A recombinant mussel adhesive protein (type III), nucleic acid molecule, fermentation strain, preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and discloses a recombinant mussel adhesive protein (type III), a nucleic acid molecule, a fermentation strain, and preparation methods and applications thereof. The recombinant mussel adhesive protein provided by the present invention comprises a type III mussel adhesive protein and a biofunctional peptide segment (such as those shown in SEQ ID NO: 1, 5, and / or 8) connected by peptide bonds. By introducing the biofunctional peptide, common engineering bacteria such as Escherichia coli can directly utilize dopa for dopa incorporation expression in cells, obtaining a recombinant mussel adhesive protein with a high dopa content, which can effectively solve the problem of reduced biosafety caused by the introduction of tyrosinase in the preparation of existing mussel adhesive proteins; meanwhile, the recombinant mussel adhesive protein has good bioactivities such as promoting cell migration, scavenging free radicals, anti-inflammatory, antibacterial, inhibiting tyrosinase, and soothing the skin, and has great potential as an active ingredient for skin repair, and can be well applied in the medical and / or cosmetic fields.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant mussel adhesive protein (type III), a nucleic acid molecule, an engineering strain, and a preparation method and application thereof. Background Art

[0002] Mussel adhesive protein (MAP), also known as mussel byssus protein, is a special sticky protein synthesized and secreted by the foot glands of marine bivalves such as Mytilus edulis, Mytilus coruscus, and Perna viridis into the byssus; it has the functions of promoting cell adhesion and crawling, promoting wound healing, inhibiting itching, broad-spectrum adhesion, and forming a water-resistant protective film. Mussel adhesive protein has broad application prospects and industrial development prospects in the fields of medicine, food, environmental protection, materials science, etc.

[0003] Mussel adhesive protein has an extremely strong function of adhering to the substrate, which is related to its special molecular structure, DOPA (dopamine)-mediated intermolecular cross-linking, and the interaction mode with the substrate. Moreover, it also has good biocompatibility and biodegradability, and is a type of bioadhesive with great advantages and potential. Mussel adhesive protein cross-links through electrostatic interaction forces, hydrophobic interaction forces, and the cross-linking of oxidized and unoxidized multi-benzyl groups to form a microscopic biological scaffold, strongly adheres to the wound surface, and plays a role in promoting healing and relieving itching and pain by blocking water. That is, the content of DOPA in mussel adhesive protein will affect its biological activity. Therefore, tyrosinase is introduced during the preparation process of mussel adhesive protein for oxidative modification to increase the DOPA content in order to obtain a mussel adhesive protein with excellent biological activity.

[0004] However, there may be a problem of tyrosinase residue during this process, resulting in a reduction in the biosafety of the prepared mussel adhesive protein, which has great limitations. Summary of the Invention

[0005] The first object of the present invention is to provide a recombinant mussel adhesive protein. While having biological activity equivalent to or even better than that of existing mussel adhesive proteins, this recombinant mussel adhesive protein can directly utilize dopamine for dopamine incorporation and expression in the cells of common engineering bacteria such as Escherichia coli, so that the recombinant mussel adhesive protein is modified by dopamine, and there is no need for additional tyrosinase oxidation treatment, which can effectively meet the requirements of industry standards for the dopamine content of mussel adhesive protein and can effectively solve the problem of reduced biosafety caused by the introduction of tyrosinase in the preparation of existing mussel adhesive proteins.

[0006] The second object of the present invention is to provide a nucleic acid molecule.

[0007] The third object of the present invention is to provide a fermentation strain.

[0008] The fourth object of the present invention is to provide a method for preparing the above-mentioned recombinant mussel adhesive protein.

[0009] The fifth object of the present invention is to provide the application of the above-mentioned recombinant mussel adhesive protein, nucleic acid molecule and / or fermentation strain in the medical and / or cosmetic fields.

[0010] Specifically, the recombinant mussel adhesive protein provided by the present invention includes type III mussel adhesive protein and bioactive peptide segments, and the type III mussel adhesive protein and bioactive peptide segments are connected by peptide bonds; the bioactive peptide segments include one or more of fragment I, fragment II and fragment III, the amino acid sequence of fragment I is as shown in SEQ ID NO:2, the amino acid sequence of fragment II is as shown in SEQ ID NO:5, and the amino acid sequence of fragment III is as shown in SEQ ID NO:8.

[0011] Further, the amino acid sequence of the type III mussel adhesive protein is as shown in SEQ ID NO:1.

[0012] Further, the amino acid sequence of the recombinant mussel adhesive protein is as shown in SEQ ID NO:3.

[0013] Further, the amino acid sequence of the recombinant mussel adhesive protein is as shown in SEQ ID NO:6.

[0014] Further, the amino acid sequence of the recombinant mussel adhesive protein is as shown in SEQ ID NO:9.

[0015] Further, the dopamine content of the recombinant mussel adhesive protein is 0.3-1.4 wt%.

[0016] The nucleic acid molecule provided by the present invention encodes the above-mentioned recombinant mussel adhesive protein.

[0017] Further, the nucleic acid molecule includes one or more of the nucleotide fragments as shown in SEQ ID NO:4, 7 and 10.

[0018] The fermentation strain provided by the present invention synthesizes and expresses the above-mentioned recombinant mussel adhesive protein.

[0019] The method for preparing the recombinant mussel adhesive protein provided by the present invention includes: S1, introducing the above nucleic acid molecule into a host cell to obtain a fermentation strain; S2, performing fermentation culture and induction expression on the fermentation strain to obtain the recombinant mussel adhesive protein; wherein, dopamine and an induction expression agent are added in the induction expression.

[0020] Further, in step S2, the culture medium for fermentation culture is selected from one or more of LB medium, EMB medium, MAC medium and MS medium.

[0021] Further, in step S2, the inoculum size of the cells for fermentation culture is 0.5×10 5 ~1.5×10 5 cells / mL, the temperature is 25 - 37°C, and the time is 4 - 8 h.

[0022] Further, in step S2, in the induced expression, the concentration of dopamine is 0.1 - 1.0 g / L, and the concentration of the inducer is 0.1 - 1 mM.

[0023] Further, the temperature of the induced expression is 16 - 37°C, and the time is 4 - 10 h.

[0024] The present invention also provides the application of the above-mentioned recombinant mussel adhesive protein, nucleic acid molecule and / or fermentation strain in the medical and / or cosmetic fields.

[0025] Beneficial effects:

[0026] The recombinant mussel adhesive protein provided by the present invention specifically includes type III mussel adhesive protein and biofunctional peptide segments connected by peptide bonds, which can promote the intracellular dopamine incorporation expression of common engineering bacteria such as Escherichia coli, so that dopamine can be directly utilized in the cells to obtain a recombinant mussel adhesive protein with a high dopamine content. The dopamine content in the recombinant mussel adhesive protein reaches 0.3 - 1.4 wt%, meeting the requirements of the industry standard for dopamine content, effectively solving the problem of reduced biosafety caused by the introduction of tyrosinase in the preparation of existing mussel adhesive proteins, and the recombinant mussel adhesive protein has equivalent or better biological activity than the existing mussel adhesive proteins, with good application prospects. Description of the drawings

[0027] Figure 1 It is the gel electrophoresis diagram of the recombinant mussel adhesive protein provided in Examples 1 - 3 and Comparative Examples 1 - 4 of the present invention;

[0028] Figure 2 It is the experimental result diagram of the test on the cell migration promoting effect of the recombinant mussel adhesive protein provided in Example 4 of the present invention (scale is 100 μm);

[0029] Figure 3 It is the experimental result diagram of the test on the antibacterial effect of the recombinant mussel adhesive protein provided in Example 4 of the present invention;

[0030] Figure 4 It is the experimental result diagram of the test on the skin soothing effect of the recombinant mussel adhesive protein provided in Example 4 of the present invention. Detailed implementation manners

[0031] The amino acid and nucleotide sequences involved in this application are specifically shown in Table 1 as follows.

[0032] Table 1.

[0033]

[0034]

[0035]

[0036] The inventors of the present invention have found through extensive and in-depth research that by introducing a biofunctional peptide segment derived from a specific domain of fibronectin onto type III mussel adhesive protein, and enabling common engineering bacteria such as Escherichia coli to perform protein synthesis with dopa incorporation, a recombinant mussel adhesive protein with a high dopa content can be obtained. Therefore, this recombinant mussel adhesive protein does not need to additionally introduce tyrosinase for in vitro tyrosine modification, and can well meet the industry's requirements for dopa content, effectively solving the problem of reduced biosafety caused by residual tyrosinase in the prior art. Based on this, the technical solution of the present invention is obtained.

[0037] In the present invention, the recombinant mussel adhesive protein refers to a polypeptide similar to mussel adhesive protein prepared by genetic manipulation and / or modification of the gene encoding the required mussel adhesive protein using genetic recombination technology, so as to introduce a biofunctional peptide segment onto type III mussel adhesive protein; and using a plasmid or viral vector to introduce the target into an appropriate host cell for expression, and performing operations such as purification treatment.

[0038] In the present invention, the recombinant mussel adhesive protein specifically includes type III mussel adhesive protein and a biofunctional peptide segment, and the type III mussel adhesive protein and the biofunctional peptide segment are connected by a peptide bond.

[0039] In the present invention, the type III mussel adhesive protein is the type with the smallest molecular weight among the 6 types of mussel adhesive proteins, and its specific amino acid sequence is specifically shown in SEQ ID NO:1.

[0040] In the present invention, the biofunctional peptide segment includes one or more of fragment I, fragment II, and fragment III. Among them, fragment I is derived from the FN-C / H-II functional peptide segment of fibronectin, and its specific amino acid sequence is shown in SEQ ID NO:2. Fragment II is derived from the functional peptide segment of fibronectin's Type IIICS-1, and its specific amino acid sequence is shown in SEQ ID NO:5. Fragment III is derived from the functional peptide segment of fibronectin's Type IIICS-5, and its specific amino acid sequence is shown in SEQ ID NO:8.

[0041] It should be noted that the "I", "II", and "III" in the above-mentioned Fragment I, Fragment II, and Fragment III are only for distinguishing different biofunctional peptide segments for the convenience of description, and have no other meanings, nor do they affect the protection scope of the present invention.

[0042] In the present invention, the biofunctional peptide segment is specifically linked to the N-terminus and / or C-terminus of the type III mussel adhesive protein. In some embodiments, the biofunctional peptide segment is preferably linked to the C-terminus of the type III mussel adhesive protein. At this time, the recombinant mussel adhesive protein-like has high dopamine content and excellent bioactivities such as promoting cell migration, antioxidation, anti-inflammation, tyrosinase inhibition, and antibacterial.

[0043] In some specific embodiments, the amino acid sequence of the recombinant mussel adhesive protein-like is preferably as shown in SEQ ID NO: 3. At this time, the recombinant mussel adhesive protein-like has high dopamine content and excellent bioactivities such as promoting cell migration, antioxidation, anti-inflammation, tyrosinase inhibition, and antibacterial.

[0044] In some specific embodiments, the amino acid sequence of the recombinant mussel adhesive protein-like is preferably as shown in SEQ ID NO: 6. At this time, the recombinant mussel adhesive protein-like has high dopamine content and excellent bioactivities such as promoting cell migration, antioxidation, anti-inflammation, tyrosinase inhibition, and antibacterial.

[0045] In some specific embodiments, the amino acid sequence of the recombinant mussel adhesive protein-like is preferably as shown in SEQ ID NO: 9.

[0046] In the present invention, the dopamine content of the recombinant mussel adhesive protein-like is preferably 0.3 - 1.4 wt%, such as 0.3 wt%, 0.5 wt%, 0.7 wt%, 0.85 wt%, 0.88 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.18 wt%, 1.25 wt%, 1.37 wt%, 1.4 wt% or any value therebetween. At this time, the recombinant mussel adhesive protein-like has high dopamine content without in vitro modification by tyrosinase, well meeting the requirements of the industry standard for the dopamine content of the recombinant mussel adhesive protein-like, and there is no problem of low biosafety caused by tyrosinase residue.

[0047] For the purpose of preparing the above-mentioned recombinant mussel adhesive protein-like, the present invention also provides a nucleic acid molecule. The nucleic acid molecule includes a nucleotide fragment encoding the above-mentioned recombinant mussel adhesive protein-like.

[0048] In the present invention, the nucleic acid molecule preferably comprises one or more segments of the nucleotide fragments shown in SEQ ID NO: 4, 7, and 10. At this time, the nucleic acid molecule is a sequence optimized for Escherichia coli codons and can better synthesize and express the above-mentioned recombinant mussel adhesive protein in Escherichia coli cells.

[0049] In the present invention, the nucleic acid molecule is preferably constructed by introducing one or more segments of the nucleotide fragments shown in SEQ ID NO: 4, 7, and 10 into a plasmid vector. In some specific embodiments, specific examples of the plasmid vector include, but are not limited to, one or more of pET-28a, pET-30a, pET-32a, and pGEX4T-1.

[0050] For the purpose of preparing the above-mentioned recombinant mussel adhesive protein, the present invention also provides a fermentation strain. The fermentation strain can efficiently synthesize and express the above-mentioned recombinant mussel adhesive protein.

[0051] In the present invention, the fermentation strain comprises the above-mentioned nucleic acid molecule encoding the recombinant mussel adhesive protein and the corresponding protein expression system, enabling it to transcribe under the guidance of the above-mentioned nucleic acid molecule and translate using amino acids and exogenous dopamine based on the guidance of the transcription product, thereby obtaining a recombinant mussel adhesive protein with excellent biological activity and a high dopamine content.

[0052] In the present invention, the fermentation strain is a type of recipient cell commonly used in existing genetic engineering techniques, and those skilled in the art can make an adaptive selection according to actual needs. The present invention does not particularly limit its specific type, source, and culture conditions. In some specific embodiments, specific examples of the fermentation strain include, but are not limited to, one or more of Escherichia coli BL21, Escherichia coli BL21(DE3), and Escherichia coli BL21 Star(DE3).

[0053] For the purpose of preparing the above-mentioned recombinant mussel adhesive protein, the present invention also provides a method for preparing the recombinant mussel adhesive protein. The preparation method specifically includes: S1. Introduce the above-mentioned nucleic acid molecule into a host cell to obtain a fermentation strain; S2. Ferment and induce the expression of the fermentation strain to obtain the recombinant mussel adhesive protein; wherein, dopamine and an inducer are added during the induction of expression.

[0054] In the present invention, introducing the nucleic acid molecule into the host cell in step S1 is a technique commonly used in the field of genetic engineering. Those skilled in the art can make an adaptive selection according to actual needs, and the present invention does not particularly limit its specific steps, reagents, and conditions. In some specific embodiments, specific examples of the method for introducing the nucleic acid molecule into the host cell include, but are not limited to, one or more of calcium phosphate precipitation method, microinjection method, virus infection method, liposome-mediated infection method, and electroporation method.

[0055] In the present invention, the host cell is a type of recipient cell commonly used in existing genetic engineering technologies. Those skilled in the art can make an adaptive selection according to actual needs, and the present invention does not particularly limit its specific type, source, and culture conditions. In some specific embodiments, specific examples of the host cell include, but are not limited to, one or more of Escherichia coli BL21, Escherichia coli BL21(DE3), and Escherichia coli BL21 Star(DE3).

[0056] In the fermentation culture of step S2 in the present invention, those skilled in the art can make an adaptive adjustment to the culture medium and culture conditions according to the host cell used, which is a technical means commonly used in the prior art, and the present invention does not particularly limit it.

[0057] In some specific embodiments, specific examples of the culture medium used for the fermentation culture include, but are not limited to, one or more of LB medium, EMB medium, MAC medium, and MS medium.

[0058] In some specific embodiments, the conditions for the fermentation culture include that the cell inoculum amount is preferably 0.5×10 5 ~1.5×10 5 cells / mL, such as 0.5×10 5 cells / mL, 0.8×10 5 cells / mL, 1×10 5 cells / mL, 1.5×10 5 cells / mL or any value therebetween; the temperature is preferably 25 - 37°C, such as 25°C, 26°C, 28°C, 30°C, 31°C, 35°C, 37°C or any value therebetween; the time is preferably 4 - 8 h, such as 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h or any value therebetween. At this time, adopting the above fermentation culture conditions is beneficial to the synthesis and expression of proteins in the host cell, thereby obtaining recombinant mussel adhesive protein with a higher dopamine content.

[0059] In the present invention, the induced expression in step S2 specifically includes: adding dopa and an inducing expression agent to the culture solution after fermentation culture to induce the host cell to express recombinant mussel adhesive protein. Among them, the expression of the recombinant mussel adhesive protein includes normal utilization of amino acids for protein expression and dopa incorporation expression using dopa instead of tyrosine to introduce a certain content of dopa into the recombinant mussel adhesive protein.

[0060] In some specific embodiments, in the induced expression of step S2, the concentration of dopa is preferably 0.1 - 1.0 g / L, such as 0.1 g / L, 0.18 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.8 g / L, 1 g / L or any value between them. At this time, using the above dopa concentration is beneficial for the host cell to uptake and utilize dopa, so as to better achieve dopa incorporation expression, and thus obtain recombinant mussel adhesive protein with a higher dopa content.

[0061] In the present invention, in the induced expression of step S2, the inducing expression agent is a kind of substance commonly used in genetic engineering technology to induce high - level protein expression in host cells. Those skilled in the art can make adaptive selections according to actual needs, and the present invention does not particularly limit it. In some specific embodiments, specific examples of the inducer include but are not limited to: one or more of isopropyl thiogalactoside, lactose, and arabinose.

[0062] In some specific embodiments, in the induced expression of step S2, the concentration of the inducing expression agent is preferably 0.1 - 1 mM, such as 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.8 mM, 1 mM or any value between them. At this time, using the above concentration of the inducing expression agent can well induce the host cell to express recombinant mussel adhesive protein, and thus effectively improve the yield of recombinant mussel adhesive protein.

[0063] In some specific embodiments, the conditions of the induced expression include that the temperature is preferably 16 - 37 °C, such as 16 °C, 18 °C, 20 °C, 25 °C, 30 °C, 35 °C or any value between them; the time is preferably 4 - 10 h, such as 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h, 10 h or any value between them. At this time, using the above - mentioned induced expression conditions is beneficial for dopa incorporation expression in the host cell, so as to effectively increase the dopa incorporation amount, and finally obtain recombinant mussel adhesive protein with a higher dopa content.

[0064] The recombinant mussel adhesive protein provided by the present invention has good biological activities such as promoting cell migration, scavenging free radicals, anti - inflammation, antibacterial, inhibiting tyrosinase, and skin soothing, and thus can be used as an active ingredient for promoting skin repair.

[0065] The invention also provides the applications of the above recombinant mussel adhesive protein, nucleic acid molecule and / or fermentation strain in the medical and / or cosmetic fields.

[0066] Embodiments of the present invention will be described in detail below. The examples of the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. For those specific technologies or conditions not specified in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0067] Example 1

[0068] This example is used to illustrate a recombinant mussel adhesive protein and its preparation. The recombinant mussel adhesive protein includes type III mussel adhesive protein (the amino acid sequence is shown in SEQ ID NO: 1) and FN-C / H-II of fibronectin (the amino acid sequence is shown in SEQ ID NO: 2) connected by peptide bonds. The preparation of the recombinant mussel adhesive protein specifically includes:

[0069] 1. Construction of the fermentation strain:

[0070] (1) According to the amino acid sequence of the recombinant mussel adhesive protein (as shown in SEQ ID NO: 3), after codon optimization of the Escherichia coli expression system, a DNA fragment (the nucleotide is shown in SEQ ID NO: 4) capable of highly expressing the recombinant mussel adhesive protein in Escherichia coli was synthesized.

[0071] (2) After adding NdeⅠ and XhoⅠ restriction enzyme sites to both ends of the DNA fragment, it was synthesized, digested with NdeⅠ and XhoⅠ, and then recombined onto the pET-30a vector to obtain a recombinant plasmid.

[0072] (3) The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and then spread on a solid LB plate containing 100 mg / mL ampicillin (including 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15 g / L agar), and cultured overnight at 37 °C to obtain single colonies; a part of the cells in each single colony was taken for PCR identification and sequencing to obtain positive single colonies, and this positive single colony is the fermentation strain.

[0073] 2. Fermentation culture and induced expression of the fermentation strain:

[0074] (1) According to 1×10 5Pick a positive single colony with an inoculum of cfu / mL and inoculate it into LB liquid medium containing 100 mg / mL ampicillin (including 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride), and perform fermentation culture in a shaker at 37 °C and 250 r / min for 8 h to obtain a fermentation culture broth.

[0075] (2) Take IPTG and dopamine according to the addition amounts of final concentrations of 0.1 mM and 1 g / L and add them to the seed liquid, and perform induction expression in a shaker at 28 °C and 250 r / min for 10 h to obtain an induced expression broth.

[0076] 3. Purification of recombinant mussel adhesive protein:

[0077] (1) Take 50 mL of the induced expression broth and centrifuge it at 4 °C and 10000 r / min for 15 min to remove the supernatant; take 10 mL of lysis buffer (at 4 °C, including 10 mM PBS buffer (pH = 7.4), 10 mM EDTA, and 1% Triton X-100) and add it to the precipitate for resuspension to obtain a resuspended broth.

[0078] (2) Take the resuspended broth and use a high-pressure homogenizer to break it under ice bath until the broth is clear (pressure is 600 - 900 bar), then centrifuge the solution at 4 °C and 9000 r / min for 20 min to remove the supernatant to obtain protein inclusion bodies.

[0079] (3) Take a dissolution solution (including 10 mM HAc and 8 mM urea) to dissolve the protein inclusion bodies to obtain a protein dissolution solution; each time take 600 μL of the protein dissolution solution and add it to a G-25 desalting column until all the mixed solution is added, then take 10 mM PBS buffer (pH = 7.4) for elution, collect the eluate to obtain a purified recombinant mussel adhesive protein solution.

[0080] Figure 1 It is a gel electrophoresis experimental result diagram of the purified recombinant mussel adhesive protein solution. From Figure 1 it can be seen that the recombinant mussel adhesive protein provided in this example has a certain viscosity, a slower electrophoresis speed, its molecular weight is about 9.72 kD, and the obtained recombinant mussel adhesive protein has a higher purity.

[0081] Example 2

[0082] This example provides a recombinant mussel adhesive protein and its preparation. This recombinant mussel adhesive protein includes mussel adhesive protein type III (amino acid sequence as shown in SEQ ID NO:1) and fibronectin Type IIICS-1 (amino acid sequence as shown in SEQ ID NO:5) connected by peptide bonds.

[0083] The amino acid sequence of the recombinant mussel adhesive protein is as shown in SEQ ID NO: 6, and its preparation is basically the same as the preparation method provided in Example 1, except that the nucleotide of the DNA fragment used in the construction of the fermentation strain in "Step 1" is as shown in SEQ ID NO: 7, and other conditions are the same, thus obtaining the recombinant mussel adhesive protein.

[0084] Figure 1 It is the experimental result diagram of the gel electrophoresis for purifying the recombinant mussel adhesive protein solution. From Figure 1 it can be seen that the recombinant mussel adhesive protein provided in this example has a certain viscosity, a slow electrophoresis speed, its molecular weight is about 8.85 kD, and the obtained recombinant mussel adhesive protein has a high purity.

[0085] Example 3

[0086] This example provides a recombinant mussel adhesive protein and its preparation. The recombinant mussel adhesive protein includes type III mussel adhesive protein (amino acid sequence as shown in SEQ ID NO: 1) and fibronectin Type IIICS-5 (amino acid sequence as shown in SEQ ID NO: 8) connected by peptide bonds.

[0087] The amino acid sequence of the recombinant mussel adhesive protein is as shown in SEQ ID NO: 9, and its preparation is basically the same as the preparation method provided in Example 1, except that the nucleotide of the DNA fragment used in the construction of the fermentation strain in "Step 1" is as shown in SEQ ID NO: 10, and other conditions are the same, thus obtaining the recombinant mussel adhesive protein.

[0088] Figure 1 It is the experimental result diagram of the gel electrophoresis for purifying the recombinant mussel adhesive protein solution. From Figure 1 it can be seen that the recombinant mussel adhesive protein provided in this example has a certain viscosity, a slow electrophoresis speed, its molecular weight is about 8.50 kD, and the obtained recombinant mussel adhesive protein has a high purity.

[0089] Comparative Example 1

[0090] This comparative example provides a recombinant mussel adhesive protein and its preparation. The recombinant mussel adhesive protein includes type III mussel adhesive protein (amino acid sequence as shown in SEQ ID NO: 1) and fibronectin Type III-10 (amino acid sequence as shown in SEQ ID NO: 11) connected by peptide bonds.

[0091] The amino acid sequence of the recombinant mussel adhesive protein is shown in SEQ ID NO: 12, and its preparation is basically the same as the preparation method provided in Example 1, except that the nucleotides of the DNA fragment used in the construction of the fermentation strain in "Step 1" are shown in SEQ ID NO: 13, and other conditions are the same, resulting in the recombinant mussel adhesive protein.

[0092] Figure 1 It is the experimental result graph of the gel electrophoresis for purifying the recombinant mussel adhesive protein solution. From Figure 1 it can be seen that the molecular weight of the recombinant mussel adhesive protein provided in this comparative example is about 8.33 kD.

[0093] Comparative Example 2

[0094] This comparative example provides a recombinant mussel adhesive protein and its preparation. The recombinant mussel adhesive protein includes type III mussel adhesive protein (amino acid sequence shown in SEQ ID NO: 1) and FN-C / H-IV of fibronectin (amino acid sequence shown in SEQ ID NO: 14) connected by peptide bonds.

[0095] The amino acid sequence of the recombinant mussel adhesive protein is shown in SEQ ID NO: 15, and its preparation is basically the same as the preparation method provided in Example 1, except that the nucleotides of the DNA fragment used in the construction of the fermentation strain in "Step 1" are shown in SEQ ID NO: 16, and other conditions are the same, resulting in the recombinant mussel adhesive protein.

[0096] Figure 1 It is the experimental result graph of the gel electrophoresis for purifying the recombinant mussel adhesive protein solution. From Figure 1 it can be seen that the molecular weight of the recombinant mussel adhesive protein provided in this comparative example is about 9.02 kD.

[0097] Comparative Example 3

[0098] This comparative example provides a recombinant mussel adhesive protein and its preparation. The recombinant mussel adhesive protein includes type III mussel adhesive protein (amino acid sequence shown in SEQ ID NO: 1) and FN-C / H-V of fibronectin (amino acid sequence shown in SEQ ID NO: 17) connected by peptide bonds.

[0099] The amino acid sequence of the recombinant mussel adhesive protein is shown in SEQ ID NO: 18, and its preparation is basically the same as the preparation method provided in Example 1, except that the nucleotides of the DNA fragment used in the construction of the fermentation strain in "Step 1" are shown in SEQ ID NO: 19, and other conditions are the same, resulting in the recombinant mussel adhesive protein.

[0100] Figure 1This is the experimental result diagram of gel electrophoresis for purifying recombinant mussel adhesive protein solution. As can be seen from Figure 1 it, the molecular weight of the recombinant mussel adhesive protein provided in this comparative example is approximately 9.01 kD.

[0101] Comparative Example 4

[0102] This comparative example provides a recombinant mussel adhesive protein and its preparation. The amino acid sequence of the recombinant mussel adhesive protein is as shown in SEQ ID NO:1, and its preparation is basically the same as the preparation method provided in Example 1. The difference is that the nucleotides of the DNA fragment used in the construction of the fermentation strain in "Step 1" are as shown in SEQ ID NO:20, and other conditions are the same, obtaining the recombinant mussel adhesive protein.

[0103] Figure 1 This is the experimental result diagram of gel electrophoresis for purifying recombinant mussel adhesive protein solution. As can be seen from Figure 1 it, the molecular weight of the recombinant mussel adhesive protein provided in this comparative example is approximately 8.00 kD.

[0104] Example 4

[0105] In this example, the dopa content of the recombinant mussel adhesive protein provided in the above examples and comparative examples was determined according to the method specified in Appendix B of YY / T 1293.6 - 2020. The specific results are shown in Table 2.

[0106] Table 2.

[0107]

[0108]

[0109] As can be seen from the test results shown in Table 2, compared with the recombinant mussel adhesive protein provided in Comparative Example 4, the dopa content in the recombinant mussel adhesive protein provided in Examples 1 - 3 of the present invention is as high as 1.18 - 1.27 wt%, and the dopa content has increased by 2.11 - 2.45 times.

[0110] Example 5

[0111] This example is used to illustrate the biological activity of the recombinant mussel adhesive protein provided in the above examples and comparative examples. The recombinant mussel adhesive protein was mixed with PBS buffer (pH = 7.4) to prepare a protein solution of 0.5 μg / mL, and the following tests were carried out using this protein solution:

[0112] 1. Promoting cell migration effect: According to 1×10 5The Hacat cells were inoculated at an inoculum size of Figure 2 into DMEM medium (Merck, catalog number D6429) containing 10% FBS to obtain a cell suspension; 100 μL was taken and added into the cell wells, and inserts were inserted, followed by incubation at 37 °C for 24 h to form scratches; the inserts were removed, and the protein solution was taken and added into the wells at an addition amount with a final concentration of 0.2 mg / mL, and DMEM medium containing 10% FBS and PBS buffer (pH = 7.4) with equal volumes were used as the blank control and negative control respectively, and incubated at 37 °C for 24 h; the incubated cell plates were washed 3 times with PBS buffer (pH = 7.4), and the cell adhesion was observed under the microscope. The results are as

[0113] shown. Figure 2 As can be seen from the test results shown, compared with the negative control, the recombinant mussel adhesive protein provided in Examples 1-3 of the present invention has an obvious promoting effect on cell migration and can be used as an active ingredient for promoting skin repair.

[0114] 2. Free radical scavenging effect in vitro: The protein solution was taken and mixed with 0.1 mM DPPH solution at an addition amount with a final concentration of 0.8 mg / mL, and the DPPH free radical scavenging rate of the recombinant mussel adhesive protein was determined according to the method of "Cosmetics - Free Radical (DPPH) Scavenging Test Method". The results are shown in Table 3.

[0115] Table 3.

[0116] Group DPPH Free Radical Scavenging Rate (%) Example 1 72.6 Example 2 79.5 Example 3 78.9 Comparative Example 1 72.2 Comparative Example 2 66.3 Comparative Example 3 56.9 Comparative Example 4 73.4

[0117] As can be seen from the test results shown in Table 3, the recombinant mussel adhesive protein provided in Examples 1-3 of the present invention has an excellent DPPH free radical scavenging rate and can be used as an antioxidant active ingredient.

[0118] 3. Anti-inflammatory effect: RAW264.7 macrophages were used as the cell model and were treated according to the following groups:

[0119] (1) Blank control group: RAW264.7 macrophages were inoculated at an inoculum size of 1×10 5 cells / mL into DMEM medium containing 10% FBS and cultured in an incubator at 37 °C and 5% CO2 for 24 h;

[0120] (2) Inflammatory model group: On the basis of the treatment of the blank control group, LPS with a final concentration of 1 μg / mL was added to the DMEM medium for inflammation induction treatment;

[0121] (3) Experimental group: On the basis of the treatment of the inflammatory model group, the recombinant mussel adhesive protein provided in the above examples with a final concentration of 60 μg / mL was added to the DMEM medium;

[0122] (4) Comparative experimental group: On the basis of the treatment of the inflammation model group, recombinant mussel adhesive protein provided in the above comparative examples with a final concentration of 60 μg / mL was further added to the DMEM medium;

[0123] (5) Positive control group: On the basis of the treatment of the inflammation model group, dexamethasone with a final concentration of 0.1 mg / mL was further added to the DMEM medium.

[0124] After the culture was completed, the cell culture media of each group were centrifuged, the supernatant was collected, and a nitric oxide (NO) assay kit (Shanghai Xuanya Biotechnology, product number 012-1) was used to detect the NO content in the supernatant with reference to the instructions. The test results are shown in Table 4.

[0125] Table 4.

[0126]

[0127] As can be seen from the test results shown in Table 4, the NO content in the cell culture medium of the inflammation model group was significantly increased compared with that of the blank control group, indicating that the inflammatory cell model was successfully constructed. Compared with Comparative Examples 1-3, the recombinant mussel adhesive protein provided in Examples 1-3 of the present invention was used to co-treat RAW264.7 macrophages with LNP, which could significantly relieve the cell inflammation caused by the action of LNP, had excellent anti-inflammatory effects, and could be used as an active ingredient for repairing inflamed skin.

[0128] 4. Bacteriostatic effect: The protein solution was taken at an addition amount with a final concentration of 0.5 mg / mL and mixed with Propionibacterium acnes bacterial solution with a cell concentration of 1×10 5 cells / mL. DPBS solution was used as the blank control, and tetracycline with a final concentration of 0.1 mg / mL was used as the positive control. Finally, DPBS solution was added to make up to 200 μL to obtain a mixed culture solution; 150 μL of the mixed culture solution was taken for plate coating and cultured in an anaerobic incubator at 35 °C for 48 h, and the cell adhesion was observed under the microscope. The results are as Figure 3 shown.

[0129] From Figure 3 the test results shown, it can be seen that the recombinant mussel adhesive protein provided in Examples 1-3 of the present invention has excellent bacteriostatic effects on Propionibacterium acnes and can be used as an active ingredient for removing acne and inhibiting bacteria.

[0130] 5. Tyrosinase inhibition effect: The protein solution was taken at an addition amount with a final concentration of 0.8 mg / mL and mixed with 0.1 mM tyrosine solution, and the tyrosinase inhibition rate of the recombinant mussel adhesive protein was determined according to the method of "Cosmetics - Test Method for Tyrosinase Activity Inhibition". The results are shown in Table 5.

[0131] Table 5.

[0132] Group Tyrosinase Inhibition Rate (%) Example 1 81.1 Example 2 84.0 Example 3 85.5 Comparative Example 1 41.9 Comparative Example 2 50.4 Comparative Example 3 70.8 Comparative Example 4 45.8

[0133] As can be seen from the test results shown in Table 5, the recombinant mussel adhesive protein provided in Examples 1-3 of the present invention has excellent inhibitory effects on tyrosinase and can be used as a whitening active ingredient.

[0134] 6. Skin soothing effect: Drop 100 μg of 30% acetic acid solution onto a 1 cm * 2 cm cotton pad, apply it to the inner area of the subject's arm, remove the cotton pad after 5 minutes, immediately apply the same volume of protein solution, and use the untreated area as a blank control; take pictures at 0 min, 1 h, and 6 h after application. The results are as Figure 4 shown.

[0135] As Figure 4 can be seen from the test results shown, the recombinant mussel adhesive protein provided in Examples 1-3 of the present invention has good protective and accelerating recovery effects on the skin damaged by acetic acid solution stimulation and has excellent skin soothing effects.

[0136] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. Use of recombinant mussel adhesive protein in the preparation of tyrosinase inhibitors, characterized in that, The recombinant mussel adhesive protein comprises type III mussel adhesive protein and a biofunctional peptide segment, and the type III mussel adhesive protein and the biofunctional peptide segment are connected by a peptide bond; the amino acid sequence of the type III mussel adhesive protein is as shown in SEQ ID NO: 1; the biofunctional peptide segment is selected from amino acid fragments with sequences as shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 17 or SEQ ID NO: 20; the dopamine content of the recombinant mussel adhesive protein is 0.3-1.4 wt%; the sequence of the nucleic acid molecule encoding the recombinant mussel adhesive protein is as shown in SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 16 or SEQ ID NO:

19.

2. Use of the recombinant mussel adhesive protein according to claim 1 in the preparation of a tyrosinase inhibitor, characterized in that, The preparation method of the recombinant mussel adhesive protein comprises: S1. introducing the nucleic acid molecule described in claim 1 into a host cell to obtain a fermentation strain; S2. performing fermentation culture and induction expression on the fermentation strain to obtain the recombinant mussel adhesive protein; wherein, dopamine and IPTG are added in the induction expression.

3. Use of the recombinant mussel adhesive protein according to claim 2 in the preparation of a tyrosinase inhibitor, characterized in that, In step S2, the culture medium for the fermentation culture is selected from one or more of LB medium, EMB medium, MAC medium and MS medium.

4. Use of the recombinant mussel adhesive protein according to claim 2 in the preparation of a tyrosinase inhibitor, characterized in that, In step S2, the inoculum size of the cells for fermentation culture is 0.5×10 5 ~1.5×10 5 cells / mL, the temperature is 25~37 °C, and the time is 4~8 h.

5. Use of the recombinant mussel adhesive protein according to claim 2 in the preparation of a tyrosinase inhibitor, characterized in that, In step S2, in the induction expression, the concentration of dopamine is 0.1-1.0 g / L, and the concentration of IPTG is 0.1-1 mM.

6. Use of the recombinant mussel adhesive protein according to claim 2 in the preparation of a tyrosinase inhibitor, characterized in that, In step S2, the temperature of the induction expression is 16-37 °C, and the time is 4-10 h.

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