amplification overnight, the culture was amplified and protein expression was induced the next day. The bacterial cells were then collected by centrifugation.

[0015] S3. Lyse the bacterial cells with lysis buffer, centrifuge and filter, and then purify the protein;

[0016] S4. Purification and harvesting of proteins: After washing, filtration and concentration, recombinant human fibronectin stock solution with soothing complex function is harvested.

[0017] Furthermore, in step S2, 1-2% (w / v, mass / volume) of sorbitol is added to the liquid culture medium.

[0018] Furthermore, in step S2, the peptone used in the liquid culture medium is selected from Angel Yeast FP210, FP220, and / or FP108 peptone.

[0019] Furthermore, in step S2, positive monoclonal colonies are picked, inoculated into a suitable liquid culture medium, and cultured at 37°C for 10 hours. Then, they are inoculated into a new culture medium containing kanamycin at a 1:100 inoculation rate and cultured at 37°C for 3-4 hours. After that, an inducer is added, and the OD600 value during induction is 0.8-2.0. The induction temperature is set at 20-24°C, and the induction is carried out overnight.

[0020] Furthermore, the lysis buffer in step S3 is a 10-40 mM phosphate buffer with a sodium chloride concentration of 0-100 mM. 1%-2% arginine is also added to the lysis buffer and chromatography buffer to adjust the pH to 7.2-7.6.

[0021] Furthermore, the ratio of bacterial cells to lysis buffer for resuspension is 1:10-1:40 W / V, more preferably 1:20 W / V (i.e., 1g of bacteria is resuspended in 20mL of lysis buffer).

[0022] Furthermore, in step S3, protein purification refers to protein purification through affinity chromatography and anion exchange chromatography. The affinity chromatography packing material is Ni-NTA packing material with polyacrylate microspheres as the matrix. The elution conditions are a discontinuous gradient: equilibration with 20mM phosphate buffer, washing with 50mM-100mM imidazole buffer, and elution with 300mM imidazole buffer. A single purification can obtain high-purity rhHA-FN. Further, the ion exchange chromatography uses DEAE Sepharose FF with a sodium chloride concentration gradient for elution. Using anion exchange chromatography, preferably DEAE Sepharose FF packing material, for a second chromatography, a purity greater than 95% can be obtained, and the high concentration of imidazole in the first chromatography is almost completely removed.

[0023] Among them, Ni-NTA: agarose is a nickel-containing affinity resin that can be used to purify recombinant proteins containing multiple histidine (6xHis) sequences; DEAE Sepharose FF: anion exchange chromatography packing material is a weak anion exchange chromatography packing material used for rapid protein purification.

[0024] Furthermore, in step S4, a 30KD ultrafiltration membrane is used for solution concentration and replacement, which can further wash away small molecule impurities. Compared with single-step chromatography, the volume of liquid used for washing is greatly reduced after two-step chromatography, thus reducing protein loss during the washing process.

[0025] Fourthly, the application of the above-mentioned recombinant human fibronectin with soothing complex function or the recombinant human fibronectin with soothing complex function prepared by any of the above-mentioned methods in skin care products. Beneficial effects

[0026] By incorporating the hyaluronic acid-interacting domain of human fibronectin while retaining the main domains involved in cell adhesion and integrin receptor interaction, and connecting the two domains with a flexible "linker" amino acid sequence GGS, a recombinant human fibronectin with multiple functions is constructed. This recombinant human fibronectin, in addition to possessing the core characteristics of traditional recombinant fibronectin, also exhibits a soothing effect on cellular irritation. The recombinant human fibronectin prepared in this invention will have a good effect on soothing skin irritation in the field of functional active skincare products.

[0027] The recombinant human fibronectin with multiple functions obtained by the method of this invention has a molecular weight of approximately 95 kDa and a purity higher than 95%. Cell biology tests show that the recombinant human fibronectin with multiple functions provided by this invention has good activity and promotes cell adhesion and cell proliferation. Simultaneously, it exhibits good protective effects in the anti-SDS stimulation assay of keratinocytes.

[0028] This invention uses a culture medium formulation and a cell lysis buffer formulation for recombinant human fibronectin with multiple functions, which greatly improves the solubility of recombinant human fibronectin that was originally expressed mostly as polymers. This avoids the problem of low renaturation efficiency of large molecular recombinant protein inclusion bodies in industrial production. Attached Figure Description

[0029] Figure 1 is a schematic diagram of the gene structure and vector construction of recombinant human fibronectin (rhHA-FN) with soothing complex function;

[0030] Figure 2 shows the SDS-PAGE gel electrophoresis results of soluble proteins screened by peptone in the expression medium of recombinant human fibronectin with soothing complex function.

[0031] Figure 3 shows the SDS-PAGE gel electrophoresis results of soluble proteins screened for the timing of recombinant human fibronectin expression induction with soothing complex function.

[0032] Figure 4 shows the SDS-PAGE gel electrophoresis results of soluble proteins screened by sorbitol concentration in the recombinant human fibronectin expression medium with soothing complex function.

[0033] Figure 5 shows the SDS-PAGE gel electrophoresis results of soluble proteins screened by sodium chloride concentration in the recombinant human fibronectin bacterial cell lysate with soothing complex function.

[0034] Figure 6 shows the SDS-PAGE gel electrophoresis results of recombinant human fibronectin bacterial cell lysate with soothing complex function, which screened soluble proteins by pH value.

[0035] Figure 7 shows the chromatographic peaks of recombinant human fibronectin with soothing complex function under different chromatographic conditions;

[0036] Figure 8 shows the SDS-PAGE gel electrophoresis results of recombinant human fibronectin with soothing complex function.

[0037] Figure 9 shows the high-performance liquid chromatography (HPLC) results of the purity detection of recombinant human fibronectin with soothing complex function.

[0038] Figure 10 shows the cell adhesion promotion efficacy of three batches of recombinant human fibronectin samples with soothing complex function.

[0039] Figure 11 shows the cell proliferation efficacy detection of three batches of recombinant human fibronectin samples with soothing complex function;

[0040] Figure 12 shows the efficacy of recombinant human fibronectin with soothing complex in relieving SDS stimulation of keratinocytes.

[0041] Figure 13 shows the efficacy of recombinant human fibronectin with different concentrations of soothing complex in stimulating keratinocytes.

[0042] Figure 14 shows the efficacy of different concentrations of rhFN soothing SDS without HA binding region on keratinocyte stimulation;

[0043] Figure 15 shows the efficacy of different concentrations of recombinant human fibronectin (prepared by Chinese Invention Patent Publication No.: CN117586379A, invention title "A method for preparing and applying recombinant fibronectin") in relieving SDS stimulation of keratinocytes.

[0044] Figure 16 shows the efficacy of different concentrations of commercially available rhFN-1 soothing SDS on keratinocyte stimulation;

[0045] Figure 17 shows the efficacy of different concentrations of commercially available rhFN-2 soothing SDS on keratinocyte stimulation. Embodiments of the present invention

[0046] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0047] As used in this invention, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0048] The term "comprises" or "comprising" as used in this invention means "including, but not limited to," "including." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0049] The term "and / or" as used in this invention should be understood to mean any one of the options or any combination of two or more of the options.

[0050] Example 1: Design of a recombinant human fibronectin sequence with soothing complex function

[0051] A recombinant human fibronectin amino acid sequence containing the human FN protein hyaluronic acid interaction domain and the human FN protein cell adhesion and integrin receptor interaction domain was designed and named rhHA-FN, and its nucleotide sequence is shown in SEQ ID NO.1.

[0052] The sequence of the interaction region between human fibronectin, fibrin, and hyaluronic acid is shown in SEQ ID NO.2.

[0053] The sequence of the human fibronectin cell adhesion-related and integrin receptor binding region is shown in SEQ ID NO.3.

[0054] Example 2: Gene design and preparation method of recombinant human fibronectin with soothing complex function

[0055] The preparation method of recombinant human fibronectin with soothing complex function in this embodiment includes the following steps:

[0056] S1. Based on the amino acid sequence of Example 1, a gene sequence with codon optimization was designed. The optimized recombinant rhHA-FN protein coding sequence was synthesized by gene synthesis, with an NdeI restriction site added to the front of the sequence and an XhoI restriction site added to the end of the sequence. The sequence was cloned into the pET28a vector and sequenced. The vector structure is shown in Figure 1.

[0057] The gene coding sequence of recombinant human fibronectin with soothing complex function is shown in SEQ ID NO.4.

[0058] S2. Transform the correctly sequenced cloning vector into *E. coli* expression strain BL21-DE3. Pick positive single colonies and inoculate them into suitable liquid culture medium. Incubate at 37°C for 10 hours. Then, inoculate into fresh kanamycin-containing medium at a 1:100 ratio and incubate for 3-4 hours. When the OD600 value of *E. coli* reaches approximately 1.0, add 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) for induction. At this point, lower the temperature of the shaker or fermenter to 20°C and incubate overnight (approximately 8-10 hours). Collect the bacterial cells. For example, to prepare 1 L of suitable liquid culture medium, the formula is: 10 g FP-220 peptone, 5 g FM-888 yeast extract, 10 g NaCl, and 10 g sorbitol. Pour the culture medium into a 3 L shake flask with a baffle, autoclave, and then allow to cool to room temperature before use.

[0059] S3. Cell Lysis and Protein Chromatographic Purification: The lysis buffer was 20 mM phosphate buffer (PB) containing 100 mM sodium chloride and 1% arginine, with a pH of 7.4. Cells were homogenized and lysed with the lysis buffer at a ratio of 1:20 (w / v), then centrifuged to collect the supernatant for chromatographic purification. The clarified cell lysate was subjected to affinity chromatography using Ni-NTA packing material with polyacrylate microspheres as the matrix. Elution conditions were a discontinuous gradient: equilibration with 20 mM phosphate buffer, followed by washing with 5 mM-10 mM imidazole buffer and then elution with 300 mM imidazole buffer, yielding a relatively pure rhHA-FN protein peak. For rhHA-FN protein mixtures with low elution purity, anion exchange chromatography was performed using DEAE Sepharose FF with a sodium chloride concentration gradient. The resulting high-purity rhHA-FN protein was obtained through gel chromatography using the DEAE Sepharose FF ion exchange packing material.

[0060] S4. After chromatographic purification, the protein is washed, concentrated, and replaced using a 30KD ultrafiltration membrane to finally obtain recombinant human fibronectin rhHA-FN protein stock solution with soothing complex function.

[0061] Example 3: Effect of Peptone in Liquid Culture Medium on Expression Levels of Bacterial Strains

[0062] Different expression media were prepared using peptones of different types and sources (FP400, FP402, FP408, FP410, FP418, FP108, FP210, FP220, FP222, FP523, and FM885) according to the LB medium formulation. After resuscitation, *E. coli* transformed with the rhHA-FN plasmid were inoculated into different types of media at the same ratio and cultured at 37°C and 200 rpm for 3-4 hours. At this point, the OD value of the bacterial culture was approximately 0.8-2.0. Then, 0.5 mM IPTG was added and the culture was incubated overnight at 20°C. The bacteria were harvested the following day. The bacterial sludge was resuspended at a ratio of 1:10 (w / v) (1g of bacteria resuspended in 10mL of PB solution). Lysozyme was added to a final concentration of 2 mg / mL, and lysis was performed at 4°C for 2 hours, with occasional vortexing. Nuclease was added, and the mixture was treated at room temperature for 20 minutes. The mixture was then centrifuged at 12000 rpm for 5 minutes. The supernatant was collected, and an appropriate amount of SDS-PAGE (polyacrylamide gel electrophoresis) loading buffer was added. The mixture was then incubated in a boiling water bath for 5-10 minutes. SDS-PAGE gel electrophoresis was used to detect the target protein in the supernatant. As shown in Figure 2, after induction expression in FP220 medium, the amount of soluble rhHA-FN protein was the highest, while FP210, FP222, and FM885 showed relatively high amounts of soluble protein. Therefore, FP220 is the preferred peptone culture medium, with FP210, FP222, and yeast extract FM885 as alternatives.

[0063] Example 4: Effect of induction timing on protein solubility

[0064] Prepare LB medium, revive E. coli BL-21(DE3) containing the rhHA-FN plasmid, and incubate overnight at 37°C and 200 rpm. The next day, inoculate 1:100 into 100 mL of fresh medium containing kanamycin and incubate at 37°C and 200 rpm for 2.5 h. Samples are then collected to measure the OD600 value of the bacterial solution and to induce induction. Record the OD600 value of the bacterial solution at the time of collection, and take corresponding samples for induction. Collect the bacteria the next day. Resuspend the bacterial sludge at a ratio of 1:10 (w / v) (1 g of bacteria resuspended in 10 mL of PB solution), add lysozyme to a final concentration of 2 mg / mL, and lyse at 4°C for 2 hours, vortexing intermittently. Add nuclease and treat at room temperature for 20 minutes. Centrifuge at 12000 rpm for 5 minutes, collect the supernatant, add an appropriate amount of SDS-PAGE loading buffer, and incubate in a boiling water bath for 5-10 minutes. Detect the target protein in the supernatant using SDS-PAGE gel electrophoresis. As shown in Figure 3, the protein content of soluble rhHA-FN after induction was detected. It can be clearly seen that when the OD600 value of the bacterial sample was around 1.372 during induction, the expression level of soluble protein (in the supernatant) was significantly higher than at other induction times.

[0065] Example 5: Effect of sorbitol on expression levels in liquid culture medium of bacterial strains

[0066] The rhHA-FN inoculation and bacterial lysis methods in this embodiment are basically the same as those in Example 3, except that FP-220 peptone was used in the LB medium, and 1% or 2% (w / v) sorbitol was added to the medium for comparison with no sorbitol added. After lysis, the supernatant was taken for SDS-PAGE gel electrophoresis for detection. As shown in Figure 4, the medium with added sorbitol can effectively improve the expression of the target protein and the concentration of the target protein in the soluble supernatant, with the medium containing 2% sorbitol showing the best effect.

[0067] Example 6: Effect of sodium chloride concentration in lysis buffer on protein purification

[0068] The rhHA-FN inoculation and bacterial lysis methods in this embodiment are basically the same as those in Example 5. The difference lies in the concentration of sodium chloride (NaCl) in the sodium chloride-containing phosphate-buffered saline (PBS) during bacterial cell lysis: 0, 50, 100, 250, and 500 mM. After cell lysis, the supernatant was collected for SDS-PAGE gel electrophoresis detection, as shown in Figure 5. When the NaCl concentration was 0, the soluble protein concentration was the highest, while the soluble protein content was relatively ideal between NaCl concentrations of 0-100 mM. Considering that NaCl can reduce non-specific binding during chromatography to a certain extent, the NaCl concentration in the chromatography buffer was 0-100 mM.

[0069] Example 7: Effect of lysis buffer pH on protein purification

[0070] The rhHA-FN inoculation and bacterial lysis methods in this embodiment are basically the same as those in Example 5. The difference is that during bacterial cell lysis, the buffer does not contain sodium chloride, and the pH values ​​of the buffer are adjusted to 7.0, 7.4, 7.8, and 8.2, respectively. After bacterial cell lysis, the supernatant is taken for SDS-PAGE gel electrophoresis detection. As shown in Figure 6, it can be seen that the rhHA-FN content in the supernatant is highest at a pH of around 7.4, followed by a lower content at pH 7.8. Therefore, the pH of the chromatography buffer should be around 7.2-7.6.

[0071] Example 8: The effect of chromatography buffer on protein purification

[0072] The method for preparing rhHA-FN-inoculated protein in this embodiment is basically the same as that in Example 2, except that 1% arginine is added to both the lysis buffer and the chromatography buffer formulations, or arginine is not added to either the lysis buffer or the chromatography buffer formulations. Two consecutive Ni-NTA affinity chromatography analyses were performed, with a sample loading volume of 300 mL each time. A discontinuous gradient elution was performed using chromatography buffer containing imidazole, with imidazole concentrations of 100 mM, 200 mM, and 500 mM in phosphate buffer at peak elution, followed by washing with 2 M sodium chloride. As shown in Figure 7, under the chromatography conditions containing arginine, the peak elution was higher than under the chromatography conditions without arginine, and the collected liquid volume containing the target protein was larger and at a higher concentration.

[0073] Examples 3-8 above further validate the results through experimental data: the culture medium formulation and the bacterial cell lysis buffer formulation that are most suitable for the recombinant human fibronectin with multiple functions in this invention are selected, which greatly improves the solubility of the recombinant protein that was originally expressed mostly as inclusion bodies.

[0074] Comparative Example 1

[0075] A vector was constructed to express and purify the recombinant FN protein without the HA-binding region, which was named rhFN-HA-free region.

[0076] Comparative Example 2

[0077] Recombinant fibronectin containing fibronectin type III domains 8-10 was prepared according to the patent "A method for preparing and applying recombinant fibronectin" (Chinese Invention Patent Publication No.: CN117586379A).

[0078] The following examples are primarily used to verify the cell biological efficacy of rhHA-FN harvested by the methods in Examples 2-8 and the recombinant fibronectin in Comparative Examples 1-2.

[0079] Example 1: Expression and purity detection of recombinant human fibronectin with soothing complex function.

[0080] SDS-PAGE gel electrophoresis was used to detect protein expression and chromatographic purification. The specific steps were as follows: 5×SDS loading buffer was added to the bacterial sample lysis supernatant or the protein sample after chromatography. After mixing, the sample was heated at 95-98℃ for 10 minutes and briefly centrifuged. The protein was separated by electrophoresis using a prepared 10% SDS-PAGE gel. The results are shown in Figure 8, indicating that the chromatographically purified sample had high purity.

[0081] The purity of the harvested recombinant protein stock solution was determined by high-performance liquid chromatography (HPLC). The specific steps are as follows: After washing and filtration, the concentrated protein sample was filtered through a 0.22 μm filter membrane and then added to a sample vial for automated loading. The results are shown in Figure 9. A significant single main peak is visible in the chromatogram. Peak analysis shows that the area of ​​this single peak accounts for approximately 97.616%, indicating that the purity of the recombinant protein rhHA-FN is greater than 97%.

[0082] Example 2: Detection of the cellular biological activity of recombinant human fibronectin with soothing complex function.

[0083] Test 1: Detection of HaCat adhesion in human keratinocytes

[0084] In Example 1, recombinant human fibronectin rhHA-FN with soothing complex function was harvested and used to treat cell culture plates at a concentration of 20 μg / mL, with PBS treatment as a control. Each group had 6 replicates to observe the effect of FN on cell adhesion properties. The specific steps were as follows: 96-well plates were coated with 20 μg / mL of recombinant protein rhHA-FN and treated at room temperature for 1 hour. After removing the protein solution, the plates were blocked with 1% sterile bovine serum albumin (BSA) at room temperature for half an hour. HaCat cells were resuspended in serum-free medium, and 20,000 cells were seeded per well of the 96-well plate. After incubation at 37°C for 2 hours, each well was washed 3 times with PBS, and complete culture medium was added. Cell adhesion was observed and photographed. After overnight incubation, cells were incubated with a cell proliferation kit (CCK8) for 2-4 hours, and cell viability was detected using a microplate reader. The results are shown in Figure 10, indicating that rhHA-FN significantly promoted cell adhesion.

[0085] Test 2: Detection of the effect of promoting the proliferation of human keratinocytes (HaCat).

[0086] HaCat cells were seeded in 96-well plates at 4000 cells per well. Recombinant human fibronectin rhHA-FN, which possesses a soothing complex function and was obtained in Example 1, was added to the 96-well plates at a final concentration of 200 μg / mL. The control group consisted of an equal volume of PBS mixed with cell culture medium. Each group had 6 replicates. After culturing for 48 hours, CCK8 was added and incubated for 2-4 hours. Cell viability was then assessed using a microplate reader. As shown in Figure 11, rhHA-FN significantly promoted HaCat cell proliferation.

[0087] Test 3: Detection of the alleviating effect of recombinant fibronectin rhHA-FN on SDS stimulation assay in human keratinocytes.

[0088] HaCat cells were prepared into a cell suspension and seeded into 96-well plates, approximately 10,000 cells per well, and cultured for 18-24 hours. The original culture medium in each well was discarded, and MEM medium containing 0.01% SDS and 1% fetal bovine serum (FBS) was added to each well. An untreated blank control was retained. The cells were incubated at 37°C for 30 minutes. The culture medium in each well was then discarded, and the cells were washed once with PBS. The model group was added to MEM medium containing 1% FBS, the positive control group to MEM medium containing dexamethasone and 1% FBS, and the treatment group to MEM medium containing 200 μg / mL or 100 μg / mL rhHA-FN and 1% FBS. The cells were incubated at 37°C for approximately 24 hours. Then, a certain amount of MTT maintenance medium was added to each well, and the cells were incubated for 3-4 hours. The liquid was removed, and 100 μL of DMSO was added to each well. The mixture was shaken for 10-15 minutes, and the absorbance was measured at 570 nm using a microplate reader. As shown in Figure 12, the cell viability of the rhHA-FN protein experimental group was significantly improved compared with the model group, and it was basically able to reach the level of the positive group treated with dexamethasone. This indicates that the recombinant rhHA-FN protein helps keratinocytes resist SDS stimulation.

[0089] Test 4: Detection of the soothing effect of various recombinant fibronectins on SDS-stimulated human keratinocytes.

[0090] The recombinant fibronectin rhHA-FN prepared by the method described in Example 2, along with Comparative Examples 1 and 2, and two commercially available recombinant human fibronectins, commercially available rhFN-1 (rice cells expressing full-length FN) and commercially available rhFN-2 (GMP grade 65kDa), were simultaneously subjected to SDS stimulation experiments on human keratinocytes. Each well of cells was added to MEM medium containing 0.01% SDS and 1% FBS, and an untreated blank control was left. The cells were incubated at 37°C for 30 min. The culture medium in the wells was discarded, and the cells were washed once with PBS. The model group was added to MEM medium containing 1% FBS, and the treatment group was added to MEM medium containing the recombinant protein described above, containing 1% FBS. The concentration gradients of the recombinant fibronectin were 10, 25, 50, 100, and 200 ug / mL, with 5 replicates per group. The cells were incubated at 37°C for approximately 24 hours, followed by incubation with CCK8 for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the concentration gradient was used to calibrate the curves. Figures 13-17 show, in order, the efficacy of recombinant human fibronectin with combined soothing function, recombinant human fibronectin without a HA-binding region, recombinant human fibronectin prepared according to the patent "A Method for Preparing and Applying Recombinant Fibronectin" (Chinese Invention Patent Publication No.: CN117586379A), commercially available rhFN-1 protein, and commercially available rhFN-2 protein in soothing SDS-induced keratinocyte stimulation experiments. It can be seen that the protein prepared in this invention has the best effect at different concentrations.

Claims

1. A recombinant human fibronectin with a soothing complex function, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The recombinant human fibronectin with a soothing complex function according to claim 1, characterized in that, The recombinant human fibronectin has a molecular weight of 95 kDa.

3. The coding sequence of recombinant human fibronectin with a soothing complex function as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

4.

4. A method for preparing recombinant human fibronectin with a soothing complex function as described in claim 1, characterized in that, It includes the following steps: S1. Based on the amino acid sequence SEQ ID NO.1, codons for the E. coli expression system were optimized to generate the corresponding nucleotide sequence, and a protein expression vector was constructed. S2. The expression vector was transferred into the E. coli expression strain and cultured in liquid culture medium; then, after induction, the bacterial cells were collected. S3. Lyse the bacterial cells with lysis buffer, centrifuge and filter, and then purify the protein; S4. Purification and harvesting of proteins: After washing, filtration and concentration, recombinant human fibronectin stock solution with soothing complex function is harvested.

5. The method for preparing recombinant human fibronectin with a soothing complex function according to claim 4, characterized in that, In step S2, 1-2% W / V sorbitol is added to the liquid culture medium.

6. A method for preparing recombinant human fibronectin with a soothing complex function according to claim 4 or 5, characterized in that, In step S2, the peptone used in the liquid culture medium is Angel Yeast FP210, FP220 and / or FP108 peptone, with an inoculum ratio of 1:100; the OD600 value during induction is 0.8-2.0; the induction temperature is set at 20-24℃, and induction is carried out overnight.

7. The method for preparing recombinant human fibronectin with a soothing complex function according to claim 4, characterized in that, The lysis buffer in step S3 is a 10-40 mM phosphate buffer with a sodium chloride concentration of 0-100 mM.

8. The method for preparing recombinant human fibronectin with a soothing complex function according to claim 7, characterized in that, In step S3, 1%-2% arginine is added to the lysis buffer and chromatography buffer to adjust the pH to 7.2-7.

6.

9. The method for preparing recombinant human fibronectin with a soothing complex function according to claim 4, characterized in that, In step S3, protein purification refers to protein purification through affinity chromatography and anion exchange chromatography. The affinity chromatography packing material is Ni-NTA packing material with polyacrylate microspheres as the matrix material. The elution conditions are a discontinuous gradient, followed by equilibration with 20mM phosphate buffer, washing with 5mM-10mM imidazole buffer, and elution with 300mM imidazole buffer. The anion exchange chromatography uses DEAE Sepharose FF with a sodium chloride concentration gradient for elution.

10. The application of recombinant human fibronectin with a soothing complex function as described in claim 1, or recombinant human fibronectin with a soothing complex function prepared by any of the preparation methods described in claims 4-9, in skin care products.

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