A recombinant fibronectin-collagen-mussel adhesive protein fusion protein and its application

Abstract

This invention belongs to the field of genetic engineering and discloses a recombinant fibronectin-collagen-mussel adhesive protein fusion protein. The fusion protein is characterized by a structure of: fibronectin functional region - human collagen functional region - mussel adhesive protein. The fusion protein structure includes multiple fibronectin functional regions, human collagen functional regions, and mussel adhesive protein, wherein the mussel adhesive protein is selected from one or more combinations of Mfp-1, Mfp-3, Mfp-5, and Mfp-6. The fusion protein of this invention can promote cell adhesion and cell proliferation and possesses excellent whitening, anti-inflammatory, moisturizing, repairing, and sensitive skin repair functions. It can be applied to the internal and external microenvironments of the human body via topical application or microneedling, and has broad application prospects in the fields of tissue repair and biomedical materials.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a recombinant fibronectin-collagen-mussel adhesive protein fusion protein and its applications. Background Technology

[0002] Fibronectin can be divided into soluble plasma fibronectin (pFN) and insoluble cellular fibronectin (cFN). pFN is mainly synthesized by hepatocytes, while cFN is synthesized locally by cells in the extracellular matrix, and is particularly abundant in the extracellular matrix of embryos and regenerating or damaged tissues. Fibronectin comprises several modules, including 12 type I repeat units, 2 type II repeat units, and 15-17 type III repeat units. These units sequentially form structural and functional domains, mediating binding to cell surface receptors or other ECM molecules. The multi-module structure and inter-module regions give fibronectin (FN) molecules flexibility, participating in the regulation of their function. As an essential high-molecular-weight glycoprotein, fibronectin is widely present in tissues and tissue fluid. In the body's life activities, it participates in cell migration, adhesion, proliferation, hemostasis, tissue repair, and embryonic development, and has the function of a growth factor.

[0003] Collagen, a key structural component of the extracellular matrix, is the most abundant and widely distributed functional structural protein in mammals, accounting for about one-third of total protein. It is widely found in tissues such as skin, bone, tendons, ligaments, and mucous membranes. Humans have 44 collagen genes, encoding 44 α-chains and 28 types of collagen. Collagen plays a vital supporting and elastic role in skin, bones, and joints. With age, collagen levels gradually decline, which is one of the important reasons for skin aging.

[0004] Mussel adhesive proteins are natural adhesion systems secreted by the byssal glands of mussels, and are high-molecular-weight proteins rich in dopa (DOPA) groups. Mussel byssal threads are divided into proximal byssal lines and distal byssal discs, secreting six types of proteins (Mfp-1 to Mfp-6). Among these, Mfp-5 has the highest DOPA content (approximately 30%), located at the bottom layer of the byssal disc, directly adhering to solid surfaces and playing a primary adhesive role. Mussel adhesive proteins also possess cell adhesion and proliferation functions—promoting wound healing and recovery. Therefore, mussel adhesive proteins have potential repair functions for wounds on skin, mucous membranes, nerves, bones, and vascular membranes. Thus, mussel adhesive proteins show broad application prospects in medical bioadhesives, wound repair materials, implant coatings, anti-corrosion coatings, and skin barrier repair.

[0005] Most existing products express single recombinant fibronectin, human collagen, or naturally extracted mussel adhesive protein. Fibronectin is a cold-insoluble protein and is unstable at low temperatures. Recombinant collagen functional fragments or lack a complete triple helix structure, resulting in poor thermal stability and easy degradation, which limits its use and efficacy. When extracting mussel adhesive protein naturally, it usually requires about 10,000 mussels to obtain 1 mg of protein. The inefficiency and high cost of this process greatly limit the application of mussel adhesive protein. There are currently no products on the market that are temporarily expressed recombinant fibronectin-collagen-mussel adhesive protein fusion proteins. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a recombinant fibronectin-collagen-mussel adhesive protein fusion protein and its application.

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides a recombinant fibronectin-collagen-mussel adhesive protein fusion protein, the structure of which is: fibronectin functional region - human collagen functional region - mussel adhesive protein, the structure of which includes a plurality of fibronectin functional regions, human collagen functional regions and mussel adhesive protein, wherein the mussel adhesive protein is selected from one or more combinations of Mfp-1, Mfp-3, Mfp-5 and Mfp-6.

[0009] Optionally, the fibronectin functional region is selected from one or more combinations of FN III 1-15 units.

[0010] Optionally, the fibronectin functional region is selected from the sequence of human fibronectin III 9-11 units.

[0011] Optionally, the human collagen functional region can be selected from one or more combinations of human type I, type II, and type III collagen functional regions.

[0012] Optionally, the structure of the fusion protein includes two repeated mussel adhesive protein sequences, an integrin-binding region of a human collagen functional region, and a sequence of human fibronectin III 9 units.

[0013] Optionally, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.1 or SEQ ID NO.1.

[0014] In a second aspect, the present invention provides a nucleic acid encoding the fusion protein described in the first aspect, the sequence of which is shown in SEQ ID NO.3.

[0015] Thirdly, the present invention provides an expression vector containing the nucleic acid described in the second aspect, wherein the expression vector is selected from pBAD series vectors, pQE series vectors, pMAL series vectors or cold-induced pCold series vectors.

[0016] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect and / or the expression vector described in the third aspect.

[0017] Fifthly, the present invention provides the application of the recombinant fibronectin-mussel adhesive protein fusion protein described in the first aspect in the preparation of whitening and anti-inflammatory skin care products.

[0018] The beneficial effects of this invention are:

[0019] The fusion protein of this invention can promote cell adhesion and cell proliferation and has excellent functions such as whitening, anti-inflammation, moisturizing, repairing and sensitive skin repair. It can be applied to the internal and external microenvironment of the human body by means of application or microneedling, and has broad application prospects in the fields of tissue repair and biomedical materials. Attached Figure Description

[0020] Figure 1 The pFCM3 expression vector map.

[0021] Figure 2 The pFCM5 expression vector map.

[0022] Figure 3 This is due to the inhibitory effect on the inflammatory factor NO.

[0023] Figure 4 It has an inhibitory effect on the inflammatory factor IL-8.

[0024] Figure 5 SDS-PAGE detection of recombinant FCM3 and FCM5 fusion proteins.

[0025] Figure 6 The intensity of melanin signal in the head of zebrafish.

[0026] Figure 7 This refers to the melanin protein content in the head of a zebrafish. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

[0028] All reagents used in the following examples were analytical grade. Molecular cloning-related enzyme reagents were purchased from New England Biolabs. The *E. coli* strains were preserved in our laboratory. Gene synthesis, primer synthesis, and gene sequencing were performed by Suzhou Genewiz Biotechnology Co., Ltd. Unless otherwise specified in the examples, standard conditions or manufacturer-recommended conditions were followed. Reagents and instruments whose manufacturers are not specified are commercially available products. Molecular cloning experimental procedures not described in detail in the examples are based on *Molecular Cloning: A Laboratory Manual (4th Edition)* (edited by M.R. Green and J. Sambrook, translated by He Fuchu, Beijing: Science Press, 2017).

[0029] Example 1 (Construction and expression of recombinant FCM-5 fusion protein in E. coli, including expression vector and strain)

[0030] This embodiment utilizes a cold shock expression system, employing the CspA cold shock promoter to specifically, slowly, and in low-level express the target protein under low-temperature conditions. Coupled with a cold-inducible molecular chaperone, FCM-5 is slowly folded during synthesis, inhibiting inclusion body formation at its source. The sequence of human fibronectin III 9 units, the functional region of human collagen (integrin-binding region), and two repeat sequences of mussel adhesive protein mfp-5 were selected. The amino acid sequence of the fusion protein is SEQ ID NO.1. The coding gene was outsourced for optimized synthesis. NdeI and HindIII restriction enzyme sites were introduced on both sides of the coding gene to facilitate gene cloning and expression.

[0031] The amino acid sequence of the fusion protein, SEQ ID NO.1, is shown below:

[0032] ptdlrftnigpdtmrvtwapppsidltnflvryspvkneedvaelsispsdnavvltnllpgteyvvsvssvyeqhestplrgrqktgldspGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGERGETGPPGPAGFPGAPGQNGEPGGKGERG APGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKA RKYHRKGYKKYYGGGSSSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSS*.

[0033] Its nucleic acid sequence SEQ ID NO.3 is shown below:

[0034]

[0035] Vector and exogenous fragment digestion: The empty expression vector pCold II plasmid was extracted and digested with enzymes. The digestion reaction system consisted of 84 μL pCold II, 3 μL NdeI, 3 μL HindIII, and 10 μL 10*cutsmart. Digestion was carried out at 37°C for 1.5 h. Separation was achieved by 1.0% agarose gel electrophoresis, with approximately 4.4 kb of the vector band excised from the gel. Purification was performed using a gel extraction kit. The gene-encoding plasmid was extracted and digested with enzymes. The digestion reaction system consisted of 59 μL gene, 2 μL NdeI, 2 μL HindIII, and 7 μL 10*cutsmart. Digestion was carried out at 37°C for 1.5 h. Separation was achieved by 1.0% agarose gel electrophoresis, yielding approximately 1 kb of band. The band was excised from the gel and recovered using a gel extraction kit.

[0036] Ligation of the vector with the exogenous fragment: The ligation system consisted of 1 μL of the pCold II fragment digested with NdeI / HindIII, 2 μL of the synthesized gene fragment digested with NdeI / HindIII, 0.5 μL of T4 ligase, 1 μL of 10* ligase buffer, and purified water to a final volume of 5.5 μL. The mixture was digested at 25°C for 0.5 h to obtain the ligation product. The ligation product was transformed into DH5α to obtain the recombinant expression vector, named pFCM5. The pFCM5 recombinant expression vector map is shown below. Figure 2 .

[0037] The host cells were Escherichia coli Stbl3 competent cells. The transformation method was as follows: the competent cells were taken out of the -80℃ freezer and placed on ice. After the competent cells thawed, about 100-200 ng of pFCM5 was added. The cells were slowly stirred with a pipette tip and then placed on ice for 15 minutes. The cells were then heat-shocked at 42℃ for 90 seconds, placed on ice for 3 minutes, and 500 μL of LB medium was added. The cells were incubated at 37℃ and 220 rpm for 45 minutes. 50 μL of the incubator was evenly spread on LB solid medium containing 50 μg / mL Amp and cultured upside down overnight to obtain single clones. After expansion, the cells were stored at -80℃ with 20% final concentration glycerol. After sequencing analysis confirmed that the cells were correct, the recombinant expression transformant was obtained and named FCM5.

[0038] 5 μL of glycerol bacteria from the recombinant expression transformant FCM5 was inoculated into 5 mL of LB liquid medium containing 50 μg / ml Amp and cultured at 37°C and 220 rpm for 16 h. The bacterial culture was then inoculated into LB liquid medium containing 50 μg / ml Amp at a bacterial-to-medium ratio of 1:100 (v:v) and cultured at 37°C and 220 rpm until the OD600 reached approximately 0.8. The culture was then cooled to 15°C and incubated for 10 min. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2 mM, and induction was performed at 15°C for 20 h. After fermentation, the cells were collected, resuspended in 1 / 2 volume of 50 mM Tris (pH 8.5), and the cells were sonicated to disrupt the cell structure. The supernatant was collected by centrifugation and analyzed by SDS-PAGE. Figure 5 Soluble expression of the FCM-5 fusion protein can be achieved using a cold shock expression system.

[0039] Example 2 (Construction and expression of recombinant FCM-3 fusion protein in E. coli, including expression vector and strain)

[0040] In this embodiment, the sequence of human fibronectin III 9 units, the functional region of human collagen (integrin binding region), and two repeat sequences of mussel adhesive protein mfp-3 were selected. The amino acid sequence of the fusion protein is SEQ ID NO.2. The coding gene was outsourced for optimized synthesis. NdeI and HindIII restriction enzyme sites were introduced on both sides of the coding gene to facilitate gene cloning and expression.

[0041] The amino acid sequence of the fusion protein, SEQ ID NO.2, is shown below:

[0042] ptdlrftnigpdtmrvtwapppsidltnflvryspvkneedvaelsispsdnavvltnllpgteyvvsvssvyeqhestplrgrqktgldspGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGERGETGPPGPAGFPGAPGQNGEP GGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERNNISVAVLVALVLIGSFAVQSDAADYYGPKYGPPRRYGGGNYNRYGRRYGGYKGWNNGWKRGRWGRKYYNNISVAVLVALVLIGSFAVQSDAADYYGPKYGPPRRYGGGNYNRYGRRYGGYKGWNNGWKRGRWGRKYY*.

[0043] Its nucleic acid sequence SEQ ID NO.4 is shown below:

[0044]

[0045] Vector and exogenous fragment digestion: The empty expression vector pET28a-EGFP plasmid was extracted and digested with enzymes. The digestion reaction system consisted of 84 μL pET28a-EGFP, 3 μL NdeI, 3 μL HindIII, and 10 μL 10*cutsmart. Digestion was carried out at 37℃ for 1.5 h, followed by separation by 1.0% agarose gel electrophoresis. Approximately 5 kb of the vector was excised from the gel and purified using a gel extraction kit. The gene-encoding plasmid was extracted and digested with enzymes. The digestion reaction system consisted of 59 μL gene, 2 μL NdeI, 2 μL HindIII, and 7 μL 10*cutsmart. Digestion was carried out at 37℃ for 1.5 h, followed by separation by 1.0% agarose gel electrophoresis, yielding approximately 1 kb of band. This band was excised from the gel and recovered using a gel extraction kit.

[0046] Ligation of the vector with the exogenous fragment: The ligation system consisted of 1 μL of the digested (NdeI / HindIII) pET28a-EGFP fragment, 2 μL of the synthesized gene fragment digested (NdeI / HindIII), 0.5 μL of T4 ligase, 1 μL of 10* ligase buffer, and purified water to a final volume of 5.5 μL. The mixture was digested at 25°C for 0.5 h to obtain the ligation product. The ligation product was transformed into DH5α to obtain the recombinant expression vector, named pFCM3. The pFCM3 recombinant expression vector map is shown below. Figure 1 .

[0047] The host cells were Escherichia coli BL21(DE3) competent cells. The transformation method was as follows: the competent cells were taken out of the -80℃ freezer and placed on ice. After the competent cells thawed, about 100-200 ng of pFM3 was added. The cells were slowly stirred with a pipette tip and then placed on ice for 15 minutes. The cells were then heat-shocked at 42℃ for 90 seconds, placed on ice for 3 minutes, and 500 μL of LB medium was added. The cells were incubated at 37℃ and 220 rpm for 45 minutes. 50 μL of the incubator was evenly spread on LB solid medium containing 50 μg / mL kan. The cells were incubated upside down overnight to obtain single clones. After expansion, the cells were stored at -80℃ with 20% final concentration glycerol. After sequencing analysis confirmed that the cells were correct, the recombinant expression transformant was named FCM3.

[0048] 5 μL of glycerol bacteria from the recombinant expression transformant FCM3 was inoculated into 5 mL of LB liquid medium containing 50 μg / mL Kan and cultured at 37°C and 220 rpm for 16 h. Then, the bacterial culture was inoculated into LB liquid medium containing 50 μg / mL Kan at a bacterial-to-medium ratio of 1:100 (v:v) and cultured at 37°C and 220 rpm until the OD600 reached approximately 0.8. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2 mM, and induction was performed at 30°C for 20 h. After fermentation, the cells were collected, resuspended in 1 / 2 volume of 50 mM Tris (pH 8.5), and the cells were sonicated to disrupt the cell structure. The supernatant was collected by centrifugation and analyzed by SDS-PAGE. Figure 5 Soluble expression of the FCM-3 fusion protein can be achieved using conventional pET vectors and fermentation methods.

[0049] Example 3 (Tyrosinase conversion reaction and chromatographic purification of recombinant FCM3 / FCM5 fusion protein)

[0050] After fermentation, the fermentation broth was centrifuged, and FCM3 and FCM5 cells were collected separately. The cells were resuspended in 1 / 2 volume of pH 8.5 50 mM Tris, and the cells were sonicated to disrupt the cell structure. The supernatant was collected by centrifugation as the crude fusion protein. The crude fusion protein was analyzed by SDS-PAGE. Figure 5 .

[0051] This example tested the conversion reaction of different concentrations of the fusion protein:

[0052] The crude FCM3 fusion protein was diluted with 1mM CuCl2 and 20mM PB buffer at pH 8.5 to final concentrations of 0.5g / L, 1g / L, 2g / L, and 4g / L, respectively. 0.1% tyrosinase (commercially available tyrosinase was used in this example) was added, and the mixture was incubated at 30°C for 3 hours. After the reaction was completed, chromatography purification was performed.

[0053] The crude FCM5 fusion protein was diluted with 1 mM CuCl2 and 20 mM PB buffer at pH 8.5 to final concentrations of 0.5 g / L, 1 g / L, 2 g / L, and 4 g / L, respectively. 0.1% tyrosinase (commercially available tyrosinase was used in this example) was added, and the mixture was incubated at 30°C for 3 h. After the reaction was completed, chromatography purification was performed.

[0054] The fusion protein was purified using Ni-NTA column affinity chromatography on an AKTA protein purification system. First, the Ni-NTA column was equilibrated with two column volumes of equilibration buffer (50 mmol / L Tris-HCl, 150 mmol / L NaCl, pH 7.5). Then, the crude protein sample was loaded and eluted with two column volumes of elution buffer A (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 30 mmol / L imidazole, pH 7.5) to remove impurities. Finally, elution was performed with two column volumes of elution buffer B (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 250 mmol / L imidazole, pH 7.5), and the elution was collected.

[0055] Example 4 (Detection of dopa group content)

[0056] In this embodiment, the content of DOPA group was determined by a modified quantitative method, namely, DOPA oxidized by nitrite, and the absorbance value at A490~A520 nm was measured by an ELISA reader to calculate the content of DOPA in the recombinant FCM-3 and FCM-5 fusion protein.

[0057] Plotting the standard curve: ① Preparation of DOPA standard solution: Prepare a 10 mM DOPA stock solution and dilute it to 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM. ② Take 25 µL of DOPA standard solution, add 50 µL of 0.5 M HCl, mix thoroughly, then add 75 µL of 100 g / L sodium nitrite and 100 g / L sodium molybdate solution, mix thoroughly, let stand for 30 s, add 100 µL of 1 M NaOH, and immediately scan the spectrum from A490 to A520 nm using a microplate reader. Plot the standard curve with DOPA concentration on the x-axis and absorbance on the y-axis.

[0058] Recombinant protein sample detection: Take 25 µL of the sample to be tested, add 50 µL of 0.5 M HCl, mix thoroughly, then add 75 µL of 100 g / L sodium nitrite and 100 g / L sodium molybdate solution, mix thoroughly, let stand for 30 s, add 100 µL of 1 M NaOH, and immediately scan the spectrum at A490~A520 nm using an ELISA reader. Calculate the dopa content of the sample based on the measured absorbance of the sample solution. If the absorbance of the test solution exceeds the highest value of the standard curve, the sample needs to be diluted.

[0059] As shown in Table 1, in this embodiment, the lower the concentration of the fusion protein, the higher the dopa content of the fusion protein sample. Since FCM-3 has less tyrosine than FCM-5, the optimal reaction concentrations of FCM-3 and FCM-5 are 1 g / L and 0.5 g / L, respectively.

[0060] Table 1. Determination of dopa content in recombinant FCM-3 and FCM-5 fusion proteins

[0061]

[0062] Example 5 (Cell Proliferation Assay)

[0063] The cell proliferation-promoting effect of the recombinant fusion protein was detected by the MTT assay. The cells used in the experiment were human skin fibroblasts (HSF).

[0064] Cells were cultured in DMEM medium containing 10% fetal bovine serum and penicillin-antibiotic PS at 37°C and 5% CO2, with a cell concentration controlled at 5.0 × 10⁶ cells / ml. 5 ~7.0×10 5 Cells were passaged and used for biological activity assays 24 hours later. 96-well culture plates were used to prepare 5 × 10⁶ HSF solutions of cultured fibroblasts. 3 100 μL of cell suspension was seeded into each well and incubated in a 5% CO2 incubator at 37 ℃ for 24 h. The original culture medium was discarded, and 100 μL of the experimental group (0.5 mg / L, 1 g / L) solution was added to each well. A negative control group (cell culture medium only) and a positive control group (4% DMSO) were also set up, with three replicates for each. Measurements were performed at 48 h and 96 h. 50 μL of MTT (thiazolyl blue) was added to each well, and the cells were incubated at 37 ℃ for 2 h. The original culture medium was then discarded, and 150 μL of dimethyl sulfoxide was immediately added to each well. The cells were incubated at room temperature with gentle shaking for 10–15 min. The absorbance (A) of each well was measured at 490 nm using a microplate reader, and the relative cell proliferation rate (RGR) was calculated (%).

[0065] Table 2, showing the cell proliferation rates at 48 h and 96 h, indicates that the recombinant FCM-3 and FCM-5 fusion protein samples exhibited cell proliferation-promoting effects at concentrations ranging from 0.5 to 1 g / L, with the best effect observed at a concentration of 0.5 g / L. Therefore, the recombinant FCM-3 and FCM-5 fusion protein can promote cell proliferation, possesses good cell compatibility, and meets the requirements for use as a biomaterial.

[0066] Table 2. Cell proliferation rate assay of recombinant FCM-3 and FCM-5 fusion protein at 48 h and 96 h.

[0067]

[0068] Example 6 (Cell Adhesion Assay)

[0069] This embodiment uses recombinant FCM-3 and FCM-5 fusion proteins with high dopamine content for testing.

[0070] Human skin fibroblast (HSF) cell lines were selected and cultured in complete medium containing 10% fetal bovine serum (FBS) in DMEM with double antibiotic PS at 37°C and 5% carbon dioxide, maintaining a cell concentration of 5.0 × 10⁶ cells per ml. 5 ~1.0×10 6 Cells were passaged and used for biological activity assays 24–36 hours later.

[0071] The recombinant FCM3 / FCM5 fusion protein prepared in Example 5 was dissolved in PBS to a final concentration of 0.1 mg / ml. 50 μL of the recombinant fibronectin-collagen fusion protein solution was added to each well of a 96-well cell culture plate and incubated at 37°C for 2 h. PBS was added to the control wells.

[0072] Discard the culture medium in the culture flask, digest and collect the cells with trypsin, and add 5 × 10⁵ cells to each well. 5 Cells were cultured at 37°C in a 5% CO2 incubator for 2 hours, washed three times with PBS to remove unadhered cells, and then 200 μL of LMEM medium was added. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 2 hours. The absorbance of the 96-well plate was read at 450 nm and 630 nm using a microplate reader. Using 630 nm as the reference wavelength, the absorbance was measured at 450 nm, and the results were recorded.

[0073] Cell adhesion rate = (absorbance value of experimental group at 450nm - absorbance value of negative control group at 450nm) / absorbance value of negative control group at 450nm × 100%.

[0074] The results showed that, at a concentration of 0.1 mg / ml, the cell adhesion rate of recombinant FCM3 fusion protein to HSF cells was 57.6%, and that of recombinant FCM5 fusion protein to HSF cells was 64.7%, indicating that both recombinant FCM3 and FCM5 fusion proteins of the present invention have good cell adhesion-promoting activities, with recombinant FCM5 fusion protein showing better performance.

[0075] Example 7 (Testing of Soothing and Anti-inflammatory Efficacy)

[0076] This embodiment uses LPS-induced mouse fibroblasts L929 as an in vitro inflammatory cell model to evaluate whether the recombinant FCM-3 and FCM-5 fusion protein has in vitro soothing effects by measuring the amount of NO.

[0077] Using LPS-induced mouse macrophages RAW264.7 as an in vitro inflammatory cell model, the relative expression levels of inflammatory factor (IL-8) mRNA were measured to evaluate whether the recombinant FCM-3 and FCM-5 fusion protein had in vitro anti-inflammatory effects.

[0078] The cell lines used were L929 cells and mouse macrophage RAW264.7. The culture medium was DMEM containing 10% FBS and 1% penicillin-dextrose antibody. The testing conditions were 37°C, 5% CO2, and saturated humidity.

[0079] The corresponding cells were cultured using standard methods, and the cell suspension was seeded into 6-well cell culture plates and incubated for 18–24 h. The culture plates were then removed, and the original culture medium in the wells was discarded. Culture medium containing LPS as a stimulant was added to the test sample group and the model control group, and the plates were incubated for 18 h. Subsequently, the solution in the wells was removed. Different concentrations of FCM-3 and FCM-5 were added to the test sample group, while culture medium was added to the model control group, and the plates were incubated for 24 ± 2 h.

[0080] In the NO assay, the culture medium was aspirated, centrifuged, and the supernatant was collected. The NO content in the supernatant was tested according to the instructions of the NO assay kit. In the IL-8 assay, after the culture was completed, the cells in each group were lysed to collect RNA, which was reverse transcribed into cDNA, stored at -80℃, and finally the expression level of IL-8 was determined by real-time PCR.

[0081] When the detection concentration of FCM-3 was 10 mg / L, the NO content decreased significantly by 19.27%; when the detection concentration was 25 mg / L, the NO content decreased significantly by 26.53%; and when the detection concentration was 50 mg / L, the NO content decreased significantly by 39.97%. When the detection concentration of FCM-5 was 10 mg / L, the NO content decreased significantly by 33.92%; when the detection concentration was 25 mg / L, the NO content decreased significantly by 48.66%; and when the detection concentration was 50 mg / L, the NO content decreased significantly by 51.08%. The inhibitory effect on the inflammatory factor NO is shown in [the table below]. Figure 3 The results showed that the modified FCM-3 and FCM-5 had a significant inhibitory effect on the inflammatory factor NO at test concentrations of 10 mg / L and above, and this soothing effect was dose-dependent.

[0082] At a concentration of 10 mg / L, FCM-3 reduced the relative expression of IL-8 by 74.31%; at 25 mg / L, by 118.32%; and at 50 mg / L, by 200%. At a concentration of 10 mg / L, FCM-5 reduced the relative expression of IL-8 by 106.01%; at 25 mg / L, by 179.11%; and at 50 mg / L, by 304.59%. These results indicate that both FCM-3 and FCM-5 exhibit significant anti-inflammatory effects at different concentrations. The inhibitory effect on the inflammatory factor IL-8 is shown in [the table below]. Figure 4 .

[0083] Example 8 (Whitening Efficacy Test)

[0084] The skin-whitening efficacy of zebrafish samples was evaluated by assessing skin whiteness and melanin content. Zebrafish are transparent throughout their bodies during early development, and melanin begins to grow from the retinal epithelium at 24 hours of embryonic development. Pigment cells originate from a group of cells differentiated from the dorsal ectoderm—neural crest cells—which then proliferate, migrate, and differentiate into melanocytes. Intervention during melanin formation can inhibit melanin production.

[0085] Zebrafish skin whiteness test:

[0086] Zebrafish were randomly selected and placed in 6-well plates, 15 fish per well. Water-soluble samples were administered, and a normal control group was also included. Each well contained 3 mL of the sample. The plates were incubated at 28°C in the dark for 45 hours. Ten zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data, including the intensity (S) and efficacy (%) of melanin signal in the zebrafish head. ×100%. See test results below. Figure 6 The FCM samples showed whitening effects, specifically FCM3 reduced the melanin content in the zebrafish head by 65%, and FCM5 reduced the melanin content in the zebrafish head by 73%.

[0087] Zebrafish melanin protein content detection:

[0088] Zebrafish were randomly selected and placed in 6-well plates, 30 fish per well. Water-soluble samples were administered, with a normal control group included. Each well contained 3 mL of sample. The plates were incubated at 28°C in the dark for 45 h. Samples were collected in 1.5 mL centrifuge tubes, each containing RIPA lysis buffer. The mixture was homogenized using a grinder and the supernatant was discarded by centrifugation. 350 μL of 0.2 M NaOH solution was added to each tube, and the plates were incubated at 60°C for 1 h. A melanin standard solution was prepared using 0.2 M NaOH solution at a specific concentration. The standard solution and the sample were transferred to 96-well plates (100 μL / well). The absorbance was measured at 405 nm using a microplate reader. The melanin content (C) and whitening effect (%) of each sample group were then determined based on the standard curve. ×100%. See test results below. Figure 7 The FCM samples exhibited whitening effects, specifically FCM3 reducing melanin content by 78% and FCM5 reducing melanin content by 85%.

[0089] Comparative Example 1 (Construction and expression of recombinant FCM-5 fusion protein in E. coli, including expression vector and strain)

[0090] In this embodiment, the sequence of human fibronectin III 9-unit, the integrin binding region of human type III collagen, and two repeating sequences of mussel adhesive protein mfp-5 were selected. The amino acid sequence of the fusion protein is SEQ ID NO.1. The coding gene was outsourced for optimized synthesis. NdeI and HindIII restriction enzyme sites were introduced on both sides of the coding gene to facilitate gene cloning and expression.

[0091] Vector and exogenous fragment digestion: The empty expression vector pET28a-EGFP plasmid was extracted and digested with enzymes. The digestion reaction system consisted of 84 μL pET28a-EGFP, 3 μL NdeI, 3 μL HindIII, and 10 μL 10*cutsmart. Digestion was carried out at 37℃ for 1.5 h, followed by separation by 1.0% agarose gel electrophoresis. Approximately 5 kb of the vector was excised from the gel and purified using a gel extraction kit. The gene-encoding plasmid was extracted and digested with enzymes. The digestion reaction system consisted of 59 μL gene, 2 μL NdeI, 2 μL HindIII, and 7 μL 10*cutsmart. Digestion was carried out at 37℃ for 1.5 h, followed by separation by 1.0% agarose gel electrophoresis, yielding approximately 1 kb of band. This band was excised from the gel and recovered using a gel extraction kit.

[0092] Ligation of the vector with the exogenous fragment: The ligation system consisted of 1 μL of the digested (NdeI / HindIII) pET28a-EGFP fragment, 2 μL of the synthesized gene fragment digested (NdeI / HindIII), 0.5 μL of T4 ligase, 1 μL of 10* ligase buffer, and purified water to a final volume of 5.5 μL. The mixture was digested at 25°C for 0.5 h to obtain the ligation product. The ligation product was transformed into DH5α to obtain the recombinant expression vector, named pFM5'. The image of the recombinant expression vector pFM5' is shown below. Figure 2 .

[0093] The host cells were Escherichia coli BL21(DE3) competent cells. The transformation method was as follows: the competent cells were taken out of the -80℃ freezer and placed on ice. After the competent cells thawed, about 100-200 ng of pFM3 was added. The cells were slowly stirred with a pipette tip and then placed on ice for 15 minutes. The cells were then heat-shocked at 42℃ for 90 seconds, placed on ice for 3 minutes, and 500 μL of LB medium was added. The cells were incubated at 37℃ and 220 rpm for 45 minutes. 50 μL of the incubator was evenly spread on LB solid medium containing 50 μg / mL kan. The cells were incubated upside down overnight to obtain single clones. After expansion, the cells were stored at -80℃ with 20% final concentration glycerol. After sequencing analysis confirmed that the cells were correct, the recombinant expression transformant was named FM5'.

[0094] 5 μL of glycerol bacteria from the recombinant expression transformant FM5' was inoculated into 5 mL of LB liquid medium containing 50 μg / ml Kan and cultured at 37°C and 220 rpm for 16 h. Then, the bacterial culture was inoculated into LB liquid medium containing 50 μg / ml Kan at a bacterial-to-medium ratio of 1:100 (v:v) and cultured at 37°C and 220 rpm until the OD600 reached approximately 0.8. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2 mM, and induction was performed at 30°C for 20 h. After fermentation, the cells were collected, resuspended in 1 / 2 volume of pH 8.5 50 mM Tris, and the cells were sonicated to disrupt the cell structure. The supernatant was collected by centrifugation, and SDS-PAGE analysis revealed no soluble expression of the FCM-5 fusion protein using conventional methods.

[0095] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A recombinant fibronectin-collagen-mussel adhesive protein fusion protein, characterized in that, The structure of the fusion protein is: fibronectin functional region - human collagen functional region - mussel adhesive protein. The structure of the fusion protein includes multiple fibronectin functional regions, human collagen functional regions and mussel adhesive proteins, wherein the mussel adhesive proteins are selected from one or more combinations of Mfp-1, Mfp-3, Mfp-5 and Mfp-6.

2. The fusion protein according to claim 1, characterized in that, The fibronectin functional region is selected from one or more combinations of FN III 1-15 units.

3. The fusion protein according to claim 2, characterized in that, The fibronectin functional region is selected from the sequence of human fibronectin III 9-11 units.

4. The fusion protein according to claim 1, characterized in that, The human collagen functional region can be selected from one or more combinations of human type I, type II, and type III collagen functional regions.

5. The fusion protein according to claim 1, characterized in that, The structure of the fusion protein includes two repeating mussel adhesive protein sequences, an integrin-binding region of the human collagen functional domain, and a sequence of human fibronectin III 9 units.

6. The fusion protein according to any one of claims 1-5, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.1 or SEQ ID NO.

1.

7. A nucleic acid encoding the fusion protein of claim 1, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO.

3.

8. An expression vector containing the nucleic acid of claim 7, characterized in that, The expression vector is selected from pBAD series vectors, pQE series vectors, pMAL series vectors, or cold-induced pCold series vectors.

9. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 7 and / or the expression vector of claim 8.

10. The application of the recombinant fibronectin-mussel adhesive protein fusion protein according to claim 1 in the preparation of whitening and anti-inflammatory skin care products.

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