Fusion protein with high repairing capability, preparation method and application thereof

Abstract

The invention provides a fusion protein with high repair capacity, a preparation method and application thereof, and belongs to the technical field of hybrid peptides. The amino acid sequence of the fusion protein is shown as SEQ ID NO.1; the fusion protein is obtained by sequentially connecting the following modules in series: a functional domain 1 of fibronectin, a hydrophilic region 1 of elastin, a functional domain 1 of mussel mucin, a hydrophilic region 2 of elastin, a functional domain 2 of fibronectin, a hydrophilic region 3 of elastin, a functional domain 2 of mussel mucin, a hydrophilic region 4 of elastin and an integrin binding region of elastin. The fusion protein of fibronectin, elastin and mussel mucin is constructed, and the nucleotide sequence of the fusion protein is optimized, so that the relative migration rate of keratinocytes can be obviously improved, the expression quantity of repair-related genes of HaCat cells can be improved, the secretion quantity of skin stimulating factors can be reduced, and the inhibition rate on hyaluronidase can be improved; the skin repairing and relieving capability is improved.

Description

Technical Field

[0001] The invention relates to a fusion protein with high repair ability, a preparation method and application thereof, and belongs 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's foot. At present, at least 13 types of mussel adhesive proteins have been studied and identified. Mussel adhesive protein contains lysine and dopa, which can form a water-insulating microscopic biological scaffold with positive charge, adsorb negatively charged epidermal cells, fibroblasts, etc., quickly adhere to the wall, crawl and grow, promote wound healing, and can be used to repair skin injuries such as burns and laser surgery; it can also be used in skin care products to play a physical isolation role and reduce dust irritation to the skin.

[0003] The traditional preparation method of mussel mucin is mainly chemical extraction, which consumes a lot of manpower, material and financial resources, has low production capacity and low purity, and affects biological activity.

[0004] Fibronectin (FN) is a macromolecular glycoprotein that is widely present in plasma, cell surfaces and cell matrix. As an important adhesion molecule, it binds to at least 11 integrin receptors and plays a very important role in the interaction between cells and between cells and matrix. A large number of studies have shown that fibronectin is involved in wound healing, tissue repair, embryonic differentiation, immune response and other processes, and plays an irreplaceable and important role in the regeneration and repair of chronic and difficult-to-heal wounds. Fibronectin is used as a raw material for cosmetics and has a repairing effect on sensitive skin, red blood streaks, skin damage after medical aesthetics, acne, acne and other problematic skin.

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

[0006] Currently, the bioremediation raw materials on the market are all non-fusion proteins represented mainly by mussel mucin, fibronectin, collagen, elastin, etc., and their repair capabilities are limited. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a fusion protein with high repair ability, a preparation method and application thereof, to achieve the following invention objectives: fibronectin, elastin, and mussel mucin are constructed to obtain a fusion protein, and the repair and soothing ability of the fusion protein is improved.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions: A fusion protein with high repair ability, the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0009] The fusion protein is obtained by serially connecting the following modules: functional domain 1 of fibronectin, hydrophilic region 1 of elastin, functional domain 1 of mussel mucin, hydrophilic region 2 of elastin, functional domain 2 of fibronectin, hydrophilic region 3 of elastin, functional domain 2 of mussel mucin, hydrophilic region 4 of elastin, and integrin binding region of elastin.

[0010] The nucleotide sequence of the functional domain 1 of the fibronectin is shown in SEQ ID NO.3; the nucleotide sequence of the hydrophilic region 1 of the elastin is shown in SEQ ID NO.4; the nucleotide sequence of the functional domain 1 of the mussel mucin is shown in SEQ ID NO.5; the nucleotide sequence of the hydrophilic region 2 of the elastin is shown in SEQ ID NO.6; the nucleotide sequence of the functional domain 2 of the fibronectin is shown in SEQ ID NO.7; the nucleotide sequence of the hydrophilic region 3 of the elastin is shown in SEQ ID NO.8; the nucleotide sequence of the functional domain 2 of the mussel mucin is shown in SEQ ID NO.9; the nucleotide sequence of the hydrophilic region 4 of the elastin is shown in SEQ ID NO.10; the nucleotide sequence of the integrin binding region of the elastin is shown in SEQ ID NO.11.

[0011] The nucleotide sequence of the fusion protein is used to construct a recombinant plasmid, which is then transferred into competent E. coli cells to construct a genetically engineered bacterium expressing the fusion protein, and then the fusion protein is obtained through culture and induction expression.

[0012] The fusion protein is used in preparing skin repair and / or soothing products.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention constructs a fusion protein of fibronectin, elastin and mussel mucin. Compared with single fibronectin, elastin or mussel mucin, the fusion protein can improve the relative mobility of keratinocytes, increase the expression of repair-related genes in HaCat cells, reduce the secretion of IL-6, TNF-α and NO in RAW264.7 cells, and improve the inhibition rate of hyaluronidase, and has good repair and soothing effects.

[0014] The present invention optimizes the nucleotide sequence of the fusion protein. The fusion protein after the nucleotide sequence optimization can significantly improve the relative mobility of keratinocytes, increase the expression of repair-related genes in HaCat cells, reduce the secretion of IL-6, TNF-α and NO in RAW264.7 cells, and improve the inhibition rate of hyaluronidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The SDS-PAGE electrophoretogram of Example 4 of the present invention; Wherein, 1 is the electrophoretic band of the bacterial lysis supernatant before induction expression after optimization of BL21-pET28a(+)-MAP / FN / ELN; M is Marker; 2 is the electrophoretic band of the bacterial lysis supernatant after optimization of BL21-pET28a(+)-MAP / FN / ELN after induction expression; 3 is the electrophoretic band of the bacterial lysis supernatant before optimization of BL21-pET28a(+)-MAP / FN / ELN before induction expression; 4 is the electrophoretic band of the bacterial lysis supernatant after optimization of BL21-pET28a(+)-MAP / FN / ELN after induction expression; Figure 2 This is a microscope image of cell migration in Example 7 of the present invention; Figure 3 is a bar graph of the relative migration rate of cells in Example 7 of the present invention; Figure 4 A bar graph showing the relative expression of cell repair-related mRNA in Example 8 of the present invention; Figure 5 is a bar graph of the IL-6 content in the cell supernatant of Example 9 of the present invention; Figure 6 is a bar graph of the TNF-α content in the cell supernatant of Example 9 of the present invention; Figure 7 It is a bar graph of the NO content in the cell supernatant of Example 9 of the present invention. DETAILED DESCRIPTION

[0016] Example 1 Sequence design of fusion protein The present invention constructs a fusion protein from fibronectin, elastin and mussel mucin, 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.

[0017] The fusion protein is obtained by sequentially connecting the following modules: The functional domain 1 of fibronectin, the nucleotide sequence of which is shown in SEQ ID NO.3; The hydrophilic region 1 of elastin, the nucleotide sequence of which is shown in SEQ ID NO.4; Mussel mucin functional domain 1, the nucleotide sequence of which is shown in SEQ ID NO.5; The hydrophilic region 2 of elastin, the nucleotide sequence of which is shown in SEQ ID NO.6; The functional domain 2 of fibronectin, the nucleotide sequence of which is shown in SEQ ID NO.7; The hydrophilic region 3 of elastin, the nucleotide sequence of which is shown in SEQ ID NO.8; Mussel mucin functional domain 2, the nucleotide sequence of which is shown in SEQ ID NO.9; The hydrophilic region 4 of elastin, the nucleotide sequence of which is shown in SEQ ID NO.10; The integrin binding region of elastin has a nucleotide sequence as shown in SEQ ID NO.11.

[0018] The nucleotide sequence of the fusion protein before optimization is shown in SEQ ID NO.12.

[0019] In the SEQ ID NO.2, the first 6 bases are NcoI restriction site, the last 6 bases are HindIII restriction site, and the 3 bases before the HindIII restriction site are the stop codon.

[0020] In the SEQ ID NO.12, the first 6 bases are NcoI restriction site, the last 6 bases are SacI restriction site, and the 3 bases before the SacI restriction site are the stop codon.

[0021] Example 2 Construction of recombinant plasmid The nucleotide sequences of the fusion proteins shown in SEQ ID NO.2 and SEQ ID NO.12 were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene synthesis to obtain the PUC57-MAP / FN / ELN plasmids before and after optimization.

[0022] pET28a(+) and PUC57-MAP / FN / ELN before optimization plasmids were double-digested with NcoI and SacI enzymes, respectively, and PUC57-MAP / FN / ELN after optimization plasmids were double-digested with NcoI and HindIII enzymes, respectively. After the digestion, the recovered double-digestion products were connected with Solution I ligase at 16°C for 3h to obtain recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli DH5α competent cells, and positive transformants were selected for culture after resistance screening, and sequenced after PCR identification; 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, and the plasmids were extracted to obtain the recombinant plasmids pET28a(+)-MAP / FN / ELN before optimization and pET28a(+)-MAP / FN / ELN after optimization.

[0023] Example 3 Construction of recombinant fusion protein engineering bacteria Take 1 μL of recombinant plasmid pET28a(+)-MAP / FN / ELN before optimization and pET28a(+)-MAP / FN / ELN after optimization (concentration of 1 μg / μL) and mix with 100 μL of thawed E. coli BL21(DE3) competent cell suspension, ice bath for 30 minutes, heat shock for 90 seconds in a 42℃ water bath, ice bath for 5 minutes again, then add 400 μL of LB medium without resistance, and culture at 37℃ and 190 rpm for 40 minutes. Pipette 200 μL of bacterial solution and evenly spread it on LB agar plate containing 50 μg / mL kanamycin, invert and culture overnight at 37℃. The next day, a single transformed colony was picked and inoculated into a test tube containing 5 mL of LB liquid culture medium (the final concentration of Kana was 50 μg / mL). After overnight culture at 37°C and 180 rpm, the plasmid of the bacterial solution was extracted. After sequencing, the recombinant fusion protein engineering bacteria BL21-pET28a(+)-MAP / FN / ELN before optimization and BL21-pET28a(+)-MAP / FN / ELN after optimization were obtained.

[0024] Example 4 Inducible expression of recombinant fusion protein engineering bacteria Pick the correct sequenced BL21-pET28a(+)-MAP / FN / ELN before optimization and BL21-pET28a(+)-MAP / FN / ELN after optimization, inoculate them into 5mL LB medium (final concentration of Kana is 50μg / mL), and culture them at 37℃ and 180rpm overnight. Then take 300μL of the overnight bacterial solution and inoculate it into a triangular flask containing 30mL LB medium (final concentration of Kana is 50μg / mL) and culture it for 2.5h until the initial OD 600When the concentration of IPTG reaches 0.65, the inducer IPTG is added to make the final concentration 0.2 mM, the induction temperature is 37°C, the induction time is 5 h, and the induction is completed.

[0025] Collect a small amount of the above OD 600 When the concentration of the bacterial solution reached 0.65, the bacterial solution without the addition of the inducer was centrifuged to collect the bacterial precipitate, and the supernatant obtained by lysis was diluted 2 times and then analyzed by 12% SDS-PAGE electrophoresis; a small amount of bacterial solution after the induction was collected, the bacterial precipitate was centrifuged to collect the bacterial precipitate, and the supernatant obtained by lysis was diluted 2 times and then analyzed by 12% SDS-PAGE electrophoresis; the results are shown in Figure 1 .

[0026] Figure 1 It can be seen that the fusion protein MAP / FN / ELN has a specific band at around 43.4KD, and the expression level of the fusion protein MAP / FN / ELN after BL21-pET28a(+)-MAP / FN / ELN optimization is much higher than that before BL21-pET28a(+)-MAP / FN / ELN optimization.

[0027] Example 5 Purification of fusion protein After the induction, the bacterial suspension of BL21-pET28a(+)-MAP / FN / ELN before optimization and after optimization was centrifuged at 8000 rpm for 30 min, and the precipitated bacteria were taken out. According to 1 g of bacteria, 10 mL of 20 mM acetic acid-sodium acetate buffer (pH 4.0) containing 0.15 M sodium chloride was added, and the suspension was resuspended for 30 min. The suspension was centrifuged at 4°C and 8000 rpm for 20 min, and the supernatant was discarded. The precipitate was resuspended in 20 mM acetic acid-sodium acetate buffer (pH 4.0) containing 1 M sodium chloride for 30 min. The bacteria were broken once at 400 bar and three times at 800 bar using a high-pressure sterilizer. The bacterial suspension after breaking was centrifuged at 4°C and 8000 rpm for 20 min, and the supernatant was taken out. 20 mM acetic acid-sodium acetate (pH 4.0), adjusted the conductivity to 30±2mS / cm, and purified the fusion protein stock solution after filtering through a 0.45μm membrane.

[0028] Example 6 Lyophilization of fusion protein The fusion protein stock solutions before and after MAP / FN / ELN optimization were freeze-dried using a vacuum freeze dryer from Haier Bio. The freeze-drying procedure is shown in Table 1 to obtain freeze-dried fusion protein powders, which were marked as before and after MAP / FN / ELN optimization.

[0029] Table 1 Vacuum freeze-drying process of fusion protein stock solution

[0030] Example 7 Fusion protein promoted cell migration experiment HaCat cells in the logarithmic growth phase were cultured at 8×10 5 The cells were inoculated into 6-well plates at a density of 100 μL / well and incubated overnight in an incubator (37°C, 5% CO2). The scratch test was performed when the plating rate reached more than 80%. Use a 10 μL pipette tip to make two vertical scratches in the six-well plate. The vertical scratch is used as the baseline (the pipette tip is perpendicular to the edge of the ruler), and the scratch spacing is kept at 2 cm; after the vertical scratch, make a horizontal scratch perpendicular to the baseline near the central axis of the six-well plate. After the scratch, wash the cells three times with PBS. For the MAP / FN / ELN pre-optimization group, the MAP / FN / ELN post-optimization group, the commercial mussel mucin group, the commercial human fibronectin group, and the commercial elastin group, the protein lyophilized powder was prepared into a 0.1 mg / mL protein solution with DMEM culture medium. The dosage per well was 2 mL, and 2 replicates were set for each group. DMEM culture medium was added to the NC group, and the culture was continued in the incubator (37°C, 5% CO2) for 24 hours. After taking pictures of the migration groups using an inverted microscope ( Figure 2 ), the migration rate of cells in 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 .

[0031] Table 2 Cell migration results

[0032] The results show that the optimized fusion protein MAP / FN / ELN with high repair ability of the present invention can significantly improve the relative mobility of keratinocytes compared with MAP / FN / ELN before optimization, commercial mussel mucin, commercial human fibronectin, and commercial elastin, that is, it has better cell repair ability.

[0033] Example 8 Fusion protein promotes cell repair experiment HaCat cells in the logarithmic growth phase were cultured at 5×10 5 The cells were inoculated into 6-well plates at a density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2). When the cell plating rate in the 6-well plates reached 50%, the freeze-dried powders of MAP / FN / ELN before optimization, MAP / FN / ELN after optimization, commercial mussel mucin, commercial human fibronectin, and commercial elastin were prepared into 0.1 mg / mL protein solutions with DMEM culture medium, and the dosage per well was 2 mL. DMEM culture medium was added to the NC group. After the drug administration, the 6-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 h.

[0034] Total RNA was extracted from cells and the purity of the extracted RNA was detected using an ultra-micro spectrophotometer. 260 / OD 280The values ​​are all between 1.8 and 2.0, and the next step of reverse transcription experiment can be carried out.

[0035] The RNA was reverse transcribed to obtain the reverse transcription product, and the real-time fluorescence quantitative PCR experiment was performed to detect the expression levels of cell repair-related genes h-COL1A1, h-AQP3, h-CLDN1, h-COL17A1, h-FLG and h-LOR. The results of real-time fluorescence quantitative PCR gene detection are shown in Table 3 and the bar graph is shown in Figure 4 .

[0036] Table 3 Results of fluorescent quantitative PCR gene detection

[0037] The results show that after optimization, the fusion protein MAP / FN / ELN with high repair ability of the present invention can significantly increase the expression levels of h-COL1A1, h-AQP3, h-CLDN1, h-COL17A1, h-FLG and h-LOR genes in HaCat cells compared with MAP / FN / ELN before optimization, commercial mussel mucin, commercial human fibronectin and commercial elastin, indicating that the fusion protein MAP / FN / ELN of the present invention has a certain effect on promoting cell repair after optimization.

[0038] Example 9 Soothing Experiment of Fusion Protein The contents of factors such as IL-6, TNF-α and NO can reflect the irritation state of the skin. By detecting the secretion levels of these factors, the soothing ability of the fusion protein can be evaluated.

[0039] RAW264.7 cells suspended in 10 vol% FBS-DMEM medium were cultured at 4 × 10 5 Each well was inoculated with 100 cells / well in a 6-well plate and incubated overnight in an incubator (37°C, 5% CO2). Fresh culture medium was added to the blank group (BC group), culture medium containing 0.5 μg / mL LPS was added to the model group (PC group), and culture medium containing 0.5 μg / mL LPS and 0.1 mg / mL of MAP / FN / ELN before optimization, MAP / FN / ELN after optimization, commercial mussel mucin, commercial human fibronectin, and commercial elastin was added to the experimental group, 2 mL per well. After incubation in an incubator (37°C, 5% CO2) for 24 hours, the cell supernatant was collected and centrifuged at 4°C, 1000g for 15 minutes to obtain the supernatant. According to the operating instructions of the kit, the ELISA kit was used to detect the contents of IL-6, TNF-α and NO in the cell supernatant.

[0040] Depend on Figure 5-7It can be seen that after optimization, the fusion protein MAP / FN / ELN with high repair ability of the present invention can significantly reduce the secretion of IL-6, TNF-α and NO compared with MAP / FN / ELN before optimization, commercial mussel mucin, commercial human fibronectin and commercial elastin, which is beneficial to relieve skin irritation and help the skin return to normal.

[0041] Example 10 Hyaluronidase inhibition rate experiment of fusion protein (1) Reagent preparation Hyaluronidase solution: concentration is 500U / mL, prepared and used immediately, using acetate buffer as solvent; Sodium hyaluronate solution: concentration is 0.5 mg / mL, using acetate buffer as solvent; 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 a pH of 5.6; Acetylacetone solution: Mix 50mL 1.0mol / L sodium carbonate solution and 3.5mL acetylacetone evenly (prepare before use); P-DAB color developer: 0.8 g p-dimethylaminobenzaldehyde is dissolved in a mixture of 15 mL concentrated hydrochloric acid and 15 mL anhydrous ethanol, and mixed evenly; The mass concentration of the concentrated hydrochloric acid is 37%; CaCl2 solution: concentration 2.5 mol / L; NaOH solution: concentration is 5 mol / L.

[0042] (2) Experimental methods The freeze-dried powders of MAP / FN / ELN before and after optimization, commercial mussel mucin, commercial human fibronectin, and commercial elastin were prepared into 1 mg / mL sample solutions with distilled water, and the solutions were tested according to the preparation table in Table 4.

[0043] Table 4 Solution preparation table

[0044] The hyaluronidase inhibition rate of the sample was calculated according to the following formula: Hyaluronidase inhibition rate (%) = [(CD)-(AB)] / (CD) × 100%; Calculation showed that the inhibition rates of MAP / FN / ELN before and after optimization, commercial mussel mucin, commercial human fibronectin and commercial elastin on hyaluronidase at a concentration of 1 mg / mL were 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.

2. A fusion protein with high repair ability according to claim 1, characterized in that: The fusion protein is obtained by serially connecting the following modules: functional domain 1 of fibronectin, hydrophilic region 1 of elastin, functional domain 1 of mussel mucin, hydrophilic region 2 of elastin, functional domain 2 of fibronectin, hydrophilic region 3 of elastin, functional domain 2 of mussel mucin, hydrophilic region 4 of elastin, and integrin binding region of elastin.

3. A fusion protein with high repair ability according to claim 2, characterized in that: The nucleotide sequence of the functional domain 1 of the fibronectin is shown in SEQ ID NO.3; the nucleotide sequence of the hydrophilic region 1 of the elastin is shown in SEQ ID NO.4; the nucleotide sequence of the functional domain 1 of the mussel mucin is shown in SEQ ID NO.5; the nucleotide sequence of the hydrophilic region 2 of the elastin is shown in SEQ ID NO.6; the nucleotide sequence of the functional domain 2 of the fibronectin is shown in SEQ ID NO.7; the nucleotide sequence of the hydrophilic region 3 of the elastin is shown in SEQ ID NO.8; the nucleotide sequence of the functional domain 2 of the mussel mucin is shown in SEQ ID NO.9; the nucleotide sequence of the hydrophilic region 4 of the elastin is shown in SEQ ID NO.10; and the nucleotide sequence of the integrin binding region of the elastin is shown in SEQ ID NO.

11.

4. The method for preparing the fusion protein according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of the fusion protein is used to construct a recombinant plasmid, which is then transferred into competent E. coli cells to construct a genetically engineered bacterium expressing the fusion protein, and then the fusion protein is obtained through culture and induction expression.

5. Use of the fusion protein according to any one of claims 1 to 3 in the preparation of skin repair and / or soothing products.

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