Preparation method of high-purity recombinant mussel mucin

Abstract

The invention discloses a preparation method of high-purity recombinant mussel mucoprotein, and belongs to the technical field of recombinant protein preparation, the idea of improving target protein expression by adopting fusion protein is adopted, and fusion expression is performed on partial peptide fragments of an amino acid sequence of mytilus coruscus Mfp-3 and an amino acid sequence of common mussel preCol-D to obtain the high-purity recombinant mussel mucoprotein. The recombinant mussel mucin which is high in purity, low in endotoxin content and good in adhesion performance is obtained by combining improvement of induction, tyrosinase modification and separation and purification processes, a new thought is provided for industrial production of the recombinant mussel mucin through successful preparation of the recombinant mussel mucin, raw material support is provided for multi-field application of the recombinant mussel mucin, and the method is suitable for industrial production of the recombinant mussel mucin. The customized sequence recombinant mussel mucoprotein is prepared according to application requirements, and the application of the mussel mucoprotein in different fields is promoted.

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) is produced and stored in the glands of mussel feet. It is also called mussel foot protein. It enables mussels to adhere to the water and survive. It has high strength, high toughness, waterproofness, biodegradability and super strong adhesion. It has a very broad prospect in the field of biotechnology applications. The content of DOPA [3,4-dihydroxyphenylalanine (3,4-dihydroxyphenylalanine, DOPA)] is crucial to the adhesion function of mussel adhesive protein, and its non-toxicity and non-immunogenicity make it have broad application potential in the medical and aesthetic fields. 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. It can be widely used in surface chemistry, biomedicine, marine engineering, daily chemical products and other fields.

[0003] At present, the most direct way to obtain mussel mucin is to directly extract natural adhesive protein components from the mussel foot gland. However, due to the low secretion of mussel foot protein, only about 1 mg of adhesive protein can be extracted from 10,000 mussels. This directly extracted adhesive product is expensive and easy to solidify, which restricts its application range. Genetic engineering is another effective way to obtain mussel mucin, but the obtained mussel mucin also has low expression and unsatisfactory adhesion performance. Therefore, how to obtain mussel mucin with high purity, good biosafety and good adhesion performance is an important research direction in the future. Summary of the invention

[0004] The purpose of the invention is to provide a method for preparing high-purity recombinant mussel mucin in view of 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. According to the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system, and the designed nucleotide sequence was sent to Shanghai Bioengineering Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then connected to the pET28a empty plasmid;

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

[0009] S3, transforming the obtained pET28a-Mfp3D expression vector into the expression host BL21 (DE3) competent cells, and obtaining single colonies through Kana resistance plate screening, and the positive strains verified by PCR gel electrophoresis and sequenced correctly are the engineering bacteria expressing recombinant mussel viscose protein;

[0010] S4, inoculating the recombinant mussel mucin expression engineering bacteria into LB medium for fermentation to obtain recombinant bacterial fermentation liquid;

[0011] S5, shaking culture the obtained recombinant bacterial fermentation liquid until OD600=0.6-0.8, adding inducer, placing in a shaker for induction culture, collecting the bacterial cells after the induction culture, resuspending with buffer A and ultrasonically disrupting, collecting the supernatant after centrifugation, passing the supernatant through a nickel column twice, eluting impurities with buffer B, and finally recovering the target protein with buffer C;

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

[0013] S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified by Ni-NTA column affinity chromatography, 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 thick-shelled mussel Mfp-3 and the amino acid sequence of blue mussel 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 described in step S1 is named Mfp-3D, and has a theoretical molecular weight of 34.52 kDa and a theoretical isoelectric point of 9.72.

[0018] Preferably, 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.

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

[0020] Preferably, the inducing agent 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, 28-32° C., and shaking the bacteria at 200-220 rpm for 10-16 hours.

[0022] Preferably, the buffer A in step S5: 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 disruption 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 modifier described in step S6 is composed of: 20-26 μM copper sulfate, 10-12 mM ascorbic acid, 60-70 mM Chinese herbal extract, and 1-2% tyrosinase in terms of substrate volume.

[0028] Preferably, the preparation method of the traditional Chinese medicine extract is: deep-cold crush 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 at -100 to -60°C, add water to each of them, stir well and mix, then heat and boil, keep boiling for 10 to 16 minutes, simmer on low heat for 8 to 10 hours, filter and concentrate the filtrate to 1 to 2 g / mL.

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

[0030] The present invention adopts the idea of ​​improving the expression of target protein by fusion protein, fuses and expresses partial peptide segments of the amino acid sequence of thick-shelled mussel Mfp-3 and the amino acid sequence of blue mussel preCol-D, and combines the improvement of induction, tyrosinase modification and separation and purification process to obtain recombinant mussel mucin with high purity, low endotoxin content and good adhesion performance. The DOPA content is 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, adopts the fusion expression method, realizes the industrial production of various types of mussel mucins by Escherichia coli expression system, thereby developing more recombinant mussel mucins that can realize industrial production and have more efficacy and application prospects, provides raw material support for the multi-field application of recombinant mussel mucin, realizes the preparation of customized sequence recombinant mussel mucin according to application requirements, and promotes 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 the 2 mg / mL sample prepared from the recombinant mussel mucin lyophilized powder obtained in Example 2. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are 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. According to the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system, and the designed nucleotide sequence was sent to Shanghai Bioengineering Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then connected to the pET28a empty plasmid;

[0036] The recombinant mussel mucin is composed of the amino acid sequence of thick-shelled mussel Mfp-3 (GenBank: ADB79747.1) and a partial peptide segment of the amino acid sequence of blue mussel 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 obtained by screening on Kana resistance plates. The plasmid was extracted, and the plasmid with the correct sequencing was the constructed pET28a-Mfp3D expression vector;

[0039] S3, transforming the obtained pET28a-Mfp3D expression vector into the expression host BL21 (DE3) competent cells, and obtaining single colonies through Kana resistance plate screening, and the positive strains verified by PCR gel electrophoresis and sequenced correctly are the engineering bacteria expressing recombinant mussel viscose protein;

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

[0041] The formula of the LB medium is 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 liquid was cultured by shaking until OD600 = 0.6-0.8, 0.6 mM lactose was added, 1% glycerol was used as the carbon source, and the mixture was placed in a shaker at 28°C, 200 rpm for induction culture for 10 h. After the induction culture, the bacteria were collected, resuspended with buffer A and ultrasonically disrupted for 8 min, 4°C, 11000 rpm, and the supernatant was collected after centrifugation for 20 min. After the supernatant passed through the nickel column twice, the impurities were eluted with buffer B, and finally the target protein was recovered with buffer C;

[0043] The 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, adding a modifying agent to the buffer C containing the target protein in step S5, reacting at room temperature for 6 hours, and collecting the modified enzyme reaction solution;

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

[0048] Preferably, the preparation method of the Chinese medicine extract is: 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 deep-cold-crushed at -100°C, respectively added with water, stirred and mixed, then heated and boiled, kept boiling for 10 minutes, boiled 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 by Ni-NTA column affinity chromatography, 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. According to the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system, and the designed nucleotide sequence was sent to Shanghai Bioengineering Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then connected to the pET28a empty plasmid;

[0053] The recombinant mussel mucin is composed of the amino acid sequence of thick-shelled mussel Mfp-3 (GenBank: ADB79747.1) and a partial peptide segment of the amino acid sequence of blue mussel 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 obtained by screening on Kana resistance plates. The plasmid was extracted, and the plasmid with the correct sequencing was the constructed pET28a-Mfp3D expression vector;

[0056] S3, transforming the obtained pET28a-Mfp3D expression vector into the expression host BL21 (DE3) competent cells, and obtaining single colonies through Kana resistance plate screening, and the positive strains verified by PCR gel electrophoresis and sequenced correctly are the engineering bacteria expressing recombinant mussel viscose protein;

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

[0058] The formula of the LB medium is 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 liquid was cultured by shaking until 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 bacteria were collected, resuspended with buffer A and ultrasonically disrupted for 9 min, 4°C, 12000 rpm, and the supernatant was collected after centrifugation for 28 min. After the supernatant passed through a nickel column twice, impurities were eluted with buffer B, and finally the target protein was recovered with buffer C;

[0060] The buffer A: imidazole 9mM, urea 8M, NaH2PO4 101mM, Tris-HCl 10mM, 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, adding a modifying agent to the buffer C containing the target protein in step S5, reacting at room temperature for 6 hours, and collecting the modified enzyme reaction solution;

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

[0065] Preferably, the preparation method of the Chinese medicine extract is: 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 deep-cold crushed at -80°C, respectively added with water, stirred and mixed, then heated and boiled, kept boiling for 13 minutes, boiled on low heat for 9 hours, filtered, and the filtrate was concentrated to 1.5 g / mL;

[0066] S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified by Ni-NTA column affinity chromatography, 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. According to the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system, and the designed nucleotide sequence was sent to Shanghai Bioengineering Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then connected to the pET28a empty plasmid;

[0070] The recombinant mussel mucin is composed of the amino acid sequence of thick-shelled mussel Mfp-3 (GenBank: ADB79747.1) and a partial peptide segment of the amino acid sequence of blue mussel 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 obtained by screening on Kana resistance plates. The plasmid was extracted, and the plasmid with the correct sequencing was the constructed pET28a-Mfp3D expression vector;

[0073] S3, transforming the obtained pET28a-Mfp3D expression vector into the expression host BL21 (DE3) competent cells, and obtaining single colonies through Kana resistance plate screening, and the positive strains verified by PCR gel electrophoresis and sequenced correctly are the engineering bacteria expressing recombinant mussel viscose protein;

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

[0075] The formula of the LB medium is 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 liquid was cultured by shaking until OD600 = 0.6-0.8, 1.0 mM lactose was added, 2% glycerol was used as the carbon source, and the mixture was placed in a shaker at 32°C, 220 rpm for 16 h. After the induction culture, the bacteria were collected, resuspended with buffer A and ultrasonically disrupted for 10 min, 4°C, 13000 rpm, and the supernatant was collected after centrifugation for 36 min. After the supernatant passed through the nickel column twice, the impurities were eluted with buffer B, and finally the target protein was recovered with buffer C;

[0077] The 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, adding a modifying agent to the buffer C containing the target protein in step S5, reacting at room temperature for 6 hours, and collecting the modified enzyme reaction solution;

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

[0082] Preferably, the preparation method of the Chinese medicine extract is: 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 deep-cold crushed at -60°C, respectively added with water, stirred and mixed, then heated and boiled, 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 by Ni-NTA column affinity chromatography, 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 scheme is consistent with the technical scheme 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 scheme is consistent with the technical scheme of Example 2.

[0088] 1. Endotoxin content determination

[0089] The pure recombinant mussel mucin freeze-dried powder of the above Examples 1 to 3 and Comparative Examples 1 to 2 was tested, and 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 10EU.

[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 labeled sensitivity of the horseshoe crab reagent, the unit is EU·mL -1) Take 1 tube of bacterial endotoxin working standard, dissolve it with endotoxin test water and dilute it to a standard solution containing 2λ of endotoxin. 2λ / test solution: dissolve 0.05g of recombinant mussel mucin in 10mL of 2λ / test water solution. Endotoxin-free / test water. Prepare 2 tubes for all samples for endotoxin determination.

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

[0092] Table 1 Endotoxin test results

[0093]

[0094] It can be concluded from Table 1 above 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 T / CASME 530-2023 standard.

[0095] 2. Determination of DOPA Content

[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 make up to 100 mL. 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL were taken into a 10 mL volumetric flask, and diluted to the scale with 0.012 mol / L hydrochloric acid. Shake well 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 to 50 mL with 0.012 mol / L hydrochloric acid to obtain a recombinant mussel mucin solution with a concentration of 0.5 mg / mL.

[0099] Finally, take the series of standard solutions and recombinant mussel mucin solution and place them in test tubes, add 0.5mL 0.516mol / L hydrochloric acid to each test tube, and then add 1.5mL nitrite reagent to each tube in turn; 5min later, add 2mL 1mol / L sodium hydroxide and shake well, and measure the absorbance at a wavelength of 500nm. Origin 9.0 software was used for data analysis, and a standard curve was made with DOPA concentration as the horizontal axis and absorbance as the vertical axis, and then the DOPA content W of the sample was calculated based on the measured absorbance value of the sample solution.

[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 each group of recombinant mussel mucin

[0104] DOPA content (%) <![CDATA[Dopamine concentration (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] It can be seen from Table 2 above that the recombinant mussel mucin prepared by the method of the present invention has a higher DOPA content, which indicates that the recombinant mussel mucin prepared by the present invention has good adhesion performance.

[0106] In addition, by comparing Examples 1 to 3 with Comparative Examples 1 to 2, it was found that the DOPA content of 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, showing 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 monitoring role in the quality attributes of the products.

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

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

[0111] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing high-purity recombinant mussel mucin, characterized in that: The steps include: S1. According to the amino acid sequence of the recombinant mussel mucin, codon optimization was performed for the host system, and the designed nucleotide sequence was sent to Shanghai Bioengineering Co., Ltd. for full gene synthesis. The synthesized sequence was double-digested with restriction endonucleases NcoI / SacI and then connected to the pET28a empty plasmid; S2. The recombinant product was transformed into E. coli TOP10 competent cells, and single colonies with positive bands were obtained by screening on Kana resistance plates. The plasmid was extracted, and the plasmid with the correct sequencing was the constructed pET28a-Mfp3D expression vector; S3, transforming the obtained pET28a-Mfp3D expression vector into the expression host BL21 (DE3) competent cells, and obtaining single colonies through Kana resistance plate screening, and the positive strains verified by PCR gel electrophoresis and sequenced correctly are the engineering bacteria expressing recombinant mussel viscose protein; S4, inoculating the recombinant mussel mucin expression engineering bacteria into LB medium for fermentation to obtain recombinant bacterial fermentation liquid; S5, shaking culture the obtained recombinant bacterial fermentation liquid until OD600=0.6-0.8, adding inducer, placing in a shaker for induction culture, collecting the bacterial cells after the induction culture, resuspending with buffer A and ultrasonically disrupting, collecting the supernatant after centrifugation, passing the supernatant through a nickel column twice, eluting impurities with buffer B, and finally recovering the target protein with buffer C; S6, adding a modifying agent to the buffer C containing the target protein in step S5, reacting at room temperature for 6 hours, and collecting the modified enzyme reaction solution; S7. The recombinant mussel mucin in the enzyme reaction solution is eluted and purified by Ni-NTA column affinity chromatography, and finally freeze-dried to obtain a pure freeze-dried powder.

2. A method for preparing a high-purity recombinant mussel mucin according to claim 1, characterized in that: The amino acid sequence of the recombinant mussel mucin described in step S1 is shown in SEQ ID NO.

1.

3. A method for preparing high-purity recombinant mussel mucin according to claim 1, characterized in that: The recombinant mussel mucin in step S1 is composed of a partial peptide segment of the amino acid sequence of Mfp-3 of thick-shelled mussel and the amino acid sequence of preCol-D of blue mussel; The GenBank of Mfp-3 is ADB79747.1, and the GenBank of preCol-D is AAB96638.

1.

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

72.

5. A method for preparing high-purity recombinant mussel mucin according to claim 1, characterized in that: 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.

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

7. A method for preparing high-purity recombinant mussel mucin according to claim 1, characterized in that: The inducing agent 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, 28-32° C., and shaking the bacteria at 200-220 rpm for 10-16 hours.

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

9. A method for preparing high-purity recombinant mussel mucin according to claim 1, characterized in that: The composition of the modifier described in step S6 is: 20-26 μM copper sulfate, 10-12 mM ascorbic acid, 60-70 mM Chinese herbal extract, and 1-2% tyrosinase in substrate volume.

10. A method for preparing high-purity recombinant mussel mucin according to claim 9, characterized in that: The preparation method of the traditional Chinese medicine extract is as follows: 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 are respectively deep-cold-pulverized at -100--60°C, water is added to each of them, stirred and mixed, and then heated and boiled, kept boiling for 10-16 minutes, boiled on low heat for 8-10 hours, filtered, and the filtrate is concentrated to 1-2 g / mL.

Images