A fusion protein with high repair ability, preparation method thereof and application thereof

Abstract

The present invention provides a fusion protein with high repair ability, a preparation method thereof and an application thereof, belonging to the technical field of hybrid peptides. The amino acid sequence of the fusion protein is as shown in SEQ ID NO.1. The fusion protein is obtained by sequentially connecting the following modules in series: functional domain 1 of fibronectin, hydrophilic region 1 of elastin, functional domain 1 of mussel adhesive protein, hydrophilic region 2 of elastin, functional domain 2 of fibronectin, hydrophilic region 3 of elastin, functional domain 2 of mussel adhesive protein, hydrophilic region 4 of elastin, and integrin-binding region of elastin. The present invention constructs a fusion protein of fibronectin, elastin and mussel adhesive protein, optimizes the nucleotide sequence of the fusion protein, can significantly improve the relative migration rate of keratinocytes, increase the expression level of repair-related genes of HaCat cells, reduce the secretion amount of skin irritation factors, increase the inhibition rate of hyaluronidase, and improve skin repair and soothing ability.

Description

Technical Field

[0001] The present invention relates to a fusion protein with high repair ability, a preparation method thereof and an application thereof, belonging to the technical field of hybrid peptides. Background Art

[0002] Mussel adhesive protein (MAP) is a protein complex secreted by the secretory glands of the mussel foot. Currently, at least 13 mussel adhesive proteins have been studied and identified. Because mussel adhesive protein contains lysine and dopa, it can form a water-repellent and positively charged microscopic biological scaffold, adsorb negatively charged epidermal cells, fibroblasts, etc., adhere, crawl and grow quickly, promote wound healing, and can be used for the repair of skin injuries such as burns and after laser surgery; it can also be used in skin care products to play a physical isolation role and reduce the irritation of dust to the skin.

[0003] The traditional preparation method of mussel adhesive protein is mainly chemical extraction method, which requires a large amount of manpower, material resources and financial resources, has low productivity and low purity, and affects biological activity.

[0004] Fibronectin (FN) is a macromolecular glycoprotein widely present in plasma, cell surfaces and cell matrices. As an important adhesion molecule, it binds to at least 11 integrin receptors and plays an extremely important role in the interaction between cells and between cells and the matrix. A large number of studies have shown that fibronectin is involved in processes such as wound healing, tissue repair, embryonic differentiation, immune response, etc., and plays an irreplaceable and important role in the regenerative repair treatment of chronic non-healing wounds. Fibronectin is used as a cosmetic raw material and has a repair effect on problem skins such as sensitive skin, telangiectasia, skin damage after medical aesthetics, acne, and acne.

[0005] Elastin (ELN) is an important extracellular matrix protein, and its main component is elastic fiber, which provides elasticity and flexibility for tissues. Elastin plays an important role in regulating various cell functions, including promoting cell adhesion, proliferation, differentiation, chemotaxis and migration, etc., and is considered an ideal raw material for preparing medical materials. Although elastin only accounts for about 2% of the total protein in the dermis, it plays an important role in skin elasticity.

[0006] Currently, the biological repair raw materials on the market are all non-fusion proteins mainly represented by mussel adhesive protein, fibronectin, collagen, elastin, etc., and their repair ability is limited. Summary of the Invention

[0007] In view of the deficiencies in the prior art, the present invention provides a fusion protein with high repair ability, a preparation method thereof, and an application thereof, achieving the following invention objectives: constructing a fusion protein from fibronectin, elastin, and mussel adhesive protein to improve the repair and soothing ability of the fusion protein.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A fusion protein with high repair ability, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.1.

[0010] The fusion protein is obtained by sequentially connecting the following modules in series: functional domain 1 of fibronectin, hydrophilic region 1 of elastin, functional domain 1 of mussel adhesive protein, hydrophilic region 2 of elastin, functional domain 2 of fibronectin, hydrophilic region 3 of elastin, functional domain 2 of mussel adhesive protein, hydrophilic region 4 of elastin, and integrin-binding region of elastin.

[0011] The nucleotide sequence of functional domain 1 of fibronectin is as shown in SEQ ID NO.3; the nucleotide sequence of hydrophilic region 1 of elastin is as shown in SEQ ID NO.4; the nucleotide sequence of functional domain 1 of mussel adhesive protein is as shown in SEQ ID NO.5; the nucleotide sequence of hydrophilic region 2 of elastin is as shown in SEQ ID NO.6; the nucleotide sequence of functional domain 2 of fibronectin is as shown in SEQ ID NO.7; the nucleotide sequence of hydrophilic region 3 of elastin is as shown in SEQ ID NO.8; the nucleotide sequence of functional domain 2 of mussel adhesive protein is as shown in SEQ ID NO.9; the nucleotide sequence of hydrophilic region 4 of elastin is as shown in SEQ ID NO.10; the nucleotide sequence of the integrin-binding region of elastin is as shown in SEQ ID NO.11.

[0012] Construct a recombinant plasmid with the nucleotide sequence of the fusion protein, transfer it into Escherichia coli competent cells, construct a genetically engineered bacterium expressing the fusion protein, and then obtain the fusion protein through culture and induced expression.

[0013] The application of the fusion protein in the preparation of skin repair and / or soothing products.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention constructs a fusion protein of fibronectin, elastin, and mussel adhesive protein. Compared with single fibronectin, elastin, or mussel adhesive protein, it can increase the relative migration rate of keratinocytes, increase the expression level of repair-related genes in HaCat cells, reduce the secretion of IL-6, TNF-α, and NO in RAW264.7 cells, and increase the inhibition rate of hyaluronidase, showing good repair and soothing effects.

[0016] The present invention optimizes the nucleotide sequence of the fusion protein. The fusion protein with optimized nucleotide sequence can significantly increase the relative migration rate of keratinocytes, increase the expression level of repair-related genes in HaCat cells, reduce the secretion of IL-6, TNF-α, and NO in RAW264.7 cells, and increase the inhibition rate of hyaluronidase. Brief Description of the Drawings

[0017] Figure 1 It is the SDS-PAGE electrophoresis diagram of Example 4 of the present invention;

[0018] Among them, 1 is the electrophoresis band of the supernatant of the lysed bacteria before induced expression of BL21-pET28a(+)-MAP / FN / ELN after optimization; M is Marker; 2 is the electrophoresis band of the supernatant of the lysed bacteria after induced expression of BL21-pET28a(+)-MAP / FN / ELN after optimization; 3 is the electrophoresis band of the supernatant of the lysed bacteria before induced expression of BL21-pET28a(+)-MAP / FN / ELN before optimization; 4 is the electrophoresis band of the supernatant of the lysed bacteria after induced expression of BL21-pET28a(+)-MAP / FN / ELN before optimization;

[0019] Figure 2 It is the microscopic diagram of cell migration in Example 7 of the present invention;

[0020] Figure 3 It is the bar chart of the relative migration rate of cells in Example 7 of the present invention;

[0021] Figure 4 It is the bar chart of the relative expression level of cell repair-related mRNA in Example 8 of the present invention;

[0022] Figure 5 It is the bar chart of the IL-6 content in the cell supernatant of Example 9 of the present invention;

[0023] Figure 6 It is the bar chart of the TNF-α content in the cell supernatant of Example 9 of the present invention;

[0024] Figure 7 It is the bar chart of the NO content in the cell supernatant of Example 9 of the present invention. Detailed Embodiments

[0025] Sequence Design of the Fusion Protein in Example 1

[0026] In the present invention, a fusion protein is constructed from fibronectin, elastin, and mussel adhesive protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence of the fusion protein is optimized. The optimized nucleotide sequence is shown in SEQ ID NO.2.

[0027] The fusion protein is obtained by sequentially connecting the following modules:

[0028] Functional domain 1 of fibronectin, whose nucleotide sequence is shown in SEQ ID NO.3;

[0029] Hydrophilic region 1 of elastin, whose nucleotide sequence is shown in SEQ ID NO.4;

[0030] Functional domain 1 of mussel adhesive protein, whose nucleotide sequence is shown in SEQ ID NO.5;

[0031] Hydrophilic region 2 of elastin, whose nucleotide sequence is shown in SEQ ID NO.6;

[0032] Functional domain 2 of fibronectin, whose nucleotide sequence is shown in SEQ ID NO.7;

[0033] Hydrophilic region 3 of elastin, whose nucleotide sequence is shown in SEQ ID NO.8;

[0034] Functional domain 2 of mussel adhesive protein, whose nucleotide sequence is shown in SEQ ID NO.9;

[0035] Hydrophilic region 4 of elastin, whose nucleotide sequence is shown in SEQ ID NO.10;

[0036] Integrin-binding region of elastin, whose nucleotide sequence is shown in SEQ ID NO.11.

[0037] The nucleotide sequence of the fusion protein before optimization is shown in SEQ ID NO.12.

[0038] In SEQ ID NO.2, the first 6 bases are the NcoI restriction enzyme site, the last 6 bases are the HindIII restriction enzyme site, and the 3 bases in front of the HindIII restriction enzyme site are the stop codon.

[0039] In SEQ ID NO.12, the first 6 bases are the NcoI restriction enzyme site, the last 6 bases are the SacI restriction enzyme site, and the 3 bases in front of the SacI restriction enzyme site are the stop codon.

[0040] Example 2 Construction of Recombinant Plasmids

[0041] The nucleotide sequences of the fusion proteins shown in SEQ ID NO.2 and SEQ ID NO.12 were commissioned to Genewiz (Suzhou) Inc. for gene synthesis to obtain the plasmids PUC57-MAP / FN / ELN before optimization and PUC57-MAP / FN / ELN after optimization.

[0042] pET28a(+) and the plasmid PUC57-MAP / FN / ELN before optimization were double digested with NcoI and SacI enzymes, and the plasmid PUC57-MAP / FN / ELN after optimization was double digested with NcoI and HindIII enzymes. After the digestion, the recovered double-digested products were ligated with Solution I ligase at 16°C for 3 h to obtain recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli DH5α competent cells. After resistance screening, positive transformants were picked for culture, identified by PCR and then sequenced; if the sequencing results were correct, the recombinant plasmids pET28a(+)-MAP / FN / ELN before optimization and pET28a(+)-MAP / FN / ELN after optimization were successfully constructed. The plasmids were extracted by miniprep to obtain the recombinant plasmids pET28a(+)-MAP / FN / ELN before optimization and pET28a(+)-MAP / FN / ELN after optimization.

[0043] Example 3 Construction of Recombinant Fusion Protein Engineering Bacteria

[0044] 1 μL each of the recombinant plasmids pET28a(+)-MAP / FN / ELN before optimization and pET28a(+)-MAP / FN / ELN after optimization (concentration: 1 μg / μL) was mixed with 100 μL of the thawed Escherichia coli BL21(DE3) competent cell suspension. After ice-bathing for 30 min, it was heat-shocked in a 42°C water bath for 90 s, then ice-bathed again for 5 min, and then 400 μL of antibiotic-free LB medium was added. It was cultured on a shaker at 37°C and 190 rpm for 40 min. 200 μL of the bacterial solution was evenly spread on an LB agar plate containing 50 μg / mL kanamycin and incubated overnight at 37°C in an inverted position. The next day, the transformed single colonies were picked and inoculated into a test tube containing 5 mL of LB liquid medium (the final concentration of Kana was 50 μg / mL) and cultured overnight at 37°C and 180 rpm. The plasmids of the bacterial solution were extracted. After correct sequencing, the recombinant fusion protein engineering bacteria BL21-pET28a(+)-MAP / FN / ELN before optimization and BL21-pET28a(+)-MAP / FN / ELN after optimization were obtained respectively.

[0045] Example 4 Induced Expression of Recombinant Fusion Protein Engineering Bacteria

[0046] Respectively pick single colonies of correctly sequenced BL21-pET28a(+)-MAP / FN / ELN before optimization and BL21-pET28a(+)-MAP / FN / ELN after optimization, inoculate them into 5 mL of LB medium (the final concentration of Kana is 50 μg / mL), and culture them overnight at 37 °C with shaking at 180 rpm. Then pipette 300 μL of the overnight bacterial solution and inoculate it into a triangular flask containing 30 mL of LB medium (the final concentration of Kana is 50 μg / mL), and culture for 2.5 h to make the initial OD 600 reach 0.65. Add the inducer IPTG to make its final concentration 0.2 mM, the induction temperature is 37 °C, and the induction time is 5 h, then the induction ends.

[0047] Collect a small amount of the above-mentioned bacterial solution with OD 600 reaching 0.65 without adding the inducer. Centrifuge to collect the bacterial cell precipitate, and the supernatant obtained after lysis is diluted 2-fold and then analyzed by 12% SDS-PAGE electrophoresis; collect a small amount of the bacterial solution after the induction ends, centrifuge to collect the bacterial cell precipitate, and the supernatant obtained after lysis is diluted 2-fold and then analyzed by 12% SDS-PAGE electrophoresis; the results are shown in Figure 1 .

[0048] Figure 1 It can be seen that the fusion protein MAP / FN / ELN has a specific band at about 43.4 KD. After optimization, the expression level of the fusion protein MAP / FN / ELN in BL21-pET28a(+)-MAP / FN / ELN is much higher than that before optimization.

[0049] Example 5 Purification of the fusion protein

[0050] After the induction ends, centrifuge the bacterial solutions of BL21-pET28a(+)-MAP / FN / ELN before optimization and BL21-pET28a(+)-MAP / FN / ELN after optimization at 8000 rpm for 30 min, take the precipitated bacterial cells, and resuspend them in 10 mL of 20 mM acetic acid-sodium acetate buffer solution (pH 4.0) containing 0.15 M sodium chloride according to 1 g of bacterial cells for 30 min. Centrifuge at 4 °C and 8000 rpm for 20 min, discard the supernatant, resuspend the precipitate in 20 mM acetic acid-sodium acetate buffer solution (pH 4.0) containing 1 M sodium chloride for 30 min, use a high-pressure cell disruptor to disrupt the cells once at 400 bar and three times at 800 bar. After disrupting the bacterial solution, centrifuge at 4 °C and 8000 rpm for 20 min, take the supernatant, add 20 mM acetic acid-sodium acetate (pH 4.0) to the supernatant to adjust the conductivity to 30 ± 2 mS / cm, filter through a 0.45 μm membrane, and purify to obtain the stock solution of the fusion protein.

[0051] Lyophilization of the fusion protein in Example 6

[0052] The original solutions of the fusion protein before and after the optimization of MAP / FN / ELN were lyophilized using a vacuum freeze dryer from Haier Biology. The lyophilization procedure is shown in Table 1, and the lyophilized powder of the fusion protein was obtained, labeled as before the optimization of MAP / FN / ELN and after the optimization of MAP / FN / ELN.

[0053] Table 1 Vacuum freeze-drying process of the original solution of the fusion protein

[0054]

[0055] Example 7 Cell migration promotion experiment of the fusion protein

[0056] HaCat cells in the logarithmic growth phase were seeded into a 6-well plate at a density of 8×10 5 cells / well and incubated overnight in an incubator (37 °C, 5% CO2). When the plating rate reached over 80%, a scratch assay was performed. Two longitudinal scratches were made in the 6-well plate with a 10 μL pipette tip, and the vertical scratch served as the baseline (the pipette tip was perpendicular to the edge of the ruler), with the distance between the scratches maintained at 2 cm. After making the vertical scratches, perpendicular to the baseline, horizontal scratches were made near the central axis of the 6-well plate. After the scratching was completed, the cells were washed 3 times with PBS. For the group before the optimization of MAP / FN / ELN, the group after the optimization of MAP / FN / ELN, the commercial mussel adhesive protein group, the commercial human fibronectin group, and the commercial elastin group, the lyophilized powder of the protein was respectively formulated into a 0.1 mg / mL protein solution with DMEM medium, and the dosage per well was 2 mL. Two replicate wells were set for each group. The NC group was added with DMEM medium, and the cells were continued to be cultured in an incubator (37 °C, 5% CO2) for 24 h. After taking pictures of each migration group with an inverted microscope ( Figure 2 ), the cell migration rate of the negative control group was normalized, and the relative migration rate of each group was calculated. The results are shown in Table 2 and Figure 3 .

[0057] Table 2 Cell migration results

[0058]

[0059] The results showed that the optimized MAP / FN / ELN fusion protein with high repair ability of the present invention could significantly increase the relative migration rate of keratinocytes compared with that before the optimization of MAP / FN / ELN, commercial mussel adhesive protein, commercial human fibronectin, and commercial elastin, that is, it had good cell repair ability.

[0060] Example 8 Cell repair promotion experiment of the fusion protein

[0061] HaCat cells in the logarithmic growth phase were seeded at 5×10 5Inoculate into a 6-well plate at an inoculation density of cells per well, and incubate overnight in an incubator (37 °C, 5% CO2). When the cell seeding rate in the 6-well plate reaches 50%, the lyophilized powders of MAP / FN / ELN before optimization, MAP / FN / ELN after optimization, commercial mussel adhesive protein, commercial human fibronectin, and commercial elastin are respectively prepared into protein solutions with a concentration of 0.1 mg / mL using DMEM medium. The dosage per well is 2 mL, and the NC group is added with DMEM medium. After dosing, place the 6-well plate in an incubator (37 °C, 5% CO2) and incubate for 24 h.

[0062] Extract the total RNA of the cells, and use a ultra-micro spectrophotometer to detect the purity of the extracted RNA. The OD 260 / OD 280 values are all between 1.8 and 2.0, and the next reverse transcription experiment can be carried out.

[0063] Perform reverse transcription on the RNA to obtain the reverse transcription product, and conduct a real-time fluorescence quantitative PCR experiment to detect the expression levels of genes related to cell repair, namely h-COL1A1, h-AQP3, h-CLDN1, h-COL17A1, h-FLG, and h-LOR. The results of the real-time fluorescence quantitative PCR gene detection are shown in Table 3, and the column chart is shown in Figure 4 .

[0064] Table 3 Results of fluorescence quantitative PCR gene detection

[0065]

[0066] The results show that after optimization of the fusion protein MAP / FN / ELN with high repair ability of the present invention, compared with MAP / FN / ELN before optimization, commercial mussel adhesive protein, commercial human fibronectin, and commercial elastin, it can significantly increase the expression levels of h-COL1A1, h-AQP3, h-CLDN1, h-COL17A1, h-FLG, and h-LOR genes in HaCat cells, indicating that the optimized fusion protein MAP / FN / ELN of the present invention has a certain effect on promoting cell repair.

[0067] Example 9 Soothing experiment of the fusion protein

[0068] The contents of factors such as IL-6, TNF-α, and NO can reflect the stimulation state of the skin. By detecting the secretion levels of these factors, the soothing ability of the fusion protein can be evaluated.

[0069] RAW264.7 cells suspended in 10 vol% FBS-DMEM culture medium were seeded at 4×10 5Inoculate into a 6-well plate at a density of [number] per well and incubate overnight in an incubator (37 °C, 5% CO2). In the blank control group (BC group), add fresh culture medium. In the model group (PC group), add culture medium containing 0.5 μg / mL LPS. In the experimental groups, add culture medium containing 0.5 μg / mL LPS and 0.1 mg / mL of pre-optimized MAP / FN / ELN, post-optimized MAP / FN / ELN, commercial mussel adhesive protein, commercial human fibronectin, and commercial elastin, 2 mL per well. After incubating for 24 h in an incubator (37 °C, 5% CO2), collect the cell supernatant, centrifuge at 4 °C and 1000 g for 15 min to obtain the supernatant, and according to the operation instructions of the kit, use an ELISA kit to detect the contents of IL-6, TNF-α, and NO in the cell supernatant.

[0070] It can be seen from Figures 5-7 that after optimization of the fusion protein MAP / FN / ELN with high repair ability of the present invention, compared with pre-optimized MAP / FN / ELN, commercial mussel adhesive protein, commercial human fibronectin, and commercial elastin, it can significantly reduce the secretion levels of IL-6, TNF-α, and NO, which is beneficial to alleviating skin irritation and helping the skin to return to a normal state.

[0071] Example 10 Hyaluronidase Inhibition Rate Experiment of the Fusion Protein

[0072] (1) Reagent Preparation

[0073] Hyaluronidase solution: Concentration is 500 U / mL, freshly prepared and used immediately, with acetate buffer as the solvent;

[0074] Sodium hyaluronate solution: Concentration is 0.5 mg / mL, with acetate buffer as the solvent;

[0075] Acetate buffer: 4.8 mL of 0.2 mol / L acetic acid and 45.2 mL of 0.2 mol / L sodium acetate, mixed and diluted to 100 mL to prepare an acetate buffer with pH = 5.6;

[0076] Acetylacetone solution: Mix 50 mL of 1.0 mol / L sodium carbonate solution and 3.5 mL of acetylacetone evenly (prepared immediately before use);

[0077] P-DAB chromogenic reagent: Dissolve 0.8 g of p-dimethylaminobenzaldehyde in a mixed solution of 15 mL of concentrated hydrochloric acid and 15 mL of absolute ethanol, and mix evenly;

[0078] The mass concentration of the concentrated hydrochloric acid is 37%;

[0079] CaCl2 solution: Concentration is 2.5 mol / L;

[0080] NaOH solution: Concentration is 5 mol / L.

[0081] (2) Experimental method

[0082] Take the freeze-dried powders of MAP / FN / ELN before optimization, MAP / FN / ELN after optimization, commercial mussel adhesive protein, commercial human fibronectin, and commercial elastin, and prepare sample solutions with a concentration of 1 mg / mL using distilled water respectively, and conduct tests according to the preparation table in Table 4.

[0083] Table 4 Preparation table of solutions

[0084]

[0085] Calculate the hyaluronidase inhibition rate of the sample according to the following formula:

[0086] Hyaluronidase inhibition rate (%) = [(C - D) - (A - B)] / (C - D) × 100%;

[0087] It can be obtained through calculation that the hyaluronidase inhibition rates of MAP / FN / ELN before optimization, MAP / FN / ELN after optimization, commercial mussel adhesive protein, commercial human fibronectin, and commercial elastin at a concentration of 1 mg / mL are 72.3%, 79.9%, 65.2%, 68.4%, and 62.7% respectively.

Claims

1. A fusion protein with high repair ability, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO.1; The fusion protein is obtained by sequential tandem connection of the following modules: functional domain 1 of fibronectin, hydrophilic region 1 of elastin, functional domain 1 of mussel adhesive protein, hydrophilic region 2 of elastin, functional domain 2 of fibronectin, hydrophilic region 3 of elastin, functional domain 2 of mussel adhesive protein, hydrophilic region 4 of elastin, integrin-binding region of elastin; The nucleotide sequence of functional domain 1 of fibronectin is shown in SEQ ID NO.3; the nucleotide sequence of hydrophilic region 1 of elastin is shown in SEQ ID NO.4; the nucleotide sequence of functional domain 1 of mussel adhesive protein is shown in SEQ ID NO.5; the nucleotide sequence of hydrophilic region 2 of elastin is shown in SEQ ID NO.6; the nucleotide sequence of functional domain 2 of fibronectin is shown in SEQ ID NO.7; the nucleotide sequence of hydrophilic region 3 of elastin is shown in SEQ ID NO.8; the nucleotide sequence of functional domain 2 of mussel adhesive protein is shown in SEQ ID NO.9; the nucleotide sequence of hydrophilic region 4 of elastin is shown in SEQ ID NO.10; the nucleotide sequence of integrin-binding region of elastin is shown in SEQ ID NO.

11.

2. The preparation method of the fusion protein according to claim 1, characterized in that: The nucleotide sequence of the fusion protein is used to construct a recombinant plasmid, which is then transferred into Escherichia coli competent cells to construct a genetically engineered bacterium expressing the fusion protein. The fusion protein is obtained after culturing and induced expression.

3. Use of the fusion protein according to claim 1 in the preparation of skin repair and / or soothing products.

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