Preparation method of recombinant mussel mucin rich in dopa

Abstract

The invention discloses a preparation method of recombinant mussel mucin rich in dopa, and belongs to the technical field of genetic engineering. When the recombinant mussel mucoprotein is co-expressed and modified, a certain amount of modifier is fed in the induction process according to the growth of thalli and the expression condition of protein, so that on one hand, the component concentration of a co-expression modification system is controlled, and a favorable environment is provided for the recombinant tyrosinase to oxidize tyrosine into DOPA; on the other hand, an environment is created for the synthesized recombinant mussel mucin containing dopa, the situation that dopa residues are oxidized into unnecessary substances such as dopaquinone and melanin due to long co-expression modification time is avoided, glutamic acid with a certain concentration in a modification auxiliary system serves as energy supply to promote modification, and the dopa oxidation rate and modification efficiency are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular is a method for preparing DOPA-rich recombinant mussel mucin. Background Art

[0002] Mussel adhesive protein (MAP) is produced and stored in the glands of mussel feet, also known as mussel foot protein, which has high strength, high toughness, waterproofness and super strong adhesion. Studies have shown that mussel adhesive protein can adhere to almost all solid material surfaces. The key to its high adhesion lies in the rich DOPA (i.e. 3,4-dihydroxyphenylalanine, DOPA) groups in its structure. At present, 13 mussel adhesive proteins have been identified, including 8 adhesive proteins, namely Mfp-1, Mfp-2, Mfp-3F, Mfp-3S, Mfp-4, Mfp-5, Mfp-6, Mfp-7, and 5 foot fiber skeleton proteins, namely preCOL-D, preCOL-P, preCOL-NG, PTMP-1, TMP-1. Among them, preCOL-D, preCOL-P, and preCOL-NG mainly control the core skeleton and extension of mussel foot fiber. Mfp-2-6 is mainly located in the byssus disc of mussels and is the main protein component for the formation of strong adhesion of mussels.

[0003] So far, six types of mussel adhesive proteins related to adhesion function have been identified, Mfp-1 to Mfp-6 proteins, which show functional localization and perform different functions. Among them, Mfp2-6 are the main adhesion functional molecules. Among them, Mfp-3 has the smallest molecular weight among the six adhesion proteins, but its DOPA content is high, so it can directly attach to the surface of the medium and produce strong surface interactions. Mfp-5 has the highest DOPA content among all mussel adhesive proteins, and also has a high positive charge. It is one of the key proteins in the mussel foot thread adhesive interface.

[0004] Since the secretion of natural mussel mucin is very low, about 10,000 mussels are needed to extract 1 mg of mussel mucin, so direct extraction has low production efficiency, high cost and high price. Moreover, the DOPA content of these protein products ranges from 0.2 mol% to 30 mol%, and the molecular weight distribution ranges from 5-7 kDa to 108 kDa, which is heterogeneous, resulting in the limitation of natural mussel mucin in practical application. It is highly feasible to produce mussel mucin with the help of chassis bacteria modified by genetic engineering technology. At the same time, compared with yeast chassis bacteria, Escherichia coli as chassis bacteria has the advantages of short growth cycle, high efficiency, no need for methanol as an inducer, and environmental friendliness.

[0005] The recombinant mussel mucin obtained by genetic engineering technology has solved the problem of poor uniformity of molecular weight distribution of extracted mussel mucin to a certain extent, but its adhesion is relatively weak. After searching: Chinese invention patent: A method for producing recombinant mussel mucin rich in DOPA (publication number: CN116947996A, publication date: 2023.10.27), the application scheme adopts a yeast expression system to re-modify the recombinant protein after expression and translation in vitro to achieve the purpose of increasing DOPA oxidation rate and adhesion. However, the yeast expression system in this patent requires methanol induction, serious environmental pollution, and a long fermentation period.

[0006] In addition, some studies have proposed to use tyrosinases from different sources to perform synergistic oxidative modification of tyrosine residues of recombinant mussel mucin in vivo and in vitro in prokaryotic cells (Yuan Sheng. Expression and adhesion properties of mussel foot protein Mfp-5 and its fusion protein in Escherichia coli. Huazhong University of Science and Technology, 2021. DOI: 10.27157 / d.cnki.ghzku.2021.004489), but the dopa oxidation rate of recombinant mussel mucin is insufficient, and the oxidized tyrosine mostly exists in the form of dopaquinone.

[0007] Tyrosinase is a multifunctional copper-containing oxidoreductase with a binuclear Cu 2+ Center, each Cu 2+ The surrounding chelating domain is formed by three histidines. When in contact with specific substrates, it provides oxygen atoms by forming a side oxygen bridge structure, thereby achieving oxidation of the substrate. Although there are reports of DOPA modification of tyrosine residues in the host during prokaryotic expression of mussel mucin, the co-expression induced fermentation time of the prokaryotic host is short and the enzyme catalytic efficiency is low, resulting in insufficient oxidation in the DOPA modification process. In addition, the oxidized tyrosine mostly exists in the form of DOPAquinone, which affects the molecular weight distribution uniformity and purity of the modified protein, and is not conducive to the subsequent application of recombinant mussel mucin. Summary of the invention

[0008] 1. Technical problem to be solved by the invention

[0009] The purpose of the present invention is to solve the problem that the recombinant mussel mucin obtained by the existing recombinant mussel mucin preparation process has insufficient dopa oxidation rate and the oxidized tyrosine mostly exists in the form of dopaquinone.

[0010] 2. Technical solution

[0011] In order to achieve the above object, the technical solution provided by the present invention is:

[0012] A method for preparing DOPA-rich recombinant mussel mucin of the present invention comprises the following steps:

[0013] S100, construct expression vector,

[0014] S200, transform to obtain host bacteria;

[0015] S300, inoculation and culture;

[0016] S400, induction culture and fermentation;

[0017] S500, separation and purification;

[0018] The expression vector in step S100 includes constructing expression vectors corresponding to recombinant mussel mucin M3G and tyrosinase: wherein the expression vector of recombinant mussel mucin includes a fusion gene sequence of Mfp protein and a protein of the mussel byssus fiber skeleton and a tyrosinase gene sequence.

[0019] Preferably, the amino acid sequence of the recombinant mussel mucin M3G in the step S100 is composed of the amino acid sequence of thick-shelled mussel Mfp-3 and a partial peptide selected from purple mussel preCol-P, and the sequence is shown in SEQ.ID.NO.1; the tyrosinase is the tyrosinase of Bacillus megaterium, and the amino acid sequence is shown in SEQ.ID.NO.2, and the Mfp protein is one of natural mussel Mfp1, Mfp2, Mfp3, Mfp4, Mfp5, Mfp6 or Mfp7; the protein of the mussel byssus fiber skeleton is part or all of the Gly-XY structural region peptides contained in natural mussel preCOL-D, preCOL-P, preCOL-NG, PTMP-1, and TMP-1.

[0020] Preferably, the step S100 specifically comprises designing a corresponding nucleotide sequence according to the sequence and the codon preference of Escherichia coli, adding an Xhol restriction endonuclease cleavage site CTCGAG at the 3' end, adding a stop codon and an Ncol restriction endonuclease cleavage site CCATGG at the 5' end, and the recombinant mussel mucin M3G carries a 6×HIS tag; performing full gene synthesis on the designed nucleotide sequence (SEQ.ID.NO.3), and connecting the synthesized sequence to the Escherichia coli vector pETDuet through double restriction digestion of Xhol and Ncol to obtain the recombinant mussel mucin expression vector pETDuet-M3G-TRY.

[0021] Preferably, the step S200 specifically comprises introducing the recombinant mussel mucin expression vector pETDuet-M3G-TRY into the host bacteria competent cells through electroporation, screening the correct positive strains for transformation through ampicillin antibiotics, identifying the expression of the positive strains by shaking flasks, and selecting the optimal co-expression recombinant strain as the recombinant mussel mucin M3G genetic engineering bacteria, wherein the host bacteria is selected from any one of yeast, Escherichia coli, and Bacillus subtilis.

[0022] Preferably, the step S300 specifically comprises inoculating the seed liquid of the recombinant mussel mucin M3G genetically engineered bacteria into the fermentation medium for fermentation culture, and supplementing with glycerol and continuing the fermentation culture after the carbon source is exhausted.

[0023] Preferably, the step S400 specifically includes adding an inducer and a modifier to perform induction culture when the OD of the fermentation liquid of the co-expressed recombinant Escherichia coli reaches 30-60OD, continuing to ferment and culture the co-expressed recombinant strain, and using the co-expressed tyrosinase and the tyrosinase modification auxiliary system during the fermentation and culture process to modify the tyrosine residues in the recombinant mussel mucin into DOPA groups, thereby generating DOPA-rich recombinant mussel mucin in the fermentation and culture system.

[0024] Preferably, the inducer includes 15-150uM copper sulfate, 5-10mM ascorbic acid ethyl ether and 1-3mM glutamic acid, and the inducer is slowly added to the fermentation broth system at the beginning of induction; the modifier includes 15-150uM metal copper ions, 5-10mM antioxidant and 1-3mM amino acid.

[0025] Preferably, the step S500 is specifically to centrifuge the fermentation broth obtained by fermentation culture, collect the bacteria for washing and resuspending, crush the bacterial suspension by a high-pressure homogenizer, collect the supernatant after crushing by centrifugation, and then sequentially perform nickel ion exchange chromatography and molecular exclusion chromatography, ion exchange chromatography or hydrophobic chromatography, and then the sample is ultrafiltered, concentrated, and then freeze-dried after liquid exchange to obtain a pure product of DOPA-rich recombinant mussel mucin; the elution reagent used in the nickel ion exchange chromatography is a 20mMPB solution containing 0.5-1.5MNaCl; the equilibrium solution used in the hydrophobic exchange chromatography is a 20mMPB solution containing 0.5-1.5MNaCl, and the elution reagent is a 20mMPB solution (pH7.5).

[0026] Preferably, the fermentation culture conditions include: controlling the pH at 6.5-7.5, the temperature at 30.0-37.0°C, and controlling the dissolved oxygen at 20%-50% by the rotation speed and ventilation volume; the induction culture conditions include: adjusting the pH to 6.8-7.1 by using ammonia water at the beginning of the induction culture, adjusting the rotation speed, ventilation volume and flow modifier during the induction culture, controlling the temperature at 16.0-33.0°C, and the induction culture time is 4-20h; adding ascorbic acid ethyl ether with a final concentration of 5-10mM, copper sulfate of 15-150uM and glutamic acid of 1-3mM to the Escherichia coli culture system after the expanded culture, and adding the modifier during the induction culture.

[0027] Preferably, the flow acceleration rate of the flow modifier is controlled as follows: 2 hours before the start of induction, the modifier flow acceleration rate is 0.4 mL / L / h, after more than 2 hours of induction until 6 hours, the modifier flow acceleration rate is 1.6 mL / L / h, and after more than 6 hours of induction until the end, the modifier flow acceleration rate is 2.2 mL / L / h.

[0028] 3. Beneficial effects

[0029] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0030] The invention discloses a method for preparing a recombinant mussel mucin rich in DOPA, wherein the induction method uses a relatively low temperature induction, and the induction time is extended in combination with the specific growth rate, which not only solves the problem of insufficient DOPA oxidation rate and low modification efficiency caused by the short prokaryotic co-expression time, but also overcomes the problem of easy generation of inclusion bodies in the prokaryotic co-expressed recombinant mussel mucin. The ascorbic acid ethyl ether modification auxiliary system used controls the quantitative conversion of DOPA, avoids excessive oxidation to form dopaquinone, and at the same time weakens the inhibition of tyrosinase by other antioxidants during the modification process, and weakens the cross-linking and degradation of the recombinant mussel mucin that may occur during the modification process, thereby generating a recombinant mussel mucin rich in DOPA. During the co-expression modification of recombinant mussel mucin, a certain amount of modifier is added during the induction process according to the growth of the bacteria and the expression of the protein. On the one hand, the concentration of the components of the co-expression modification system is controlled to provide a favorable environment for the recombinant tyrosinase to oxidize tyrosine to DOPA. On the other hand, an environment is created for the synthesized recombinant mussel mucin containing DOPA to avoid the oxidation of DOPA residues into dopaquinone, melanin and other unnecessary substances caused by the long co-expression modification time. In the modification auxiliary system, a certain concentration of glutamate is contained as an energy supply to promote the modification, thereby improving the DOPA oxidation rate and modification efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the plasmid map of PACYCDuet-M3G-TYR;

[0032] Figure 2 The electrophoresis diagram of tyrosinase (TYR) and recombinant mussel mucin (M3G) obtained by co-expression shake flask fermentation;

[0033] Figure 3 The electrophoresis diagram of the fermentation products of recombinant mussel mucin M3G at different induction temperatures;

[0034] Figure 4 This is the electrophoresis diagram of the purified product from the fermentation broth of recombinant mussel mucin M3G;

[0035] Figure 5 This is the DOPA color image of recombinant mussel mucin M3G;

[0036] Figure 6 This is the identification diagram of the DOPA content of the recombinant mussel mucin M3G;

[0037] Figure 7 Schematic diagram of DPPH radical scavenging rate of recombinant mussel mucin M3G. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0039] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequential order. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present application described here. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0040] In the present application, the terms "upper", "lower", "left", "right", "front", "back", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings. These terms are mainly used to better describe the present application and its embodiments, and are not used to limit the indicated devices, elements or components to have a specific orientation, or to be constructed and operated in a specific orientation.

[0041] In addition, some of the above terms may be used to express other meanings in addition to indicating orientation or positional relationship. For example, the term "on" may also be used to express a certain dependency or connection relationship in some cases. For those of ordinary skill in the art, the specific meanings of these terms in this application can be understood according to specific circumstances.

[0042] In addition, the terms "installed", "set", "provided with", "connected", "connected", and "socketed" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral structure; it can be a mechanical connection, or an electrical connection; it can be a direct connection, or an indirect connection through an intermediate medium, or it can be an internal connection between two devices, elements, or components. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0043] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0044] Example 1

[0045] See attached Figure 1-7 , a method for preparing DOPA-rich recombinant mussel mucin of this embodiment comprises the following steps:

[0046] S100, construct expression vector,

[0047] S200, transform to obtain host bacteria;

[0048] S300, inoculation and culture;

[0049] S400, induction culture and fermentation;

[0050] S500, separation and purification;

[0051] In order to obtain the DOPA-rich recombinant mussel mucin, Escherichia coli was used to express the recombinant mussel mucin M3G (the recombinant mussel mucin was composed of the fusion of Mfp-3 of natural mussel and part of the protein peptide chain constituting the skeleton of mussel byssus fiber), and tyrosinase from Bacillus filamentosus was co-expressed in Escherichia coli. The co-expression fermentation process of the heterologously expressed tyrosinase (recombinant tyrosinase) and the recombinant mussel mucin M3G was used to achieve DOPA modification of the recombinant mussel mucin in vivo, and then the DOPA-rich recombinant mussel mucin M3G was obtained after purification.

[0052] The expression vector in step S100 includes constructing expression vectors corresponding to recombinant mussel mucin M3G and tyrosinase: wherein the expression vector of recombinant mussel mucin includes a fusion gene sequence of Mfp protein and a protein of the mussel byssus fiber skeleton and a tyrosinase gene sequence.

[0053] S100, construct expression vector,

[0054] (1) Sequence structure

[0055] The amino acid sequence of the recombinant mussel mucin (named M3G) is composed of the amino acid sequence of the thick-shelled mussel Mfp-3 and a partial peptide selected from the blue mussel preCol-P. The amino acid sequence of M3G is specifically shown in SEQ.ID.NO.1. The amino acid sequence of the tyrosinase of Bacillus megaterium is specifically shown in SEQ.ID.NO.2.

[0056] (2) Construction of recombinant mussel mucin expression vector

[0057] According to the amino acid sequence of M3G and the amino acid sequence of tyrosinase (named TYR) of Bacillus megaterium (SEQ.ID.NO.1), the corresponding nucleotide sequence (i.e., the coding sequence of M3G and TRY) was designed according to the codon preference of Escherichia coli. At the same time, for the subsequent molecular operations, the nucleotide sequence added the Xhol restriction endonuclease cleavage site CTCGAG at the 3' end, and added the stop codon and Ncol restriction endonuclease cleavage site CCATGG at the 5' end. At the same time, M3G was labeled with a 6×HIS tag; the designed nucleotide sequence (SEQ.ID.NO.3) was fully synthesized. This synthesized sequence was double-digested with Xhol and Ncol and connected to the Escherichia coli vector pETDuet to obtain a recombinant mussel mucin expression vector, named pETDuet-M3G-TRY. The plasmid map is shown in Figure 1 .

[0058] S200, transform to obtain host bacteria;

[0059] The expression vector pETDuet-M3G-TRY was introduced into the competent cells of the Escherichia coli host bacteria BL21 by electroporation. The positive strains with correct transformation were selected by ampicillin antibiotics. The expression of the positive strains was identified by shaking flasks. The optimal co-expression recombinant strain was selected as the recombinant mussel mucin M3G genetic engineering strain. The SDS-PAGE electrophoresis results of the expression product of the engineering strain are shown in Figure 2 As shown. The theoretical molecular weight of recombinant mussel mucin is 35.98 KDa.

[0060] S300, inoculation and culture;

[0061] The recombinant mussel mucin M3G genetic engineering bacteria obtained by screening were fermented, using TB culture medium as the base material, controlling the pH to 7.0, the temperature to 37.0°C, and the dissolved oxygen to 20-50%. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol (50% glycerol aqueous solution by mass) was added to supplement the carbon source;

[0062] S400, induction culture and fermentation;

[0063] When the OD of the bacteria in the fermentation tank (3L) reaches 30-60, IPTG induction is started, the induction temperature is adjusted to 30℃, the pH is controlled to 7.0, and the dissolved oxygen is controlled to 20-50%. At the beginning of the induction, a final concentration of 8mM ascorbic acid ethyl ether, 0.02mM copper sulfate and 4mM glutamic acid are added at one time, and a certain proportion of modifiers are added during the induction process. The copper ions are used as tyrosinase reaction substrates, and the addition of ascorbic acid ethyl ether can inhibit the conversion of the formed L-DOPA to dopaquinone. Among them, the modifier formula is: 25mM ascorbic acid ethyl ether, 0.8mM copper sulfate and 4.5mM glutamic acid, and the volume is fixed with sterile deionized water; the flow rate is controlled as follows:

[0064] 2h before the start of induction, the modifier flow rate is 0.4mL / h, after more than 2h of induction until 4h, the modifier flow rate is 1.6mL / / h, after more than 4h of induction until the end, the modifier flow rate is 2.2mL / h. After 5-12 hours of fermentation induction, the fermentation liquid is released into the tank to complete the fermentation production and tyrosinase modification of recombinant mussel mucin dopa;

[0065] S500, separation and purification;

[0066] The fermentation broth is centrifuged to collect the bacteria, and the cell fragments are removed after high-pressure homogenization. Finally, the supernatant rich in DOPA recombinant mussel mucin M3G is purified by column gradient elution using a nickel ion exchange chromatography column, and the eluate containing DOPA recombinant mussel mucin M3G is collected. The eluate is then purified using a hydrophobic column, and the eluate is collected and freeze-dried to obtain DOPA-rich recombinant mussel mucin M3G with a purity of ≥95%. The electrophoresis results of the samples taken during the fermentation process are as follows: Figure 3 As shown in A, the protein content was detected by BCA protein quantification method.

[0067] Comparative Example 1

[0068] This comparative example is the same as Example 1 except that the induction temperature and the amount of the inducer are different:

[0069] For the fermentation and in vivo modification of recombinant mussel mucin M3G, at the beginning of induction, the induction temperature was adjusted to 25°C, the pH was controlled at 7.0, the dissolved oxygen was controlled at 20-50%, and the inducer concentration was 0.5mM IPTG. At the same time, a final concentration of 8mM ascorbic acid ethyl ether, 0.02mM copper sulfate and 7mM glutamic acid were added at one time at the beginning of induction, and a certain proportion of modifiers were added during the induction process. After 6-14 hours of induction, the fermentation broth was put into the tank, the fermentation broth was centrifuged to collect the bacteria, and the cell fragments were removed after high-pressure homogenization and crushing. Finally, the recombinant mussel mucin M3G was purified with a nickel ion exchange chromatography column and a hydrophobic column, and the eluate was collected for freeze-drying to obtain DOPA-rich recombinant mussel mucin M3G with a purity of ≥95%. The results of electrophoresis of samples taken during the fermentation process are shown in the following figure. Figure 3 As shown in B, the protein content was detected by BCA protein quantification method.

[0070] Comparative Example 2

[0071] This comparative example is the same as Example 1 except that the induction temperature and the amount of the inducer are different:

[0072] For the fermentation and in vivo modification of recombinant type III mussel mucin, at the beginning of induction, the induction temperature was adjusted to 16°C, the pH was controlled at 7.0, the dissolved oxygen was controlled at 20-50%, and the inducer concentration was 0.2mM IPTG. At the same time, a final concentration of 8mM ascorbic acid ethyl ether, 0.02mM copper sulfate and 7mM glutamic acid were added at one time at the beginning of induction, and a certain proportion of modifiers were added during the induction process. After 9-16 hours of induction, the fermentation broth was put into the tank, the bacteria were collected by centrifugation, and the cell fragments were removed after high-pressure homogenization and crushing. Finally, the recombinant mussel mucin M3G was purified by nickel ion exchange chromatography column and hydrophobic column, and the eluate was collected for freeze-drying to obtain DOPA-rich recombinant mussel mucin M3G with a purity of ≥95%. The results of electrophoresis of samples taken during the fermentation process are as follows Figure 3 As shown in C, the protein content was detected by BCA protein quantification method.

[0073] Experimental process and results

[0074] 1. Output comparison

[0075] The purified samples were subjected to BCA protein quantification. In Example 1, the yield of the recombinant mussel mucin M3G formed under the induced modification conditions at 30°C was about 300 mg / L. In Comparative Example 1, the yield of the recombinant mussel mucin M3G formed under the induced modification conditions at 25°C was about 500 mg / L. In Comparative Example 1, the yield of the recombinant mussel mucin M3G formed under the induced modification conditions at 16°C was about 600 mg / L.

[0076] 2. Purification of recombinant mussel mucin M3G

[0077] The fermentation broth was centrifuged to collect the bacterial precipitate, and then resuspended with 20mMPB, and the resuspended solution was passed through a high-pressure homogenizer (850bar, 3 cycles) for cell disruption, and the disrupted liquid was filtered through a 0.45μm micromembrane, and the filtrate was collected. The recombinant mussel mucin M3G in the filtrate was then purified by column gradient elution using a nickel ion exchange chromatography column, wherein the equilibrium solution was 20mMPB, pH7.5, the washing solution was 20mMPB, 150mMNaCl, pH7.5, the eluent was 20mMPB, 1MNaCl, pH7.5, and the regeneration solution was 20mMPB, 150mMNaCl, 500mM imidazole, pH7.5. The eluate containing the recombinant mussel mucin M3G in the peak range of 100-max-100mAU / mL was collected, and then the eluate was purified by hydrophobic chromatography. The recombinant mussel mucin with a purity of ≥95% and rich in dopa groups was obtained by collecting the eluate. The electrophoresis test results of the purified recombinant mussel mucin are shown in Figure 4 .

[0078] 3. Identification and content determination of recombinant mussel mucin M3G DOPA

[0079] 3.1 DOPA group identification

[0080] 3.1.1 Basis: YY / T1293.6-2020 Contact Wound Dressings, Appendix A of "Part 6 Mussel Mucin Dressings".

[0081] 3.1.2 Principle of identification experiment: In the presence of alkaline and glycine as a reducing agent, the 1,2-dihydroxybenzene of the DOPA residue in the protein molecule can be oxidized and converted into quinone compounds. After adding nitro blue tetrazolium chloride (NBT), NBT reacts with quinone compounds to generate insoluble blue-purple crystalline formazan.

[0082] 3.1.3 Experimental procedures

[0083] (1) The purified recombinant mussel mucin M3G was prepared into a 1 mg / mL liquid with purified water, and 2 μL of the sample was placed on a 5 cm×5 cm 0.2 μm nitrocellulose membrane (NC membrane), and the sample position was marked.

[0084] (2) After the sample was absorbed by the NC membrane, the NC membrane carrying the sample was placed in a 500 mL beaker, 300 mL of pure water was added, and ultrasonic treatment was performed for 10 min.

[0085] (3) Take out the NC membrane and place it in a culture dish. Add NBT staining solution and stain for 45 minutes in the dark.

[0086] (4) After taking out the NC membrane and rinsing it twice with boric acid solution, store it in sodium borate solution overnight, and then rinse it three times with purified water to observe whether there are blue-purple spots generated at the sample mark.

[0087] 3.1.4 Experimental results

[0088] from Figure 5 It can be seen that the recombinant mussel mucin M3G obtained by inducing modification of the co-expressed recombinant strain at 25°C and 30°C has obvious purple spots on the NC membrane (B and C), while the purple spots of the recombinant mussel mucin M3G co-expressed at 16°C (A) are not obvious.

[0089] 3.2 Determination of DOPA group content

[0090] 3.2.1 Basis: YY / T1293.6-2020 Contact Wound Dressings, Appendix B of "Part 6 Mussel Mucin Dressings".

[0091] 3.2.2 Principle: Substances containing 3,4-dihydroxyphenylalanine (DOPA) structure are yellow under acidic conditions and turn into deep orange when excess alkali is added.

[0092] 3.2.3 Results

[0093] After testing the obtained multiple recombinant mussel mucins M3G, the content of dopa groups was positively correlated with the temperature of induction modification. M3G-30℃ reached 15.04mol%, close to the dopa content of natural mussels, in line with industry standards, and significantly higher than the commercial mussel mucin-1. The commercial mussel mucin-2 with Mfp-1 as the main component contained almost no dopa groups (see Appendix Figure 6 ).

[0094] 4. Free radical scavenging ability test of recombinant mussel mucin M3G

[0095] 4.1 Test principle: 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517nm. When there is a free radical scavenger, the light absorption of DPPH ethanol solution is weakened due to pairing with its single electron. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the size of the antioxidant activity.

[0096] 4.2 Test methods:

[0097] (1) Sample preparation: Take the supernatant of the cell lysis solution for later use. If the effective concentration in the sample is high, it can be appropriately diluted with the extraction solution.

[0098] (2) Prepare vitamin C solution: dissolve a 10 mg vial of vitamin C in 1 ml of nitrogen free radical extract to make a 10 mg / ml vitamin C solution. This solution can be divided into 0.1 ml portions and stored at -20°C.

[0099] (3) Preparation of Vc standard working solution: If linearity measurement is required, it is recommended to dilute 10 mg / ml vitamin C solution to 50, 40, 30, 20, 10, or 5 μg / ml Vc standard working solution using nitrogen free radical extract. If a positive control with a clearance rate greater than 90% is required, it is recommended to use a Vc standard working solution greater than 50 ug / ml.

[0100] (4) Turn on the spectrophotometer and preheat for 30 minutes, adjust the wavelength to 517 nm, and set the zero value to anhydrous ethanol.

[0101] (5) DPPH addition: Set up blank tube, sample test tube, sample control tube, and positive control tube according to the table below. Add the solutions in order, mix well, and let stand at room temperature in the dark for 30 min.

[0102]

[0103] 4.3 OD value determination: Add the solution in each tube into the cuvette in turn, and use a spectrophotometer to detect the absorbance value of each tube, which are recorded as A0, A1, A2, and A3 in turn.

[0104] calculate:

[0105] Positive control·DPPH clearance rate (%) = (A0-A3) / A0×100%;

[0106] Test sample·DPPH clearance rate (%) = [A0-(A1-A2)] / A0×100%;

[0107] Note: A0 = absorbance value of blank tube A1 = absorbance value of sample tube;

[0108] A2 = absorbance value of the sample control tube A3 = absorbance value of the positive control tube;

[0109] The free radical scavenging rate of the sample at each tested concentration was calculated according to the formula, and the standard deviation (SD) of the scavenging rate between the parallel tubes in each group was calculated. The SD value must be ≤3% to consider the parallelism of the test valid.

[0110] 4.4. Experimental results

[0111] After comparing the free radical scavenging ability of the recombinant mussel mucin M3G obtained in the experiment with the commercial mussel mucin products, the SD values ​​of all test groups were ≤3%, and the parallelism of the experiment was effective. Among them, the free radical scavenging rate of the recombinant mussel mucin M3G induced and modified at 30℃ was the highest, and the scavenging ability was positively correlated with the induced modification temperature. The free radical scavenging ability of M3G-30℃ was significantly higher than that of the commercial mussel mucin-1, and the commercial mussel mucin-2 with Mfp-1 as the main component had almost no free radical scavenging ability (see Appendix Figure 7 ).

[0112] 5. Dissolution and compounding test of recombinant mussel mucin M3G

[0113] This experiment mainly examined the solubility stability of the recombinant mussel mucin M3G obtained above and the commercially available mussel mucin under different pH conditions and their compatibility with different excipients.

[0114] 5.1 Dissolution stability

[0115] Using buffer solutions of different pH values ​​as solvents, the recombinant mussel mucin M3G of the present invention and commercially available mussel mucin were respectively prepared into solutions with a mass fraction of 0.1%, and the test samples were placed in a 37°C incubator for 24 hours after dissolution, and the state of the solution was observed. The results are shown in Table 1. The results show that the recombinant mussel mucin M3G of the present invention is stably dissolved at pH 5.0-8.0 and is not easy to precipitate, while the commercially available mussel mucin can be dissolved stably under acidic conditions of pH 3.0 and 4.0, but will precipitate and precipitate during standing as the pH increases.

[0116] Table 1. Observation results of the test solution after standing at 37°C for 24 hours ① Recombinant mussel mucin of the present invention

[0117]

[0118]

[0119] ② Commercially available mussel mucin

[0120]

[0121] 5.2 Compatibility

[0122] At room temperature, 0.1% mass fraction of carbomer 940, carbomer U20, xanthan gum, carboxymethyl cellulose, hydroxyethyl cellulose, sodium hyaluronate, sodium polyacrylate solution was prepared respectively, and then 0.1wt% of the recombinant mussel mucin M3G of the present invention or commercial mussel mucin was added respectively, and the solution state was observed, and the results are shown in Table 2. The results show that the recombinant mussel mucin M3G of the present invention can be compounded with carbomer 940, carbomer U20, xanthan gum, carboxymethyl cellulose, hydroxyethyl cellulose, sodium hyaluronate, and sodium polyacrylate, and there is no precipitation reaction and the solution is transparent, but the commercial mussel mucin has no precipitation reaction when compounded only with hydroxyethyl cellulose, and flocculent precipitation occurs when compounded with other excipients.

[0123] Table 2. Observation results after compounding the test sample and auxiliary materials ① Recombinant mussel mucin of the present invention

[0124]

[0125] ② Commercially available mussel mucin

[0126]

[0127]

[0128] It can be seen from the above comparative experimental results that the recombinant mussel mucin of the present invention adopts the Mfp protein and the partial protein structure of the mussel foot silk, which gives the recombinant mussel mucin toughness and elasticity, and the Mfp protein is positively charged and rich in dopa groups at physiological pH, and has excellent adhesion, anti-oxidation and inflammation-reducing abilities. In addition, the solubility of the recombinant mussel mucin is significantly improved, and it can also stably exist at different pH values ​​(including at neutral pH), dissolve stably, and is not easy to precipitate, and can be combined with external preparation excipients such as carbomer, xanthan gum, carboxymethyl cellulose, sodium hyaluronate, and sodium polyacrylate.

[0129] The preparation method of the recombinant mussel mucin of the present invention utilizes the co-expression principle, so that the DOPA modification of its tyrosine residues is achieved while the recombinant mussel mucin is expressed, thereby obtaining a recombinant mussel mucin rich in DOPA groups. The process is simple and the yield is high, which can meet the application in the fields of medicines, medical devices and cosmetics.

[0130] The host bacteria in the present invention is Escherichia coli, and IPTG, lactose or arabinose are used as inducers, thereby solving the problems of high waste liquid treatment costs and environmental pollution caused by using methanol as an inducer when yeast is used as a host bacteria.

[0131] The recombinant mussel mucin and tyrosinase gene sequences of the present invention are constructed on one expression vector, thus solving the problem that prokaryotic co-expression requires two expression vectors, resulting in high bacterial growth pressure and difficult expression.

[0132] The induction method of the present invention adopts lower temperature induction and prolongs the induction time in combination with the specific growth rate, which not only solves the problems of insufficient DOPA oxidation rate and low modification efficiency caused by short prokaryotic co-expression time, but also overcomes the problem of easy generation of inclusion bodies in prokaryotic co-expression of recombinant mussel mucin.

[0133] The ascorbic acid ethyl ether modification auxiliary system adopted in the present invention controls the quantitative conversion of DOPA, avoids excessive oxidation to form dopaquinone, and at the same time weakens the inhibition of tyrosinase by other antioxidants in the modification process, weakens the cross-linking and degradation of recombinant mussel mucin that may occur in the modification process, thereby generating recombinant mussel mucin rich in DOPA.

[0134] During the co-expression modification of the recombinant mussel mucin, a certain amount of modifier is added according to the growth of the bacteria and the expression of the protein during the induction process. On the one hand, the concentration of the components of the co-expression modification system is controlled to provide a favorable environment for the recombinant tyrosinase to oxidize tyrosine into DOPA. On the other hand, an environment is created for the synthesized recombinant mussel mucin containing DOPA to avoid oxidation of DOPA residues into dopaquinone, melanin and other unnecessary substances caused by a long co-expression modification time. In the modification auxiliary system, a certain concentration of glutamic acid is used as an energy supply to promote the modification, thereby improving the DOPA oxidation rate and modification efficiency.

[0135] The above-described embodiments only express a certain implementation mode of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

[0136] SEQ.ID.NO.1

[0137] VQSAGYGYDLGYNAPWPYNNGYYGYNGYNGYHGRYGWNKGWNSGPWGGSYYGNKGYLYGRPGPSGAPGNPGAPGALGAPGPRGSPGFVGLPGPRGSPGEPGNQGPIGGPGYPGPRGPQGPDGAMGPQGPCGDRGAPGVPGKQGPVGGQGPAGPRGPRGDEGPVGPKGEPGARGADGKPGDKGPDGETGPQGPAGPKGQVGDQGKPGAKGETGDQGARGEAGKAGEQGPGGIQGPKGPVGGQGPAGPAGPLGPQGPMGERGPQGPTGSEGPVGAPGPKGSVGDQGAQGDQGATGADGKKGEPGERGQQGAAGPVGRPGPRGDRGAKGIQGSRGRPGGMGRRGNRGSQGAVGPRGETGPDGNQGQRGEQGAP

[0138] SEQ.ID.NO.2

[0139] MSNKYRVRKNVLRLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQLQDPSQSQIWSADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLDALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS

[0140] SEQ.ID.NO.3

[0141]

Claims

1. A method for preparing DOPA-rich recombinant mussel mucin, characterized in that: The steps include: S100, construct expression vector, S200, transform to obtain host bacteria; S300, inoculation and culture; S400, induction culture and fermentation; S500, separation and purification; The expression vector in step S100 includes constructing expression vectors corresponding to recombinant mussel mucin M3G and tyrosinase: wherein the expression vector of recombinant mussel mucin includes a fusion gene sequence of Mfp protein and a protein of the mussel byssus fiber skeleton and a tyrosinase gene sequence.

2. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 1, characterized in that: The amino acid sequence of the recombinant mussel mucin M3G in step S100 is composed of the amino acid sequence of thick-shelled mussel Mfp-3 and a partial peptide selected from purple mussel preCol-P, and the sequence is shown in SEQ.ID.NO.1; the tyrosinase is the tyrosinase of Bacillus megaterium, and the amino acid sequence is shown in SEQ.ID.NO.2; the Mfp protein is one of natural mussel Mfp1, Mfp2, Mfp3, Mfp4, Mfp5, Mfp6 or Mfp7; the protein of the mussel byssus fiber skeleton is part or all of the Gly-XY structural region peptides contained in natural mussel preCOL-D, preCOL-P, preCOL-NG, PTMP-1, and TMP-1.

3. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 2, characterized in that: The step S100 specifically designs the corresponding nucleotide sequence according to the sequence and the codon preference of Escherichia coli, adds the Xhol restriction endonuclease cleavage site CTCGAG at the 3' end, adds the stop codon and the Ncol restriction endonuclease cleavage site CCATGG at the 5' end, and the recombinant mussel mucin M3G carries a 6×HIS tag; the designed nucleotide sequence (SEQ.ID.NO.3) is subjected to full gene synthesis, and the synthesized sequence is connected to the Escherichia coli vector pETDuet through double restriction digestion of Xhol and Ncol to obtain the recombinant mussel mucin expression vector pETDuet-M3G-TRY.

4. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 3, characterized in that: The step S200 specifically comprises introducing the recombinant mussel mucin expression vector pETDuet-M3G-TRY into the host bacteria competent cells through electroporation, screening the correct positive strains for transformation through ampicillin antibiotics, identifying the expression of the positive strains by shaking flasks, and selecting the optimal co-expression recombinant strain as the recombinant mussel mucin M3G genetic engineering bacteria, wherein the host bacteria are selected from any one of yeast, Escherichia coli, and Bacillus subtilis.

5. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 4, characterized in that: The step S300 specifically involves inoculating the seed liquid of the recombinant mussel mucin M3G genetically engineered bacteria into a fermentation medium for fermentation culture, and supplementing with glycerol and continuing the fermentation culture after the carbon source is exhausted.

6. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 5, characterized in that: The step S400 specifically includes adding an inducer and a modifier to induce culture when the OD of the fermentation liquid of the co-expressed recombinant Escherichia coli reaches 30-60OD, continuing to ferment and culture the co-expressed recombinant strain, and using the co-expressed tyrosinase and the tyrosinase modification auxiliary system during the fermentation and culture process to modify the tyrosine residues in the recombinant mussel mucin into DOPA groups, thereby generating DOPA-rich recombinant mussel mucin in the fermentation and culture system.

7. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 6, characterized in that: The inducer comprises 15-150uM copper sulfate, 5-10mM ascorbic acid ethyl ether and 1-3mM glutamic acid, and the inducer is slowly added into the fermentation liquid system at the beginning of induction; the modifier comprises 15-150uM metal copper ion, 5-10mM antioxidant and 1-3mM amino acid.

8. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 6, characterized in that: The step S500 specifically comprises centrifuging the fermentation liquid obtained by fermentation culture, collecting the bacteria for washing and resuspending, crushing the bacterial suspension by a high-pressure homogenizer, collecting the supernatant after crushing by centrifugation, and then sequentially performing nickel ion exchange chromatography and molecular exclusion chromatography, ion exchange chromatography or hydrophobic chromatography, and then the sample is ultrafiltered, concentrated, and then freeze-dried after liquid exchange to obtain a pure product of DOPA-rich recombinant mussel mucin; the elution reagent used in the nickel ion exchange chromatography is a 20mMPB solution containing 0.5-1.5MNaCl; the equilibrium solution used in the hydrophobic exchange chromatography is a 20mMPB solution containing 0.5-1.5MNaCl, and the elution reagent is a 20mMPB solution (pH7.5).

9. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 6, characterized in that: The fermentation culture conditions include: controlling the pH at 6.5-7.5, the temperature at 30.0-37.0°C, and controlling the dissolved oxygen at 20%-50% by the rotation speed and ventilation volume; the induction culture conditions include: adjusting the pH to 6.8-7.1 by using ammonia water at the beginning of the induction culture, adjusting the rotation speed, ventilation volume and flow modifier during the induction culture, controlling the temperature at 16.0-33.0°C, and the induction culture time is 4-20h; adding ascorbic acid ethyl ether with a final concentration of 5-10mM, copper sulfate of 15-150uM and glutamic acid of 1-3mM to the Escherichia coli culture system after the expanded culture, and adding the modifier during the induction culture.

10. The method for preparing a DOPA-rich recombinant mussel mucin according to claim 9, characterized in that: The flow acceleration rate of the flow modifier is controlled as follows: 2 hours before the start of induction, the modifier flow acceleration rate is 0.4 mL / L / h, after more than 2 hours of induction until 6 hours, the modifier flow acceleration rate is 1.6 mL / L / h, and after more than 6 hours of induction until the end, the modifier flow acceleration rate is 2.2 mL / L / h.

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