CN117986340A - Novel self-assembled mussel mucin and application - Google Patents
Novel self-assembled mussel mucin and application Download PDFInfo
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- CN117986340A CN117986340A CN202410397972.2A CN202410397972A CN117986340A CN 117986340 A CN117986340 A CN 117986340A CN 202410397972 A CN202410397972 A CN 202410397972A CN 117986340 A CN117986340 A CN 117986340A
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Abstract
The invention discloses a novel self-assembled mussel mucin, which consists of a mussel mucin core functional area and a self-assembled functional area, wherein a two-embedded combined model or a three-embedded combined model is adopted in a combined mode of the two areas; the sequence of the two-embedded combined model is N-end-self-assembled functional area-mussel mucin core functional area-C end, and the sequence of the three-embedded combined model is N-end-self-assembled functional area-mussel mucin core functional area-self-assembled functional area-C end. The design and development strategy of the novel self-assembled mussel mucin provided by the invention can be expanded to the design and application of other similar proteins, can efficiently carry out biosynthesis, is simple, convenient and rapid to purify, has flexible self-assembly characteristics, and can form different protein-based materials for cell adhesion, adsorption hemostasis, tissue adhesion and other fields.
Description
Technical Field
The invention relates to the field of protein design, in particular to novel self-assembled mussel mucin and application thereof.
Background
Mussel mucins are a protein secreted by marine mussels, enabling them to adhere firmly to a variety of surfaces in a wet and dynamic marine environment. This adhesion ability is mainly due to a series of specific amino acids contained in mussel mucin, such as DOPA (3, 4-dihydroxyphenylalanine) containing catechol groups. These chemical structures impart excellent wet adhesion and water resistance properties to mussel mucins.
In view of the excellent adhesion property and biocompatibility of mussel mucin, the mussel mucin is widely developed and applied as a raw material so as to meet various application requirements, and can be applied to adhesion of surgical incisions and wounds of mucosal tissues, skin tissues, bones, soft tissues and the like in the biomedical field; in the scientific research field, the cell culture medium can be used as an additive of cell and tissue culture medium to promote the adherent growth of cultured cells; in the field of materialogy, mussel mucin hydrogels developed by various chemical modifications and crosslinking have excellent water absorption, swelling, porosity and strong adsorption capacity.
In order to meet the requirements, the natural podoglossin Mfp-1, mfp-2 and Mfp-3 are directly extracted from the podophyllo gland of marine mussels by America BIOPOLYMERS company from the beginning of the 80 th century, and are mixed to prepare a commercial adhesive 'Cell-Tak', which is an auxiliary reagent for animal Cell tissue culture. However, this method of directly extracting the adhesive protein from the marine organism has not been widely adopted and used, mainly because the direct extraction of the adhesive protein has limitations, and is a difficult, uneconomical and low-yield obtaining method. To overcome these difficulties, KITAMURA, et al first tried in 1999 to express mussel Mytilus edulis foot protein, mefp-1, using E.coli, but this protein was not available for subsequent study due to its low yield. Since 2004, advanced genetic engineering approaches have been successfully used for marine bioadhesive protein production in an attempt to increase protein yield and purity. However, the mussel mucin obtained by genetic engineering has more extreme purification conditions and low yield, and in nature, the mussel mucin plays a complex role in practice, and various high-order assembly structural characteristics are involved, and at present, the natural mussel mucin which is extracted or synthesized by genetic engineering has more single and deficient inherent characteristics, and the protein needs to be further endowed with the characteristic of multistage assembly so that the mussel mucin can be assembled into a higher-level material, thereby better improving the performance of the mussel mucin and meeting the actual requirements.
Disclosure of Invention
The invention aims at solving the problems, and aims at excavating a specific area of mussel mucin from the natural bionic point of view, introducing a proper frontal assembly area and cooperating with the functions of the specific area and the frontal assembly area to better exert respective advantages, including separation and purification and material assembly. Meanwhile, the cell factory can be utilized for high-efficiency adaptive expression, green biological production is promoted, and popularization and application of mussel mucin materials are better realized. Therefore, the invention provides a novel self-assembled mussel mucin to solve the problems of low yield and single and deficient performance of the existing recombinant mussel mucin.
In order to achieve the above object, the present invention provides the following technical solutions
A novel self-assembled mussel mucin comprises a mussel mucin core functional region (core mussel adhesive protein, cMAP) and a self-assembled functional region (self-assemble protein, SAP), wherein the two regions are combined in a two-embedded combined model or a three-embedded combined model; the sequence of the two-embedded combined model is N-end-self-assembled functional area-mussel mucin core functional area-C end, and the sequence of the three-embedded combined model is N-end-self-assembled functional area-mussel mucin core functional area-self-assembled functional area-C end.
Preferably, in the above novel self-assembled mussel adhesive protein, the two-block combined model or the three-block combined model supports the introduction of connecting links, including flexible links and rigid links.
Preferably, in the novel self-assembled mussel mucin, the amino acid sequence of the mussel mucin core functional region is shown in SEQ ID NO. 1. The mussel mucin core functional region is a section of mussel mucin key characteristic sequence obtained by coupling multidimensional information mining, wherein the multidimensional information mining covers comparison analysis of a primary sequence layer, comparison analysis of a three-dimensional structure layer and analysis of molecular evolution characteristics. Coupling analysis of multidimensional information focuses on one or more sections of local areas with consistent multidimensional information, and focuses on the composition distribution of amino acids, as well as the surface charge and the hydrophilic-hydrophobic distribution. The strategy can be popularized and applied to the excavation of related proteins of various species related to molecular evolution, including type 5, type 3, type 6 and type 1 of mussel mucin. Preferably, the mussel mucin core functional region cMAP is shown in SEQ ID NO. 1.
The specific analysis of the primary sequence layer is to download a protein sequence corresponding to a CDS region in a nucleic acid sequence of mussel mucin from NCBI, so as to obtain primary sequence information of the protein. And (3) comparing the obtained protein sequences with all the obtained mussel mucins by utilizing a ClustalW program in a MEGA program, constructing a phylogenetic tree by using a maximum likelihood method, performing a guide test of 1000 iterations, further beautifying the obtained evolutionary tree, performing motif analysis on the mussel mucin-based protein sequences by utilizing MEME Suite, checking detailed information of motif through a MEME website, and storing data. The specific analysis of the three-dimensional structure layer is to obtain the primary sequence information of all mussel mucins, model the mussel mucins through a Alphafold protein modeling platform, select a model with the highest quality from a pLDDT diagram, analyze the model through PyMod software, and analyze the surface electrostatic potential energy through APBS Tool.
The molecular evolution characteristic analysis specifically comprises the steps of downloading CDS regions in a nucleic acid sequence of mussel mucin from NCBI, obtaining first-order sequence information of a nucleic acid layer, completing multi-sequence comparison of CDS sequences based on codons through a Muscle (conda) comparison function of MEGA, constructing a phylogenetic tree, performing format purification by Easycodeml, removing branch length and Bootstrap value information, and only retaining topological structure information of the tree. A Branch Model is selected for Branch Model detection, and a Branch site Model is selected for detecting (Branch Site Model) the forward selection sites on each Branch. And sorting the detection results into an Excel table, recording the obtained selection sites by means of transposition, data sorting, functions and data screening and sorting functions of the Excel, and carrying out statistical analysis on the obtained selection sites.
Preferably, in the novel self-assembled mussel adhesive protein, the self-assembled functional region is based on a VPGXG pentapeptide basic unit, the fourth X passenger seat residue is replaced by a characteristic amino acid of a natural adhesive protein to realize bionic design, and the structural mode is [ (VPGYG) (VPG (X/Z) G) ] n and is rich in tyrosine residues; wherein V is valine, P is proline, G is glycine, Y is tyrosine; x is tyrosine or any positively charged amino acid; z is any hydrophobic amino acid with a hydrophobic fraction of more than 2.5 except proline; n is an integer greater than or equal to 1.
Preferably, in the above novel self-assembled mussel mucin, the positively charged amino acid is arginine, lysine or histidine; z is isoleucine, valine, leucine, phenylalanine, methionine or alanine; and n is 10, 15, 30, 40 or 60.
The self-assembly area SAP is characterized by being obtained through a bionic design, specifically, the kind of mucin characteristic interface adhesion amino acid represented by mussels in nature is analyzed, the mucin characteristic interface adhesion amino acid is reasonably introduced into a fourth passenger seat residue of a VPGXG basic sequence unit, meanwhile, the structural characteristics of a natural adhesion structure are considered, and relevant cooperative residues are introduced to adjust the hydrophilic and hydrophobic characteristics of the module.
Preferably, in the above novel self-assembled mussel adhesive protein, the fourth X guest seat residue in the VPGXG basic sequence unit is valine and tyrosine, the repeating unit n is 10, the final structural mode is [ (VPGYG) (VPGVG) ] 10, and the amino acid sequence is shown as SEQ ID NO. 2.
Preferably, in the novel self-assembled mussel mucin, the self-assembled functional region is located at the N-terminal end, the mussel mucin core functional region is located at the C-terminal end, and the self-assembled mussel mucin core functional region and the mussel mucin core functional region are directly arranged in series to form a two-embedded model, a linker is not needed in the middle, and the amino acid sequence of the novel self-assembled mussel mucin is shown as SEQ ID No. 3. In the present invention, the combination of the assembly region SAP and the mussel mucin core functional region cMfp is characterized by using a two-block combination or a three-block combination. Specifically, the two-embedded model protein realizes hierarchical stacking by means of the assembly modules at the N end, takes the assembly modules as cores to form a fibrous structure, the fiber surface can be exposed to display the adhesion modules, the adhesion residues can be fully enriched in a space stacking mode, the three-embedded model protein can realize non-covalent crosslinking by means of the assembly modules at the two ends, the adhesion modules are fully stretched, and the network structure is formed as a whole. The two-in-one model combination sequence may be N-end-cMfp-SAP-C end, and the three-in-one model combination sequence may be N-end-SAP-cMfp-SAP-C end, wherein the units may support the introduction of connection linker, including flexible linker such as (GGGS) N and rigid linker such as (EAAAK) N. Preferably, the assembly region SAP is located at the N-terminal end, the mussel mucin core functional region cMfp is located at the C-terminal end, and the assembly region SAP and the mussel mucin core functional region cMfp are directly arranged in series to form a two-embedded model, and a linker is not needed in the middle, and the assembly region SAP comprises an amino acid sequence shown in SEQ ID NO. 3.
Preferably, in the novel self-assembled mussel mucin, the whole gene nucleotide sequence of the novel self-assembled mussel mucin is shown as SEQ ID No. 4.
Preferably, in the novel self-assembled mussel mucin, pH is regulated and controlled by adopting an acid-base solvent to form different assembled aggregation structures with different material properties; the novel self-assembled mussel mucin sA-cMfp of the present invention can be dissolved by an acid base. Preferably, the acid-base solvent is a volatile weak acid-base solution, specifically, the basic solvent is a 1.5% ammonia solution at pH11.5, and the acidic solution at pH2.5 is a 10% acetic acid solution. The ammonia solution and the acetic acid solution with low concentration are used as weak bases of volatile weak organic acids, and the salt formed by neutralization can volatilize in the freeze-drying process. Specifically, a solution of sA-cMfp protein with a certain concentration is prepared by using 1.5% ammonia solution or 10% acetic acid solution, the solution is coated on cell culture dishes with different specifications in a certain volume, then the sA-cMfp is formed on the dishes by volatilizing an acid-base solvent to form a coating, and then cells are paved on the dishes or plates to promote cell adhesion. Preferably, the amount of sA-cMfp protein on the final unit area of dish/well is 2.5ug protein/cm 2. Preferably, the filiform adhesive with viscosity can be formed by centrifugation at 5000rpm for 1min after the pH is regulated to be about 9.8, the filiform adhesive has wiredrawing performance, emulsion can be formed by centrifugation at 5000rpm for 1min after the pH is regulated to be about 5.5, the emulsion has good film forming capability, and the emulsion can be coated on the surface of skin to form a film.
Preferably, in the novel self-assembled mussel adhesive protein, tyrosinase and proline hydroxylase are modified by hydroxylation in vitro, or in vivo by a method of coexpression of tyrosinase and proline hydroxylase in E.coli. The novel self-assembled mussel mucin sA-cMfp provided by the invention is rich in tyrosine and proline, can be subjected to hydroxylation modification by tyrosinase and proline hydroxylase in vitro, and can also be subjected to in vivo hydroxylation modification in escherichia coli by a technical method of coexpression of tyrosinase and proline hydroxylase. The dopa residue generated after hydroxylation modification can endow protein with stronger adhesion capability, and meanwhile, the excessive hydroxyl groups after hydroxylation modification can provide more modification sites, so that the flexible and adjustable performance of subsequent crosslinking is further improved. As preferable tyrosine modification conditions, 15 mmol/L ascorbic acid, 20 umol/L copper sulfate, 20 mmol/L phosphate buffer (pH 7.6) and 20U tyrosinase are adopted, stirring is carried out at room temperature for 3 h, tyrosine residues of Mfp-3P protein are modified into DOPA, and glacial acetic acid is added to adjust the pH of the feed liquid to 3.2 at the modification end point.
The preparation method of the novel self-assembled mussel mucin utilizes a genetic engineering mode to carry out biosynthesis and expression screening, the combination is [ (VPGYG) (VPGVG) ]10-cMfp, the expression vector is pET20b (+), and the expression host is escherichia coli BL21 (DE 3); the separation and purification are realized by means of temperature phase transition and washing centrifugation.
The method comprises the following steps:
(1) The cells were disrupted with a volume of buffer A. As a preferred buffer solution A, 50mM Tris-HCl (pH 8.7) was used, and the ratio of the wet weight of the cells to the volume of the buffer solution was 1 g/10 mL.
(2) Collecting the bacterial supernatant, placing in a water preppot at 40 ℃, incubating for 2min, discarding the supernatant, and centrifugally collecting the precipitate. Collecting the bacterial precipitate.
(3) The above-mentioned precipitate was resuspended in an equal volume of buffer solution B and combined, and after magnetically stirring at room temperature for a certain period of time, the supernatant was discarded and the precipitate was collected by centrifugation. As a preferred buffer solution B, 50mM Tris-HCl,1M urea, 0.5% Trixton-X100 (pH 8.7), stirring time of 1h, centrifugation speed of 10000rpm, centrifugation time of 20min was used.
(4) The precipitate collected in the previous step is resuspended with an equal volume of buffer solution C, magnetically stirred at room temperature for a certain period of time, the supernatant is discarded, and the precipitate is collected by centrifugation. As a preferred buffer solution C, 50mM Tris-HCl (pH 8.7) was used, the stirring time was 1h, the centrifugation speed was 10000rpm, and the centrifugation time was 20min.
(5) The precipitate collected in the previous step was resuspended with an equal volume of ultrapure water, magnetically stirred at room temperature for 1h, the supernatant was discarded, and the precipitate was collected by centrifugation. The stirring time is preferably 1h. The centrifugation speed was 12000rpm and the centrifugation time was 30min.
The novel self-assembled mussel mucin is applied to the preparation of medical instruments or cosmetics for promoting cell adhesion, resisting bacteria, stopping bleeding, repairing or bonding. The novel self-assembled mussel mucin sA-cMfp has good macroscopic binding capacity, and can be used for binding different materials and soft and hard tissues, including skin tissues and bone tissues. Preferably, the assembly material used for bonding is a lyophilized fiber formed after dissolution under alkaline conditions or after pH adjustment to about 9.8. Furthermore, the novel self-assembled mussel mucin sA-cMfp is prepared into an assembly body through the regulation and control of the self-assembled mussel mucin sA-cMfp, and is subjected to freeze drying for a short time, so that the self-assembled mussel mucin is sticky when wetted, the self-assembled mussel mucin sA-cMfp is timely bonded, has good adhesion hemostatic capability, and can be used in the field of wound hemostasis.
The design and development strategy of the novel self-assembled mussel mucin provided by the invention can be expanded to the design and application of other similar proteins, can efficiently carry out biosynthesis, is simple, convenient and rapid to purify, has flexible self-assembly characteristics, and can form different protein-based materials for cell adhesion, adsorption hemostasis, tissue adhesion and other fields.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
FIG. 1 is a cMAP mining analysis chart of mussel mucin core functional region;
FIG. 2 is a diagram of assembly area SAP structure design and modeling;
FIG. 3 is a diagram showing the design and self-assembly simulation analysis of novel self-assembled mussel mucin sA-cMfp;
FIG. 4 is a plasmid map of a novel self-assembled mussel mucin sA-cMfp expression vector;
FIG. 5 is a diagram showing screening electrophoresis of novel self-assembled mussel mucin sA-cMfp-expressing bacteria;
FIG. 6 shows the electrophoresis patterns of protein expression of novel self-assembled mussel mucin sA-cMfp at different culture scales;
FIG. 7 is a graph showing the purification effect of the novel self-assembled mussel mucin sA-cMfp temperature phase transition in combination with a washing centrifugation strategy;
FIG. 8 is a qualitative staining of NBT dopa after tyrosine hydroxylation of novel self-assembled mussel mucin sA-cMfp;
FIG. 9 is a diagram showing in vitro assembly detection of novel self-assembled mussel mucin sA-cMfp;
FIG. 10 is a diagram showing how novel self-assembled mussel mucin sA-cMfp forms macroscopic assemblies under the control of different ph;
FIG. 11 is a graph showing the interfacial adhesion effect of novel self-assembled mussel mucin sA-cMfp;
FIG. 12 is a graph showing the cell adhesion promoting effect of novel self-assembled mussel mucin sA-cMfp;
FIG. 13 is a graph showing the results of a CCK8 cytotoxicity assay against novel self-assembled mussel adhesive protein sA-cMfp;
FIG. 14 is a graph showing the material adhesion effect of novel self-assembled mussel mucin sA-cMfp;
FIG. 15 is a graph showing the in vitro hemostatic effect of the novel self-assembled mussel mucin sA-cMfp lyophilized fibers.
Detailed Description
The invention will be further illustrated by the following examples, which are set forth to illustrate the invention and are not to be construed as limiting the scope of the invention
Example 1: excavating mussel mucin core functional region cMAP
And sequentially selecting Animalia, mollusca, bivalvia, mytilide, mytilus species anchored to mussel genus by NCBI database under the condition of species as screening, selecting Protein reference sequence, and downloading corresponding fasta file. Selecting a nucleic acid sequence and downloading a corresponding fasta file. And uploading the collected sequence information to a SignalP server, searching the signal peptide information thereof, and deleting the signal peptide sequence in the sequence after acquiring the corresponding position number to obtain the target sequence.
The quantity and the proportion of various amino acids in the target sequence after the statistical data filtration are subjected to visual analysis on the data through a heat map drawing package TIDYHEATMAP, and meanwhile, characteristic amino acids of mussel mucin are screened out according to the content proportion.
Multi-sequence comparison of protein sequences is carried out by utilizing Mega11 software and a Muscle comparison algorithm, an ML tree is constructed and constructed, an optimal model is found to be Dayhoff+G, a MEME tool is utilized, zero or one occurrence per sequence (zoops) site contribution parameters are adopted, protein motifs are mined and analyzed, and 10 motifs of each sequence are mined
And (3) carrying out selective pressure analysis by using a PAML calculation package, selecting a branch model, and comparing the conservation of each nucleic acid sequence in a molecular evolution layer.
And carrying out structural modeling on each protein by adopting a Alphafold modeling platform, analyzing the surface structure by using pymol software, correlating the primary sequence characteristics, the three-dimensional structure characteristics and the molecular evolution site distribution, and screening to obtain a key mussel mucin core functional region cMAP, wherein the result is shown in figure 1.
Example 2: design and simulation evaluation of novel self-assembled mussel mucin sA-cMfp
Uploading the protein sequence of cMAP of the mussel mucin core functional area to a Espasy platform, and calling ProtParam tool to calculate and predict that the theoretical isoelectric point is 10.17 and the GRAVY value is-2.105. In order to achieve better self-assembly, the theoretical isoelectric point of the SAP in the self-assembly region is 7.17-4.17, and the GRAVY value is above 0.
The [ (VPGXG) (VPGXG) ] n sequence is used as a template, the fourth X guest seat residue is replaced by a key residue tyrosine of mussel mucin, the ninth X guest seat residue is replaced by valine with the hydrophobicity score of 4.2, the [ (VPGYG) (VPGVG) ] unit is finally obtained, the number of n is further adjusted to 10, the final [ (VPGXG) (VPGXG) ] 10 sequence is obtained, and the theoretical isoelectric point of the self-assembly region SAP is 5.49, and the GRAVY value is 0.6. The self-assembled region SAP was further spatially modeled by the Alphfold platform to evaluate the stacking of its individual residues.
The self-assembled region SAP is placed at the N end, and then the self-assembled region SAP is directly connected with the mussel mucin core functional region cMAP in series, so that the novel self-assembled mussel mucin sA-cMfp is obtained. Space modeling is carried out on the SAP in the self-assembled region through a Alphfold2 platform by using sA-cMfp, whether the modeled protein conformation is reasonable or not is evaluated through RAMACHANDRAN PLOT, a model with the highest model quality is selected, and stacking conditions and surface potential distribution of each residue are evaluated through pymol visualization software. And simultaneously, carrying out dimerization, trimerization and tetramerization assembly butt joint analysis by utilizing a ZDOCK platform, and analyzing the stacking mode by using pymol software, wherein the results are shown in fig. 2 and 3.
Example 3: construction and expression screening of novel self-assembled mussel mucin sA-cMfp expression vector
The codon preference of the novel self-assembled mussel mucin sA-cMfp is optimized by the codon preference of the escherichia coli, and the translation initiation amino acid methionine is added at the N end, the novel self-assembled mussel mucin sA-cMfp fragment is subjected to total gene synthesis (the nucleotide sequence is shown as SEQ ID NO. 4) by a gene synthesis company, and NdeI and EcoRI restriction enzyme sites are added at the two ends of the fragment during amplification, wherein the specific conditions are as follows: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s, extension at 72 ℃ for 10s, circulation for 30 times, extension at 72 ℃ for 10min, and heat preservation at 10 ℃.
The restriction enzyme sites NdeI and EcoRI, the obtained sA-cMfp gene fragment is inserted between NdeI and EcoRI of pET20b (+) vector to form pET20b (+) -sA-cMfp vector, the vector plasmid map is shown in figure 4; the specific process is as follows: the obtained sA-cMfp gene fragment and pET20b (+) vector are subjected to double digestion by NdeI and EcoRI and purified by a kit, a pET20b (+) -sA-cMfp vector connection product is obtained by connection according to the TIANGEL MIDI purification instruction of Takara company, the connection product is introduced into DH5 alpha competent, ampicillin resistance flat-plate culture is carried out at 37 ℃, monoclonal colonies are selected, after positive PCR identification, the monoclonal colonies are subjected to shaking in 5mL of ordinary LB culture solution containing ampicillin with a final concentration of 100 mu g/mL, and the culture solution is shaken at 37 ℃ for overnight; extracting plasmids, sequencing and detecting, transforming pET20b (+) -sA-cMfp plasmids into escherichia coli expression bacteria BL21 (DE 3) to be competent, randomly picking up 10 monoclonal bacteria, adding 5mL of common LB culture solution containing ampicillin with a final concentration of 100 mug/mL into a 20mL test tube after positive PCR identification, shaking at 37 ℃ for overnight, transferring 50ul of bacteria solution into 5mL of common LB culture solution containing ampicillin with a final concentration of 50 mug/mL according to a transfer amount of 1:100, continuously culturing at 37 ℃ for 3 hours, collecting 200ul of bacteria solution when the OD600 value reaches 0.6-0.8, centrifuging at 10000rpm for 1min, adding 1mmol/L of IPTG into the residual culture medium, continuing shaking at 37 ℃ for 3h, and finally collecting bacteria after induction and centrifuging at 10000rpm for 1 min. Adding 80uL of 5% SDS solution into thallus, boiling at 100deg.C for 10min, clarifying, adding 20uL of 5x loading buffer, and boiling at 100deg.C for 10min to complete sample preparation. The expression level and molecular weight of the recombinant protein in the expressed cells were identified by SDS-PAGE electrophoresis, and the results are shown in FIG. 5. The strain with the highest expression ratio is selected for bacteria retention and subsequent amplification culture.
Example 4: novel self-assembled mussel mucin sA-cMfp expression capability detection at different culture scales
The frozen BL21 (DE 3)/pET 20b (+) _sA-cMfp glycerol bacteria were removed from the freezer at-80℃and were prepared according to a 1:1000, 10ul was added to 10mL of ordinary LB medium containing 50. Mu.g/mL ampicillin, and the mixture was shaken at 37℃overnight. Sequentially taking 50uL, 500uL and 5000uL bacterial solutions according to the transfer amount of 1:100, respectively adding the bacterial solutions into newly prepared 5mL, 50mL and 500mL of common LB culture solution containing ampicillin with the final concentration of 100 mug/mL, shaking for 3 hours at 37 ℃, when the OD600 value reaches 0.6-0.8, taking 200uL bacterial solutions, centrifuging at 10000rpm for 1min to collect bacterial bodies before induced expression, adding 1mmol/L IPTG into the residual culture medium, continuing shaking for 3 hours at 37 ℃, finally taking 200uL bacterial solutions, centrifuging at 10000rpm for 1min to collect bacterial bodies after induced expression. After the sample preparation, SDS-PAGE electrophoresis is used for detecting the expression condition of the recombinant protein under different culture levels, and imageJ gray scale analysis is used for semi-quantitative analysis of the expression ratio, and the result is shown in FIG. 6.
The frozen BL21 (DE 3)/pET 20b (+) _sA-cMfp glycerol bacteria were removed from the freezer at-80℃and were prepared according to a 1:1000 in a ratio of 1:1000 were inoculated into 25 mL LB liquid medium containing 100. Mu.g/mL ampicillin, and cultured in an incubator at 37℃and 180rpm/min for 16: 16 h to activate the seed solution. Taking activated seed liquid according to the proportion of 1:100 were inoculated into 6 conical flasks containing 250mL of LB liquid medium 1L. And (3) culturing the secondary seed liquid under the condition that the culture condition is as before, feeding the secondary seed liquid into a tank after culturing for 4 hours, slowly feeding glucose to supplement carbon sources, magnesium sulfate and other microelements after feeding the secondary seed liquid into the tank, controlling the dissolved oxygen value in the tank to be higher than 30%, gradually increasing the stirring rotating speed to 500rpm, gradually increasing the ventilation rate while increasing the stirring speed, controlling the maximum ventilation rate to be 2.5m 3/h, controlling the internal pressure of the tank to be 0.02Mpa, and causing autolysis of bacteria due to the excessive internal pressure of the tank. After the growth of the bacteria starts to be gentle and the bacterial weight approaches the maximum value, adding IPTG to induce for 4 hours, then placing the bacteria in a tank, centrifuging at 4000rpm/min, discarding the supernatant after 30min, collecting the bacterial cells, and storing the bacterial cells in a refrigerator at the temperature of minus 20 ℃. SDS-PAGE electrophoresis detects the expression of the recombinant protein, and the results are shown in FIG. 6.
Example 5: novel self-assembled mussel mucin sA-cMfp temperature phase transition, washing, centrifugation and purification
BL21 (DE 3)/pET 20b (+) -sA-cMfp cells were weighed to 10g wet weight, 100mL of 50mM Tris-HCl (pH 8.7) was used as buffer to resuspend the cells, after sufficient resuspension, homogenized and broken 3 times at 4℃under high pressure, centrifuged at 800pa at 12000rpm for 30min, the broken supernatant was collected, placed in a water pre-pot at 40℃for 5min, the supernatant was discarded, and the pellet was collected by centrifugation. Meanwhile, collecting bacterial breaking sediment, combining the sediment,
The pellet was resuspended in 100mL of 50mM Tris-HCl (pH 8.7) buffer containing 1M urea, 0.5% Trixton-X100, magnetically stirred at room temperature for a period of time, the supernatant discarded, and the pellet was collected by centrifugation at 12000rpm for 30 min. The precipitate collected in the previous step was resuspended in 100mL of 50mM Tris-HCl (pH 8.7), magnetically stirred at room temperature for 1h, centrifuged at 12000rpm for 30min, the supernatant discarded and the precipitate collected. Finally, the sediment collected in the last step is resuspended by using 100ml of ultrapure water, after magnetic stirring for 1h at room temperature, supernatant is discarded by centrifugation at 12000rpm for 30min, and sediment is collected by centrifugation, so that the purified novel self-assembled mussel mucin sA-cMfp is obtained, and the result is shown in figure 7.
Example 6: novel in vitro hydroxylation detection of self-assembled mussel mucin
Dissolving the novel self-assembled mussel mucin sA-cMfp after freeze-drying with a little 10% acetic acid, adding 15mmol/L ascorbic acid, 20umol/L copper sulfate, 20mmol/L phosphate buffer (pH 7.6) and 20U of commercial tyrosinase, preparing into a solution with the final protein concentration of 0.5mg/mL, stirring at room temperature for 3 hours, modifying the tyrosine residue of the novel self-assembled mussel mucin sA-cMfp into DOPA, and adding acetic acid to adjust the pH of the feed liquid to 3.2 at the modification end point.
1, 2-Benzenediol of the DOPA (DOPA) residue in the DOPA protein molecule can be oxidized to a wake compound under the conditions of alkalinity and excessive glycine as a reducing agent, and insoluble blue-violet crystal formazan (Formazan) is generated after adding nitro tetrazolium chloride (NBT). Dopa is detected using this principle. Firstly, weighing 75 g glycine to dissolve in 400 mL water, regulating the pH to 10 by using solid KOH, adding water to a constant volume of 500 mL, after preparation, preserving at 2-8 ℃ to obtain glycine-potassium buffer solution (pH 10), then weighing 2.5 g sodium borate decahydrate to dissolve in 40 mL water, carrying out ultrasonic heating at 40 ℃ to obtain sodium borate solution, finally weighing 9mgNBT, adding 15 mL glycine-potassium buffer solution to dissolve, mixing uniformly to obtain NBT staining solution, and preparing the NBT staining solution immediately after use. After the preparation of the solution is completed, a2 uL sample is taken and placed on a 5cm multiplied by 5cm nitrocellulose membrane (NC membrane) with 0.2um, the position of the sample is marked, the NC membrane carrying the sample is placed in a 500 mL beaker after the sample is absorbed by the NC membrane, the solution is ultrasonically treated for 10min after 300 mL of pure water is added, the NC membrane is taken out, placed in a culture dish, and the NBT staining solution is added for 45min by shaking in a dark place. The NC membrane was taken out, washed twice with sodium borate solution, stored in sodium borate solution overnight, washed 3 times with pure water, and observed for the generation of bluish-violet spots at the sample marks, and the results are shown in FIG. 8.
Example 7: novel self-assembled mussel mucin sA-cMfp in vitro assembly characteristic detection
Taking the novel self-assembled mussel mucin sA-cMfp after freeze-drying, dissolving with a little 10% acetic acid, adding 20mmol/L phosphate buffer solution (pH 7.6) to prepare a solution with the protein concentration of 0.5mg/mL, placing the solution at 37 ℃ for incubation for 24 hours, observing short rod-shaped fiber substances in the solution, placing the solution under a bright field microscope for 100x observation of appearance, and detecting through SDS-PAGE electrophoresis, wherein the result is shown in figure 9.
Taking the novel self-assembled mussel mucin sA-cMfp after freeze-drying, respectively dissolving with a little 10% acetic acid and 1.5% ammonia solution, and diluting with ultrapure water until the protein concentration is 0.1mg/mL. Taking a clean mica sheet, respectively spotting 10uL of samples on the mica sheet, naturally airing, and detecting the appearance of the samples on the mica sheet by using an atomic force microscope, wherein the probe type is scanasyst-air, and the system is Dimension FastScan. The results are shown in FIG. 9.
Example 8: novel pH-regulated self-assembled mussel mucin sA-cMfp forming different assemblies
Taking the novel self-assembled mussel mucin sA-cMfp after freeze-drying, dissolving the novel self-assembled mussel mucin sA-cMfp by using 1.5% ammonia solution, and supplementing ultrapure water to dilute the novel self-assembled mussel mucin sA-cMfp to the protein concentration of 8mg/mL. By adding acetic acid, the pH was adjusted to 11.8, 11.0, 10.8, 10.2, 10.0, 9.8, 9.6, 9.4, 8.2, 5.4, 4.6, 4.3 in this order. Taking 1mL of each ph, observing the solution appearance at 0h and 16h respectively, and finally centrifuging at 5000rpm for 1min to collect the assemblies at different phs as shown in figure 10, wherein the assemblies formed at isoelectric points of 9.8 and surrounding ph show obvious viscose-drawn filiform appearance, and emulsion-like substances are formed at ph of 5.4 and below, and are coated on the back of the hand to form yellow films which are tightly adhered to the skin surface as shown in figure 10.
Example 9: novel adsorption capacity detection of self-assembled mussel mucin sA-cMfp on hydrophilic-hydrophobic interface
Taking novel self-assembled mussel mucin sA-cMfp after freeze-drying, dissolving with a little 10% acetic acid, adding ultrapure water to prepare a solution with protein concentration of 2.00mg/mL, 1.00mg/mL, 0.50mg/mL and 0.25mg/mL, taking 10uL respectively to be dotted on a hydrophobic plastic dish and a hydrophilic glass sheet, taking BSA protein as a reference, naturally airing for 2 hours at room temperature, washing with water, carrying out ultrasonic treatment for 5 minutes in a water bath, soaking for 10 minutes with coomassie brilliant blue dye solution, dyeing, washing out the dye solution, photographing and observing residual western blotting, and judging the adsorption capacity on the surface, wherein the result is shown in figure 11.
Example 10: novel self-assembled mussel mucin sA-cMfp cell adhesion promotion adhesion detection
Taking the novel self-assembled mussel mucin sA-cMfp after freeze-drying, respectively dissolving with a little 10% acetic acid and 1.5% ammonia solution, diluting with ultrapure water to a concentration of 1mg/mL, preparing a clean and dry dish cover of a 96-well plate, dripping 10uL of the sA-cMfp solution on the plate cover, making 4 compound holes, drying in a baking oven at 37 ℃ to form a film, washing with water for 3 times, drying in the air again for 20min, and sterilizing the ultraviolet surface for 30min to finish protein coating.
L929 cells and Hacat cells cultured for 3 days were taken and digested with pancreatin, 2mL (150 ten thousand/mL) of a cell suspension was prepared in a serum-free medium, 20uL of about 3 ten thousand cells were taken and dropped on the corresponding protein-containing membrane upper plate. The same number of cells were dropped on the cover without any treatment as a control group, and finally the culture plate was covered, after incubation at 37℃for about 1.5-2 hours, the plate was removed, and before 40X photographing as washing, after 2 gentle washes with PBS, photographing was continued as washing, and the results were compared with the front and rear changes, and are shown in FIG. 12.
Example 11: novel self-assembled mussel mucin sA-cMfp cytotoxicity detection
Taking the novel self-assembled mussel mucin sA-cMfp after freeze-drying, respectively dissolving with a little 10% acetic acid and 1.5% ammonia solution, diluting with ultrapure water to a concentration of 0.1mg/mL, coating the bottom of a 96-well plate with each solvent group respectively with the dosage of 1.0ug/cm 2(L)、2.5ug/cm2(M)、5.0ug/cm2 (H), drying to form a protein film layer, washing with water for 3 times, airing, taking a hole which is not subjected to any treatment as a control group, and irradiating the plate bottom with ultraviolet for 1H after finishing the modification.
The well-grown L929 cells and Hacat cells are digested by pancreatin, inoculated into 96 wells modified by protein and 96 wells unmodified by protein respectively according to the cell number of 5000 cells per well, and then placed in 37 ℃ for cell constant temperature culture until 24 hours, 48 hours and 72 hours. After the culture was performed to the designated time point, 10 μ LMTT solution was added to each well, the culture was continued for 4 hours, 100 μl of formazan solubilization solution was carefully added to each well, the absorbance was measured at 570nm with a microplate reader after the formazan was completely dissolved, and the absorbance curve was plotted, and the results are shown in fig. 13.
Example 12: novel self-assembled mussel mucin sA-cMfp adhesion capability detection on different materials
5Ul of sA-cMfp protein solution with the concentration of 1mg/mL is dripped on the upper part of a plastic culture dish, gun heads with different sizes are adhered on the dish, the mixture is dried for 12 hours in a 25 ℃ humid environment, and the adhesion condition is observed. The results are shown in FIG. 14.
The glass slide was dropped with 5ul of the sA-cMfp protein solution at a concentration of 1mg/mL, the tips of different sizes were adhered to a dish, dried in a wet environment at 25℃for 12 hours, and the adhesion was observed. The results are shown in FIG. 14.
A solution of sA-cMfp protein (10 ul) at a concentration of 2mg/mL was dropped onto a slide glass, and then another slide glass was covered thereon, dried in a wet environment at 25℃for 12 hours, and then a 100mL centrifuge tube was hung to see if it could lift the weight, as shown in FIG. 14
The sA-cMfp protein freeze-dried fiber is taken, a small amount of water is added to form a pasty substance, then the broken femur and skin of the rat are bonded by the sA-cMfp pasty substance, and the bonding condition is observed. The results are shown in FIG. 14.
Example 13: novel self-assembled mussel mucin sA-cMfp freeze-dried fiber in vitro hemostatic capability detection
Freeze-drying novel self-assembled mussel mucin sA-cMfp formed under different ph to obtain different freeze-dried fibers, placing the freeze-dried fibers into a 96-well plate,
50 Mu L of fresh anticoagulated whole rat blood is dripped on the surface of the sample, then 5uL of 0.2M CaCl2 solution is dripped on the sample, the sample is incubated at 37 ℃ for 5min, 150uL of distilled water is added to cause the non-coagulated red blood cells to be hemolyzed in the water, the sample is kept at the temperature of 37 ℃ for 3min, and 100uL of supernatant is taken to measure absorbance at 545 nm. The control group was a void with no lyophilized fibers and the other treatments were the same as the experimental group. The blood coagulation index calculation formula is BCI (%) =od Experimental group /OD Control group . The results are shown in FIG. 15.
Claims (10)
1. The novel self-assembled mussel mucin is characterized by comprising a mussel mucin core functional area and a self-assembled functional area, wherein a combination mode of the two areas adopts a two-embedded combination model or a three-embedded combination model; the sequence of the two-embedded combined model is N-end-self-assembled functional area-mussel mucin core functional area-C end, and the sequence of the three-embedded combined model is N-end-self-assembled functional area-mussel mucin core functional area-self-assembled functional area-C end.
2. The novel self-assembled mussel mucin according to claim 1, wherein the amino acid sequence of the mussel mucin core functional region is shown in SEQ ID No. 1.
3. The novel self-assembled mussel adhesive protein according to claim 1, wherein the self-assembled functional region is based on a VPGXG pentapeptide basic unit, and the fourth X passenger seat residue is replaced by a characteristic amino acid of a natural adhesive protein to realize bionic design, and the structural mode is [ (VPGYG) (VPG (X/Z) G) ] n, and is rich in tyrosine residues; wherein V is valine, P is proline, G is glycine, Y is tyrosine; x is tyrosine or any positively charged amino acid; z is any hydrophobic amino acid with a hydrophobic fraction of more than 2.5 except proline; n is an integer greater than or equal to 1.
4. The novel self-assembling mussel adhesive protein of claim 3, wherein the positively charged amino acid is arginine, lysine, or histidine; z is isoleucine, valine, leucine, phenylalanine, methionine or alanine; and n is 10, 15, 30, 40 or 60.
5. The novel self-assembled mussel adhesive protein according to claim 4, wherein the fourth X guest seat residue in the VPGXG basic sequence unit is valine and tyrosine, the repeating unit n is 10, the final structural mode is [ (VPGYG) (VPGVG) ] 10, and the amino acid sequence is shown in SEQ ID NO. 2.
6. The novel self-assembled mussel mucin according to claim 1, wherein the self-assembled functional region is located at the N-terminal end, the mussel mucin core functional region is located at the C-terminal end, and the self-assembled mussel mucin core functional region and the mussel mucin core functional region are directly arranged in series to form a two-embedded model without linker therebetween, and the amino acid sequence of the self-assembled mussel mucin is shown as SEQ ID No. 3.
7. The novel self-assembled mussel mucin of claim 1, wherein the novel self-assembled mussel mucin has the whole gene nucleotide sequence shown in SEQ ID No. 4.
8. The novel self-assembled mussel mucin according to claim 1, wherein the pH is adjusted using an acid-base solvent, which is a volatile weak acid-base solution, to form different assembled aggregation structures.
9. The novel self-assembled mussel adhesive protein according to claim 1, wherein tyrosinase and proline hydroxylase are modified by hydroxylation in vitro or in vivo by co-expression of tyrosinase and proline hydroxylase in escherichia coli.
10. Use of the novel self-assembled mussel mucin according to claims 1-9 for the preparation of a cell adhesion promoting, antibacterial, hemostatic, reparative or adhesive medical device or cosmetic.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160220727A1 (en) * | 2013-11-13 | 2016-08-04 | Massachusetts Institute Of Technology | Self-assembling underwater adhesives |
| US20200199199A1 (en) * | 2017-04-05 | 2020-06-25 | Industry-University Cooperation Foundation Hanyang University Erica Campus | Multiblock copolypeptides of elastin-based polypeptides and mussel foot proteins with stimuli-responsiveness and surface-adhesive, methods of preparing thereof and use thereof |
| US20200354416A1 (en) * | 2016-03-23 | 2020-11-12 | Industry-University Cooperation Foundation Hanyang University Erica Campus | Self-assembled nanostructures of elastin-and resilin-based block copolypeptides with stimuli responsiveness and resilience for drug delivery system, tissue engineering and regenerative medicine and methods of preparing the same |
| KR20220026515A (en) * | 2020-08-25 | 2022-03-04 | 한양대학교 에리카산학협력단 | Stimuli-responsive and surface-adhesive, sticky elastin and suckerin based multiblock copolypeptides, their self-assembled nanostructures and injectable hydrogels as bio-adhesives for applications |
| CN115724915A (en) * | 2022-08-17 | 2023-03-03 | 上普博源（北京）生物科技有限公司 | Elastin-like polypeptide adhesive and preparation method and application thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160220727A1 (en) * | 2013-11-13 | 2016-08-04 | Massachusetts Institute Of Technology | Self-assembling underwater adhesives |
| US20200354416A1 (en) * | 2016-03-23 | 2020-11-12 | Industry-University Cooperation Foundation Hanyang University Erica Campus | Self-assembled nanostructures of elastin-and resilin-based block copolypeptides with stimuli responsiveness and resilience for drug delivery system, tissue engineering and regenerative medicine and methods of preparing the same |
| US20200199199A1 (en) * | 2017-04-05 | 2020-06-25 | Industry-University Cooperation Foundation Hanyang University Erica Campus | Multiblock copolypeptides of elastin-based polypeptides and mussel foot proteins with stimuli-responsiveness and surface-adhesive, methods of preparing thereof and use thereof |
| KR20220026515A (en) * | 2020-08-25 | 2022-03-04 | 한양대학교 에리카산학협력단 | Stimuli-responsive and surface-adhesive, sticky elastin and suckerin based multiblock copolypeptides, their self-assembled nanostructures and injectable hydrogels as bio-adhesives for applications |
| CN115724915A (en) * | 2022-08-17 | 2023-03-03 | 上普博源（北京）生物科技有限公司 | Elastin-like polypeptide adhesive and preparation method and application thereof |
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