A method for preparing high purity recombinant mytilus edulis mucin

Abstract

The application discloses a preparation method of high-purity recombinant mussel adhesive protein and belongs to the technical field of recombinant protein preparation. The amino acid sequence of Mfp-3 of Mytilus coruscus and the partial peptide segment of the amino acid sequence of preCol-D of Mytilus edulis are fused and expressed according to the idea of improving target protein expression by using fusion protein, and the improvement of the induction, tyrosinase modification and separation and purification process is combined to obtain the recombinant mussel adhesive protein with high purity, low endotoxin content and good adhesion performance. The successful preparation of the recombinant mussel adhesive protein provides a new idea for the industrial production of the recombinant mussel adhesive protein, provides raw material support for the multi-field application of the recombinant mussel adhesive protein, realizes the preparation of the customized sequence recombinant mussel adhesive protein according to application requirements, and promotes the application of the mussel adhesive protein in different fields.

Description

Technical Field

[0001] The invention belongs to the technical field of recombinant protein preparation, and particularly relates to a method for preparing high-purity recombinant mussel mucin. Background Art

[0002] Mussel adhesive protein (MAP), also known as mussel byssin, is produced and stored in the glands of the mussel's foot. It enables mussels to adhere and survive in water. Its high strength, toughness, water resistance, biodegradability, and exceptional adhesiveness hold great promise for biotechnology applications. The content of DOPA (3,4-dihydroxyphenylalanine) is crucial to the adhesive function of mussel adhesive protein. Its non-toxic and non-immunogenic properties offer broad potential for application in medical and aesthetic medicine, including promoting cell adhesion and crawling, wound healing, inhibiting itching, achieving broad-spectrum adhesion, and forming a water-resistant protective film. It has broad applications in surface chemistry, biomedicine, marine engineering, and daily chemical products.

[0003] Currently, the most direct method for obtaining mussel mucin is to directly extract the natural adhesive protein component from the mussel foot gland. However, due to the very low secretion of mussel byssal protein, only about 1 mg of adhesive protein can be extracted from 10,000 mussels. This directly extracted adhesive product is expensive and prone to solidification, thus limiting its application. Genetic engineering is another effective method for obtaining mussel mucin, but the mussel mucin obtained also suffers from low expression levels and unsatisfactory adhesive properties. Therefore, how to obtain mussel mucin with high purity, good biosafety, and excellent adhesive properties is an important research direction for the future. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing high-purity recombinant mussel mucin in response to the existing problems.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing high-purity recombinant mussel mucin comprises the following steps:

[0007] S1. Based on the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system. The designed nucleotide sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then ligated into the pET28a empty plasmid.

[0008] S2. The recombinant product was transformed into E. coli TOP10 competent cells, and single colonies with positive bands were screened on Kana resistance plates. The plasmid was extracted and the correct plasmid was sequenced as the constructed pET28a-Mfp3D expression vector;

[0009] S3. The obtained pET28a-Mfp3D expression vector was transformed into the expression host BL21 (DE3) competent cells, and single colonies were obtained by Kana resistance plate screening. The positive strains were verified by PCR gel electrophoresis and sequenced to be the engineered bacteria expressing recombinant mussel viscose protein;

[0010] S4, inoculating the recombinant mussel mucin-expressing engineered bacteria into LB medium for fermentation to obtain a recombinant bacterial fermentation liquid;

[0011] S5. The obtained recombinant bacterial fermentation broth was shake-cultured to OD600 = 0.6-0.8, an inducer was added, and the culture was placed in a shaker for induction culture. After the induction culture, the bacteria were collected, resuspended in buffer A and ultrasonically disrupted, and the supernatant was collected after centrifugation. The supernatant was passed through a nickel column twice, and then impurities were eluted with buffer B. Finally, the target protein was recovered with buffer C;

[0012] S6. Add a modifying agent to the buffer C containing the target protein in step S5, react at room temperature for 6 hours, and collect the modified enzyme reaction solution;

[0013] S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified using a Ni-NTA column affinity chromatography method, and finally freeze-dried to obtain a pure freeze-dried powder.

[0014] Preferably, the amino acid sequence of the recombinant mussel mucin in step S1 is as shown in SEQ ID NO.1.

[0015] Preferably, the recombinant mussel mucin in step S1 is composed of partial peptide segments of the amino acid sequence of Mytilus thunbergii Mfp-3 and the amino acid sequence of Mytilus edulis preCol-D;

[0016] The GenBank of Mfp-3 is ADB79747.1, and the GenBank of preCol-D is AAB96638.1.

[0017] Preferably, the recombinant mussel mucin in step S1 is named Mfp-3D, with a theoretical molecular weight of 34.52 kDa and a theoretical isoelectric point of 9.72.

[0018] Preferably, the formula of the LB medium in step S4 is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin.

[0019] Preferably, the fermentation temperature in step S4 is 35-38°C.

[0020] Preferably, the inducer in step S5 is lactose, and the concentration of lactose is 0.6-1.0 mM;

[0021] The induction conditions are: using 1-2% glycerol as a carbon source, shaking the bacteria at 28-32° C. and 200-220 rpm for 10-16 hours.

[0022] Preferably, the buffer A in step S5 comprises: imidazole 8-10 mM, urea 6-10 M, NaH2PO4 96-106 mM, Tris-HCl 8-12 mM, pH 7.7-8.2;

[0023] Buffer B: imidazole 25-35 mM, urea 6-10 M, NaH2PO4 96-106 mM, Tris-HCl 8-12 mM, pH 6-6.8;

[0024] Buffer C: imidazole 250-350 mM, urea 6-10 M, NaH2PO4 96-106 mM, Tris-HCl 8-12 mM, pH 4.3-4.9;

[0025] The ultrasonic fragmentation time is 8 to 10 minutes;

[0026] The centrifugation conditions are 4°C, 11000-13000 rpm, and centrifugation for 20-36 min.

[0027] Preferably, the composition of the modifier in step S6 is: 20-26 μM copper sulfate, 10-12 mM ascorbic acid, 60-70 mM Chinese herbal medicine extract, and 1-2% tyrosinase by substrate volume.

[0028] Preferably, the preparation method of the traditional Chinese medicine extract is: 60-80 parts of angelica, 80-90 parts of sunflower, 30-40 parts of honeysuckle, 4-10 parts of salvia miltiorrhiza, and 1-3 parts of tripterygium wilfordii are respectively deep-cold-crushed at -100--60°C, respectively added with water, stirred evenly, and then heated to boil, kept boiling for 10-16 minutes, simmered on low heat for 8-10 hours, filtered, and the filtrate was concentrated to 1-2 g / mL.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] The present invention adopts the idea of ​​using fusion proteins to improve the expression of target proteins. The amino acid sequence of thick-shelled mussel Mfp-3 and partial peptide segments of the amino acid sequence of blue mussel preCol-D are fused and expressed, and combined with improvements in induction, tyrosinase modification, and separation and purification processes, a recombinant mussel mucin with high purity, low endotoxin content, and good adhesion performance is obtained. The DOPA content is determined to be 7.72% after identification. The successful preparation of the recombinant mussel mucin of the present invention provides a new idea for the industrial production of recombinant mussel mucin. The fusion expression method is used to achieve industrial production of various types of mussel mucin using an Escherichia coli expression system. In this way, more recombinant mussel mucins that can be industrially produced and have more functional application prospects are developed, providing raw material support for the multi-field application of recombinant mussel mucin, realizing the preparation of customized sequence recombinant mussel mucin according to application requirements, and promoting the application of mussel mucin in different fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the SDS-PAGE electrophoresis detection diagram of a 2 mg / mL sample of the recombinant mussel mucin lyophilized powder obtained in Example 2. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] Example 1

[0034] A method for preparing high-purity recombinant mussel mucin comprises the following steps:

[0035] S1. Based on the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system. The designed nucleotide sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then ligated into the pET28a empty plasmid.

[0036] The recombinant mussel mucin is composed of a partial peptide segment of the amino acid sequence of Mytilus thunbergii Mfp-3 (GenBank: ADB79747.1) and the amino acid sequence of Mytilus edulis preCol-D (GenBank: AAB96638.1), named Mfp-3D, with a theoretical molecular weight of 34.52 kDa and a theoretical isoelectric point of 9.72.

[0037] The amino acid sequence of the recombinant mussel mucin is shown in SEQ ID NO.1;

[0038] S2. The recombinant product was transformed into E. coli TOP10 competent cells, and single colonies with positive bands were screened on Kana resistance plates. The plasmid was extracted and the correct plasmid was sequenced as the constructed pET28a-Mfp3D expression vector;

[0039] S3. The obtained pET28a-Mfp3D expression vector was transformed into the expression host BL21 (DE3) competent cells, and single colonies were obtained by Kana resistance plate screening. The positive strains were verified by PCR gel electrophoresis and sequenced to be the engineered bacteria expressing recombinant mussel viscose protein;

[0040] S4, inoculating the recombinant mussel mucin-expressing engineered bacteria into LB medium, and fermenting at 35-38° C. to obtain a recombinant bacterial fermentation liquid;

[0041] The LB medium contains 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin.

[0042] S5. The obtained recombinant bacterial fermentation broth was cultured by shaking to OD600 = 0.6-0.8, 0.6 mM lactose was added, and 1% glycerol was used as a carbon source. The culture was placed in a shaker at 28°C and 200 rpm for 10 h. After the induction culture, the cells were collected, resuspended in buffer A and ultrasonically disrupted for 8 min, and then centrifuged at 4°C and 11000 rpm for 20 min. The supernatant was collected and passed through a nickel column twice, and then impurities were eluted with buffer B. Finally, the target protein was recovered with buffer C;

[0043] Buffer A: imidazole 8mM, urea 6M, NaH2PO4 96mM, Tris-HCl 8mM, pH = 7.7;

[0044] Buffer B: imidazole 25 mM, urea 6 M, NaH2PO4 96 mM, Tris-HCl 8 mM, pH = 6;

[0045] Buffer C: imidazole 250 mM, urea 6 M, NaH2PO4 96 mM, Tris-HCl 8 mM, pH = 4.3;

[0046] S6. Add a modifying agent to the buffer C containing the target protein in step S5, react at room temperature for 6 hours, and collect the modified enzyme reaction solution;

[0047] The composition of the modifier is: copper sulfate 20 μM, ascorbic acid 10 mM, Chinese herbal medicine extract 60 mM, tyrosinase 1% substrate volume;

[0048] Preferably, the preparation method of the traditional Chinese medicine extract is as follows: 60 parts of chamomile, 80 parts of sunflower, 30 parts of honeysuckle, 4 parts of salvia miltiorrhiza, and 1 part of tripterygium wilfordii are respectively crushed under -100°C, respectively added with water, stirred and mixed, and then heated to boil, kept boiling for 10 minutes, simmered on low heat for 8 hours, filtered, and the filtrate was concentrated to 1g / mL;

[0049] S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified using a Ni-NTA column affinity chromatography method, and finally freeze-dried to obtain a pure freeze-dried powder.

[0050] Example 2

[0051] A method for preparing high-purity recombinant mussel mucin comprises the following steps:

[0052] S1. Based on the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system. The designed nucleotide sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then ligated into the pET28a empty plasmid.

[0053] The recombinant mussel mucin is composed of a partial peptide segment of the amino acid sequence of Mytilus thunbergii Mfp-3 (GenBank: ADB79747.1) and the amino acid sequence of Mytilus edulis preCol-D (GenBank: AAB96638.1), named Mfp-3D, with a theoretical molecular weight of 34.52 kDa and a theoretical isoelectric point of 9.72.

[0054] The amino acid sequence of the recombinant mussel mucin is shown in SEQ ID NO.1;

[0055] S2. The recombinant product was transformed into E. coli TOP10 competent cells, and single colonies with positive bands were screened on Kana resistance plates. The plasmid was extracted and the correct plasmid was sequenced as the constructed pET28a-Mfp3D expression vector;

[0056] S3. The obtained pET28a-Mfp3D expression vector was transformed into the expression host BL21 (DE3) competent cells, and single colonies were obtained by Kana resistance plate screening. The positive strains were verified by PCR gel electrophoresis and sequenced to be the engineered bacteria expressing recombinant mussel viscose protein;

[0057] S4, inoculating the recombinant mussel mucin-expressing engineered bacteria into LB medium, and fermenting at 35-38° C. to obtain a recombinant bacterial fermentation liquid;

[0058] The LB medium contains 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin.

[0059] S5. The obtained recombinant bacterial fermentation broth was cultured by shaking to OD600 = 0.6-0.8, 0.8 mM lactose was added, 1.5% glycerol was used as a carbon source, and the mixture was placed in a shaker at 30°C, 210 rpm for 13 h. After the induction culture, the cells were collected, resuspended in buffer A and ultrasonically disrupted for 9 min, then centrifuged at 4°C, 12000 rpm for 28 min, and the supernatant was collected. The supernatant was passed through a nickel column twice, and then impurities were eluted with buffer B. Finally, the target protein was recovered with buffer C;

[0060] Buffer A: imidazole 9 mM, urea 8 M, NaH2PO4 101 mM, Tris-HCl 10 mM, pH = 7.9;

[0061] Buffer B: imidazole 30 mM, urea 8 M, NaH2PO4 101 mM, Tris-HCl 10 mM, pH = 6.4;

[0062] Buffer C: imidazole 300 mM, urea 8 M, NaH2PO4 101 mM, Tris-HCl 10 mM, pH = 4.6;

[0063] S6. Add a modifying agent to the buffer C containing the target protein in step S5, react at room temperature for 6 hours, and collect the modified enzyme reaction solution;

[0064] The composition of the modifier is: copper sulfate 23 μM, ascorbic acid 11 mM, Chinese herbal medicine extract 65 mM, tyrosinase at 1.5% substrate volume;

[0065] Preferably, the preparation method of the traditional Chinese medicine extract is as follows: 70 parts of chamomile, 85 parts of sunflower, 35 parts of honeysuckle, 7 parts of salvia miltiorrhiza, and 2 parts of tripterygium wilfordii are respectively crushed under -80°C, respectively added with water, stirred and mixed, then heated to boil, kept boiling for 13 minutes, simmered on low heat for 9 hours, filtered, and the filtrate was concentrated to 1.5g / mL;

[0066] S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified using a Ni-NTA column affinity chromatography method, and finally freeze-dried to obtain a pure freeze-dried powder.

[0067] Example 3

[0068] A method for preparing high-purity recombinant mussel mucin comprises the following steps:

[0069] S1. Based on the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system. The designed nucleotide sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then ligated into the pET28a empty plasmid.

[0070] The recombinant mussel mucin is composed of a partial peptide segment of the amino acid sequence of Mytilus thunbergii Mfp-3 (GenBank: ADB79747.1) and the amino acid sequence of Mytilus edulis preCol-D (GenBank: AAB96638.1), named Mfp-3D, with a theoretical molecular weight of 34.52 kDa and a theoretical isoelectric point of 9.72.

[0071] The amino acid sequence of the recombinant mussel mucin is shown in SEQ ID NO.1;

[0072] S2. The recombinant product was transformed into E. coli TOP10 competent cells, and single colonies with positive bands were screened on Kana resistance plates. The plasmid was extracted and the correct plasmid was sequenced as the constructed pET28a-Mfp3D expression vector;

[0073] S3. The obtained pET28a-Mfp3D expression vector was transformed into the expression host BL21 (DE3) competent cells, and single colonies were obtained by Kana resistance plate screening. The positive strains were verified by PCR gel electrophoresis and sequenced to be the engineered bacteria expressing recombinant mussel viscose protein;

[0074] S4, inoculating the recombinant mussel mucin-expressing engineered bacteria into LB medium, and fermenting at 35-38° C. to obtain a recombinant bacterial fermentation liquid;

[0075] The LB medium contains 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin.

[0076] S5. The obtained recombinant bacterial fermentation broth was cultured by shaking to OD600 = 0.6-0.8, 1.0 mM lactose was added, 2% glycerol was used as a carbon source, and the mixture was placed in a shaker at 32°C, 220 rpm for 16 h. After the induction culture, the cells were collected, resuspended in buffer A and ultrasonically disrupted for 10 min, and then centrifuged at 4°C, 13000 rpm for 36 min. The supernatant was collected and passed through a nickel column twice, and then impurities were eluted with buffer B. Finally, the target protein was recovered with buffer C;

[0077] Buffer A: imidazole 10 mM, urea 10 M, NaH2PO4 106 mM, Tris-HCl 12 mM, pH = 8.2;

[0078] Buffer B: imidazole 35 mM, urea 10 M, NaH2PO4 106 mM, Tris-HCl 12 mM, pH = 6.8;

[0079] Buffer C: imidazole 350 mM, urea 10 M, NaH2PO4 106 mM, Tris-HCl 12 mM, pH = 4.9;

[0080] S6. Add a modifying agent to the buffer C containing the target protein in step S5, react at room temperature for 6 hours, and collect the modified enzyme reaction solution;

[0081] The composition of the modifier is: copper sulfate 26 μM, ascorbic acid 12 mM, Chinese herbal medicine extract 70 mM, tyrosinase at 2% substrate volume;

[0082] Preferably, the preparation method of the traditional Chinese medicine extract is as follows: 80 parts of chamomile, 90 parts of sunflower, 40 parts of honeysuckle, 10 parts of salvia miltiorrhiza, and 3 parts of tripterygium wilfordii are respectively crushed under -60°C, respectively added with water, stirred and mixed, and then heated to boil, kept boiling for 16 minutes, simmered on low heat for 8-10 hours, filtered, and the filtrate was concentrated to 2g / mL;

[0083] S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified using a Ni-NTA column affinity chromatography method, and finally freeze-dried to obtain a pure freeze-dried powder.

[0084] Comparative Example 1

[0085] On the basis of Example 2, no Chinese herbal medicine extract is added to the modifier, and the rest of the technical solutions remain consistent with those of Example 2.

[0086] Comparative Example 2

[0087] On the basis of Example 2, copper sulfate is not added to the modifier, and the rest of the technical solutions are consistent with those of Example 2.

[0088] 1. Endotoxin Content Determination

[0089] The pure recombinant mussel mucin freeze-dried powder of Examples 1 to 3 and Comparative Examples 1 to 2 was tested. The endotoxin content was determined according to the bacterial endotoxin test method. The amount of endotoxin in each 1 mg sample should be less than 10 EU.

[0090] Weigh 0.050 g of recombinant mussel mucin into a pyrogen-free test tube and add 10 mL of bacterial endotoxin test water to a concentration of 5 mg mL -1 At the same time, prepare the control solution: 2λ / test water (λ is the sensitivity of the limulus amebocyte lysate, unit is EU·mL -1Dissolve one vial of bacterial endotoxin working standard in endotoxin test water and dilute to a standard solution containing 2λ of endotoxin. Prepare a 2λ / test solution by dissolving 0.05g of recombinant mussel mucin in 10mL of 2λ / test water. Prepare endotoxin-free / test water. Prepare two vials for each sample for endotoxin determination.

[0091] The test results are shown in Table 1 below.

[0092] Table 1 Endotoxin test results

[0093]

[0094] From Table 1 above, it can be concluded that the endotoxin content of the recombinant mussel mucin prepared by the method of the present application is less than 10EU·mg -1 , in line with the requirements for endotoxin content in the T / CASME 530-2023 standard.

[0095] 2. DOPA Content Determination

[0096] DOPA content was determined using a modified Arnow method, as follows:

[0097] First, 20 mg of levodopa standard was dissolved in 0.012 mol / L hydrochloric acid to 100 mL. 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL were respectively taken into a 10 mL volumetric flask, and diluted to the scale with 0.012 mol / L hydrochloric acid. The solution was shaken to obtain a series of standard solutions with dopa concentrations of 0, 2, 10, 20, 30, 40, 50, and 60 μg / mL.

[0098] Then, 0.025 g of pure recombinant mussel mucin freeze-dried powder was weighed and dissolved in 0.012 mol / L hydrochloric acid to a volume of 50 mL to obtain a recombinant mussel mucin solution with a concentration of 0.5 mg / mL.

[0099] Finally, the series of standard solutions and the recombinant mussel mucin solution were placed in test tubes. 0.5 mL of 0.516 mol / L hydrochloric acid was added to each test tube, followed by 1.5 mL of nitrite reagent. After 5 minutes, 2 mL of 1 mol / L sodium hydroxide was added and shaken thoroughly. The absorbance was measured at 500 nm. Data analysis was performed using Origin 9.0 software. A standard curve was constructed with DOPA concentration as the abscissa and absorbance as the ordinate. The DOPA content (W) of the sample was then calculated based on the measured absorbance values ​​of the sample solutions.

[0100]

[0101] Where, W is the percentage of DOPA in the test sample; C iis the DOPA concentration in the sample solution calculated from the standard curve, in μg·mL -1 ; M is the sample weight of recombinant mussel mucin, in mg.

[0102] The test results are shown in Table 2 below.

[0103] Table 2 DOPA content of recombinant mussel mucin in each group

[0104] Dopa content (%) <![CDATA[多巴浓度(μg·mL -1 )]]> Example 1 7.22 36.12 Example 2 7.72 38.63 Example 3 7.54 37.69 Comparative Example 1 6.25 31.26 Comparative Example 2 5.54 27.69

[0105] As can be seen from Table 2 above, the recombinant mussel mucin prepared by the method of the present invention has a high DOPA content, indicating that the recombinant mussel mucin prepared by the present invention has good adhesion performance.

[0106] In addition, when comparing Examples 1 to 3 with Comparative Examples 1 to 2, it was found that the DOPA content in Examples 1 to 2 was more significant, and there was also a certain gap between Comparative Example 1 and Comparative Example 2, indicating that the Chinese herbal extract of the present invention and copper act synergistically with each other, exhibiting a good activation effect on tyrosinase, and effectively improving the modification effect of tyrosinase.

[0107] 3. Protein Purity Analysis

[0108] Protein purity is an important testing indicator for recombinant protein products. It not only evaluates the effectiveness of the preparation process, but also plays an important role in monitoring the quality attributes of the product.

[0109] Purity was determined by SDS polyacrylamide gel electrophoresis.

[0110] The recombinant mussel mucin solution purified in Example 2 was subjected to SDS-polyacrylamide gel electrophoresis verification, and the results were as follows: Figure 1 As shown, the protein size is consistent with the predicted size of Mfp-3D protein, which is between 25 kDa and 35 kDa, close to the predicted protein molecular weight of approximately 34.52 kDa.

[0111] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing high-purity recombinant mussel mucin, characterized in that: The steps include: S1. Based on the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system. The designed nucleotide sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then ligated into the pET28a empty plasmid. The amino acid sequence of the recombinant mussel mucin is shown in SEQ ID NO.1; S2. The recombinant product was transformed into E. coli TOP10 competent cells, and single colonies with positive bands were screened on Kana resistance plates. The plasmid was extracted and the correct plasmid was sequenced as the constructed pET28a-Mfp3D expression vector; S3. The obtained pET28a-Mfp3D expression vector was transformed into the expression host BL21 (DE3) competent cells, and single colonies were obtained by Kana resistance plate screening. The positive strains were verified by PCR gel electrophoresis and sequenced to be the engineered bacteria expressing recombinant mussel viscose protein; S4, inoculating the recombinant mussel mucin-expressing engineered bacteria into LB medium for fermentation to obtain a recombinant bacterial fermentation liquid; S5. The obtained recombinant bacterial fermentation broth was shake-cultured to OD600 = 0.6-0.8, an inducer was added, and the culture was placed in a shaker for induction culture. After the induction culture, the bacteria were collected, resuspended in buffer A and ultrasonically disrupted, and the supernatant was collected after centrifugation. The supernatant was passed through a nickel column twice, and then impurities were eluted with buffer B. Finally, the target protein was recovered with buffer C; The buffer A comprises: imidazole 8-10 mM, urea 6-10 M, NaH2PO4 96-106 mM, Tris-HCl 8-12 mM, pH 7.7-8.2; Buffer B: imidazole 25-35 mM, urea 6-10 M, NaH2PO4 96-106 mM, Tris-HCl 8-12 mM, pH 6-6.8; Buffer C: imidazole 250-350 mM, urea 6-10 M, NaH2PO4 96-106 mM, Tris-HCl 8-12 mM, pH 4.3-4.9; S6. Add a modifying agent to the buffer C containing the target protein in step S5, react at room temperature for 6 hours, and collect the modified enzyme reaction solution; The modifier comprises: 20-26 μM copper sulfate, 10-12 mM ascorbic acid, 60-70 mM Chinese herbal extract, and 1-2% tyrosinase in a substrate volume; The preparation method of the traditional Chinese medicine extract comprises: deep-cold-crushing 60-80 parts of chamomile, 80-90 parts of sunflower, 30-40 parts of honeysuckle, 4-10 parts of salvia miltiorrhiza, and 1-3 parts of tripterygium wilfordii at -100--60°C, adding water to each of the mixtures, stirring evenly, and then heating and boiling for 10-16 minutes, simmering on low heat for 8-10 hours, filtering, and concentrating the filtrate to 1-2 g / mL; S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified using a Ni-NTA column affinity chromatography method, and finally freeze-dried to obtain a pure freeze-dried powder.

2. The method for preparing a high-purity recombinant mussel mucin according to claim 1, wherein The recombinant mussel mucin in step S1 is composed of a partial peptide segment of the amino acid sequence of Mytilus thunbergii Mfp-3 and the amino acid sequence of Mytilus edulis preCol-D; The GenBank of Mfp-3 is ADB79747.1, and the GenBank of preCol-D is AAB96638.

1.

3. The method for preparing a high-purity recombinant mussel mucin according to claim 1, wherein The recombinant mussel mucin described in step S1 is named Mfp-3D, with a theoretical molecular weight of 34.52 kDa and a theoretical isoelectric point of 9.

72.

4. The method for preparing a high-purity recombinant mussel mucin according to claim 1, wherein The formula of the LB medium described in step S4 is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin.

5. The method for preparing a high-purity recombinant mussel mucin according to claim 1, characterized in that: The fermentation temperature in step S4 is 35-38°C.

6. The method for preparing a high-purity recombinant mussel mucin according to claim 1, characterized in that: The inducer in step S5 is lactose, and the concentration of lactose is 0.6-1.0 mM; The induction conditions are: using 1-2% glycerol as a carbon source, shaking the bacteria at 28-32° C. and 200-220 rpm for 10-16 hours.

7. The method for preparing a high-purity recombinant mussel mucin according to claim 1, characterized in that: The ultrasonic crushing time in step S5 is 8 to 10 minutes; The centrifugation conditions are 4°C, 11000-13000 rpm, and centrifugation for 20-36 min.

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