CN108753183A - A kind of live body underwater adhesive material and application thereof - Google Patents

A kind of live body underwater adhesive material and application thereof Download PDF

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Publication number
CN108753183A
CN108753183A CN201810519230.7A CN201810519230A CN108753183A CN 108753183 A CN108753183 A CN 108753183A CN 201810519230 A CN201810519230 A CN 201810519230A CN 108753183 A CN108753183 A CN 108753183A
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biomembrane
bsla
tasa
fusion proteins
protein
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CN108753183B (en
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钟超
张琛
黄娇芳
张继聪
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University of Shanghai for Science and Technology
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University of Shanghai for Science and Technology
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Priority to CN201810519230.7A priority Critical patent/CN108753183B/en
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Priority to PCT/CN2019/088492 priority patent/WO2019228291A1/en
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Priority to US17/106,105 priority patent/US20210079049A1/en
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
    • C09J7/00—Adhesives in the form of films or foils
    • C09J7/10—Adhesives in the form of films or foils without carriers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09J105/00—Adhesives based on polysaccharides or on their derivatives, not provided for in groups C09J101/00 or C09J103/00
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
    • C09J11/00—Features of adhesives not provided for in group C09J9/00, e.g. additives
    • C09J11/08—Macromolecular additives
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09J7/00—Adhesives in the form of films or foils
    • C09J7/30—Adhesives in the form of films or foils characterised by the adhesive composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract

The present invention relates to underwater adhesive Material Fields, more particularly to a kind of live body underwater adhesive material and application thereof.The present invention provides a kind of biomembrane, and the biomembrane includes TasA fusion proteins, exocellular polysaccharide, BslA fusion proteins.Underwater adhesive provided by the present invention based on bacillus subtilis biomembrane is living materials, has the advantages such as component is adjustable, sticky ingredient expression secretion is controllable, material is renewable, to further building the underwater adhesive material of intelligent environment response type（Such as light-operated, quorum sensing, odor sensing regulation and control）It is of great significance.

Description

A kind of live body underwater adhesive material and application thereof
Technical field
The present invention relates to underwater adhesive Material Fields, more particularly to a kind of live body underwater adhesive material and application thereof.
Background technology
Underwater adhesive is explored in biological medicine, ocean as a kind of advanced function material and the fields such as reparation suffers from Extremely important application.For example, it is required for using in suture healing, the fixation etc. of bone of biomedicine field, wound Adhesive；And in marine industry, the repairing of ship, dam, sewer pipe also be unable to do without underwater adhesive.Therefore, production has Biocompatibility or specific environment adaptability, and the underwater adhesive with high intensity high viscosity is particularly important.
Currently, research is more, is mainly based upon marine organisms inspiration using wider underwater adhesive and designs bionical Material.The adhesive characteristics of common marine organisms underwater adhesive include mainly：1. from the amyloid of barnacle glue protein inspiration Biological viscosity caused by protein structure；2. caused by catechol (DOPA) structure from mussel byssus protein inspiration Biological viscosity；3. the biological viscosity caused by positive and negative charge interaction from sandcastle worm inspiration.It is sent out by many decades Exhibition, although the research of bionical underwater adhesive obtains incremental advances, these materials are often simple simulation or integration The underwater adhesive feature of one of which or two kinds of biological inspirations, lacks further integrated optimization.Review the life of nature ocean The adhesive of object then usually contains a variety of binding proteins ingredients, and its adhesion process is empty by the host organisms stringent time Between regulate and control, have many characteristics, such as multicomponent, dynamic it is controllable, environment is responded.Therefore, structure has manifold biology Inspiration underwater adhesive material, and accomplish the regulatable intelligent underwater adhesive of each component space-time, it will be expected to break through current bonding The bottleneck of technology, widen current jointing material bio-medical and commercial Application application category.
Invention content
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of biomembranes and preparation method thereof And purposes, for solving the problems of the prior art.
In order to achieve the above objects and other related objects, first aspect present invention provides a kind of composition, the composition Include TasA fusion proteins, exocellular polysaccharide, BslA fusion proteins, the TasA fusion proteins include TasA protein fragments and make a gift of Byssus protein fragments, the BslA fusion proteins include BslA protein fragments and self-coagulation binding proteins segment.
In some embodiments of the invention, the TasA protein fragments are：A) amino acid sequence such as SEQ ID No.39 Shown in polypeptide fragment；
Or, b) amino acid sequence and SEQ ID NO.39 are with 80% or more homology and with polypeptide piece a) limited The polypeptide fragment of the function of section.
In some embodiments of the invention, the mussel byssus protein is selected from Mytilus galloprovincialis byssus protein, Perna viridis foot Silk-fibroin, California mussel byssus protein.
In some embodiments of the invention, the mussel byssus protein is selected from mefp-1, mgfp-2, mefp-3, mefp- 5、mcfp-6、pvfp3、pvfp5。
In some embodiments of the invention, the mussel byssus protein segment is：C) amino acid sequence such as SEQ ID Polypeptide fragment shown in one of No.41-47；
Or, d) one of amino acid sequence and SEQ ID No.41-47 have 80% or more, 85% or more, 90% with Upper, 93% or more, 95% or more, 97% or more or 99% or more homology and has the function of the c) polypeptide fragment of restriction Polypeptide fragment.
In some embodiments of the invention, the TasA albumen sources are in bacillus subtilis.
In some embodiments of the invention, the mussel byssus protein derives from mussel.
In some embodiments of the invention, the TasA fusion proteins include TasA protein fragments successively from N-terminal to C-terminal With mussel byssus protein segment.
In some embodiments of the invention, in the composition, TasA fusion proteins, exocellular polysaccharide, BslA merge egg White mass ratio is 1-5：80-90：1-5.
In some embodiments of the invention, the exocellular polysaccharide is polyanionic polymer.
In some embodiments of the invention, the exocellular polysaccharide is the exocellular polysaccharide of metal cation chelating, the gold Belong to one or more combinations of the cation in iron ion, magnesium ion, calcium ion, manganese ion.
In some embodiments of the invention, the exocellular polysaccharide derives from bacillus.
In some embodiments of the invention, BslA protein fragments are：E) amino acid sequence is as shown in SEQ ID No.40 Polypeptide fragment；
Or, f) amino acid sequence and SEQ ID NO.40 are with 80% or more homology and with polypeptide e) limited The polypeptide fragment of function.
In some embodiments of the invention, the self-coagulation binding proteins are selected from California mussel byssus protein and spread out Raw sticky small peptide.
In some of the invention embodiments, the self-coagulation binding proteins be selected from mcfp-3s, mcfp3spep, mcfp3spep-spytag。
In some embodiments of the invention, the self-coagulation binding proteins segment is：G) amino acid sequence such as SEQ Polypeptide fragment shown in one of IDNo.48-50；
Or, h) one of amino acid sequence and SEQ ID No.48-50 have 80% or more, 85% or more, 90% with Upper, 93% or more, 95% or more, 97% or more or 99% or more homology and has the function of the g) polypeptide fragment of restriction Polypeptide fragment.
In some embodiments of the invention, the BslA albumen sources are in bacillus subtilis.
In some embodiments of the invention, the self-coagulation binding proteins derive from mussel.
In some embodiments of the invention, the BslA fusion proteins include BslA segments and oneself successively from N-terminal to C-terminal Coherency binding proteins segment.
In some embodiments of the invention, the BslA fusion proteins are modified through tyrosinase.
Second aspect of the present invention provides a kind of biomembrane, and the biomembrane includes that the TasA in composition as described above melts Hop protein, exocellular polysaccharide, BslA fusion proteins.
In some embodiments of the invention, in the biomembrane, the content of TasA fusion proteins is 1-5wt%.
In some embodiments of the invention, in the biomembrane, the content of exocellular polysaccharide is 80-90wt%.
In some embodiments of the invention, in the biomembrane, the content of BslA fusion proteins is 1-5wt%.
In some embodiments of the invention, the biomembrane derives from bacillus.
Third aspect present invention provides a kind of expression system, and the expression system can form biomembrane as described above.
In some embodiments of the invention, the expression system includes engineering bacteria.
In some embodiments of the invention, at least part of engineering bacteria, which contains, can express the TasA fusions egg The polynucleotides of the coding TasA fusion proteins of external source are integrated in white construct or genome.
In some embodiments of the invention, at least part of engineering bacteria, which contains, can express the BslA fusions egg The polynucleotides of the coding BslA fusion proteins of external source are integrated in white construct or genome.
In some embodiments of the invention, at least part of engineering bacteria can generate EPS polysaccharide, at least part of The engineering bacteria contains the construct that can express tyrosine or the multinuclear glycosides that the encoding tyrosine of external source is integrated in genome Acid.
In some embodiments of the invention, the engineering bacteria is selected from bacillus.
Fourth aspect present invention provides the preparation method of the biomembrane, including：Suitably forming the biomembrane Under the conditions of, cultivate the expression system.
The life that fifth aspect present invention provides expression system as described above or the preparation method expression as described in wound obtains Object film.
Adhesive prepares neck under water for sixth aspect present invention offer composition as described above, biomembrane as described above The purposes in domain.
Description of the drawings
Fig. 1 is shown as the explanation of the biomembrane related gene of the different bacillus subtilis strains constructed by the present invention；
Fig. 2 is shown as the expression vector collection of illustrative plates (a the present invention is based on pHT01:pHT01-tapA-sipW-tasA；b: pHT01-tapA-sipW-tasA-mefp5；c:pHT01-bslA；d:pHT01-bslA-3sp-spytag)；
Fig. 3 is shown as the transmission electron microscope image (a of nanofiber of the present invention:TD；b:TD/pHT01-tapA- sipW-tasA；c:TD/pHT01-tapA-sipW-tasA-mefp5)；
Fig. 4 is shown as the rheology test curve (a of biomembrane of the present invention (TD series):Amplitude shakes test pattern result； b:Frequency oscillation test pattern result)；
Fig. 5 is shown as the lap shear viscosity test curve (a of biomembrane of the present invention (TD series):Lap shear test dress Set schematic diagram；b:Sticky test result figure)；
Fig. 6 is shown as the rheology test curve (a of biomembrane of the present invention (TD and DD):Amplitude shakes test pattern result； b:Frequency oscillation test pattern result)；
Fig. 7 is shown as biomembrane of the present invention (TD and DD) and the mixed infrared test spectrogram of iron ion；
Fig. 8 is shown as biomembrane of the present invention (DD) and the mixed scanning electron microscope image of iron ion；
Fig. 9 is shown as biomembrane of the present invention (DD) and its adds the lap shear viscosity test result of iron ion；
Figure 10 is shown as the shows fluorescent microscopy images of present invention detection spytag and mcherry-spycatcher interactions Picture；
Figure 11 is shown as the rheology test curve (a of biomembrane of the present invention (Db series):Amplitude shakes test pattern knot Fruit；b:Frequency oscillation test pattern result)；
Figure 12 is shown as the lap shear viscosity test curve (a of biomembrane of the present invention (Db series):Lap shear test dress Set schematic diagram；b:Sticky test result figure)；
Figure 13 is shown as the structure and collection of illustrative plates of tyrosinase expression carrier of the present invention；
Figure 14 is shown as the rheology test curve of the integrated biomembrane of the present invention；
Figure 15 is shown as the final lap shear viscosity test result of the integrated biomembrane of the present invention；
Figure 16 is shown as the lap shear viscosity test result of the integrated biomembrane of the present invention under various circumstances；
Figure 17 is shown as lap shear viscosity test result of the integrated biomembrane of the present invention after passage regenerates；
Figure 18 is shown as the syringeability verification of the integrated biomembrane of the present invention；
Figure 19 is shown as the sticky applicating example on different base material of the integrated biomembrane of the present invention.
Specific implementation mode
Inventor passes through a large amount of exploitative experiments, and the composition with bacillus subtilis biofilm is basic Frame relies on TasA (amyloid protein) fusion protein, exocellular polysaccharide (exopolysaccharides, EPS) and BslA (tables Face hydrophobin) fusion protein, realize that live body jointing material viscosity component is adjustable, purpose of adhesive strength successive optimization, The present invention is completed on the basis of this.
One aspect of the present invention provides a kind of composition, and the composition may include TasA fusion proteins, and the TasA melts Hop protein may include TasA protein fragments and mussel byssus protein segment.The TasA protein fragments can be：A) amino acid Sequence polypeptide fragment as shown in SEQ ID No.39, can also be b) amino acid sequence and SEQ ID NO.39 have 80% with Upper, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more or 99% or more homology and with a) limiting Polypeptide fragment function polypeptide fragment, it is described b) in amino acid sequence refer specifically to：The ammonia as shown in SEQ ID No.39 Base acid sequence by substitution, missing or addition it is one or more (can be specifically 1-50,1-30,1-20,1-10, 1-5 or 1-3) obtained from amino acid, or in the ends N- and/or the ends C- addition one or more (can be specifically 1-50,1-30,1-20,1-10,1-5 or 1-3) obtained from amino acid, and polypeptide fragment tool of its coding Just like the amino acid sequence of the function of the encoded polypeptide fragment of amino acid sequence shown in SEQ ID No.39.The mussel foot It includes but not limited to Mytilus galloprovincialis byssus protein (mytilus edulis that silk-fibroin (mussel foot proteins), which can be selected from, Foot protein, mefp), Perna viridis byssus protein (Perna viridis foot protein, pvfp), California mussel Byssus protein (Mytilus californianus foot protein), Mediterranean mussel byssus protein (Mytilus Galloprovincialis foot protein) etc., more specifically can be selected from include but not limited to mefp-1, mgfp-2, Mefp3, mefp5, mcfp-6, pvfp3, pvfp5 or their variant etc., for example, the mussel byssus protein segment can be： C) amino acid sequence polypeptide fragment as shown in one of SEQ ID No.41-47 can also be d) amino acid sequence and SEQ One of ID No.41-47 have 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, Or 99% or more homology and have the function of c) limit polypeptide fragment polypeptide fragment, it is described d) in amino acid sequence It refers specifically to：The amino acid sequence as shown in one of SEQ ID No.41-47 is by substitution, missing or addition one or more Obtained from a (can be specifically 1-50,1-30,1-20,1-10,1-5 or 1-3) amino acid, or in N- End and/or the addition of the ends C- it is one or more (can be specifically 1-50,1-30,1-20,1-10,1-5 or 1-3) obtained from amino acid, and polypeptide fragment of its coding has the ammonia as shown in one of SEQ ID No.41-47 The amino acid sequence of the function of the encoded polypeptide fragment of base acid sequence.The TasA albumen is typically derived from bacillus subtilis (latin name：Bacillus sutilis), the mussel byssus protein is typically derived from mussel (latin name：Mytilidae).? In the embodiment of the invention, the TasA fusion proteins include TasA protein fragments and mussel foot successively from N-terminal to C-terminal Silk-fibroin segment.
Can also include exocellular polysaccharide in composition provided by the present invention.The exocellular polysaccharide is usually polyanion Polymer, typically one of the main component of biomembrane are typically the polymer substance by microorganism secretion, more specifically can be with It is natural polymers, the microorganism (that is, source of exocellular polysaccharide) for secreting the exocellular polysaccharide can include but not It is limited to bacillus (for example, bacillus subtilis), pseudomonas aeruginosa, acetobacter, Escherichia coli etc..The exocellular polysaccharide It can usually be chelated with metal cation, in the composition, the exocellular polysaccharide can also be to be chelated with metal cation Exocellular polysaccharide.The metal cation can be including but not limited to iron ion, magnesium ion, manganese ion, calcium ion, copper from One or more combinations in son, sodium ion etc., in the embodiment of the invention, the metal cation is Fe3+ Ion.Should be known to those skilled in the art by exocellular polysaccharide and the method that metal cation chelates, for example, It can be by introducing metal cation (for example, salt containing metal cation) into composition, so that exocellular polysaccharide and gold Belong to cation chelating, so as to rely on exocellular polysaccharide to build itself and Fe3+Electrostatic interaction, utilize the effect obtain viscosity Raising
Can also include BslA fusion proteins in composition provided by the present invention, the BslA fusion proteins can wrap Include BslA protein fragments and self-coagulation binding proteins segment.The BslA protein fragments can be：E) amino acid sequence such as SEQ Polypeptide fragment shown in ID No.40, can also be f) amino acid sequence and SEQ ID NO.40 have 80% or more, 85% with Upper, 90% or more, 93% or more, 95% or more, 97% or more or 99% or more homology and the polypeptide piece with e) restriction Section function polypeptide fragment, it is described f) in amino acid sequence refer specifically to：The amino acid sequence as shown in SEQ ID No.40 By substitution, missing or addition it is one or more (can be specifically 1-50,1-30,1-20,1-10,1-5 or 1-3) obtained from amino acid, or the ends N- and/or the addition of the ends C- it is one or more (can be specifically 1-50, 1-30,1-20,1-10,1-5 or 1-3) obtained from amino acid, and polypeptide fragment of its coding has such as SEQ The amino acid sequence of the function of the encoded polypeptide fragment of amino acid sequence shown in ID No.40.The self-coagulation bonds egg The white mussel byssus protein for being often referred to that itself reunion can occur usually can be the California mussel foot that itself reunion can occur Silk-fibroin and derivative sticky small peptide etc., more specifically can include but is not limited to mcfp-3s, mcfp3spep (in the application again Abbreviation 3sp), mcfp3spep-spytag etc..For example, the self-coagulation binding proteins segment can be：G) amino acid sequence The polypeptide fragment as shown in one of SEQ ID No.48-50 can also be h) amino acid sequence and SEQ ID No.48-50 One of them has 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more or 99% or more Homology and have the function of c) limit polypeptide fragment polypeptide fragment, it is described d) in amino acid sequence refer specifically to：Such as Amino acid sequence shown in one of SEQ ID No.48-50 (specifically may be used by substitution, missing or addition one or more Be 1-50,1-30,1-20,1-10,1-5 or 1-3) obtained from amino acid, or in the ends N- and/or One or more (can be specifically 1-50,1-30,1-20,1-10, the 1-5 or 1-3) amino of the ends C- addition Obtained from acid, and the polypeptide of its coding has coded by the amino acid sequence as shown in one of SEQ ID No.48-50 Polypeptide function amino acid sequence.The BslA albumen is typically derived from bacillus subtilis (latin name：Bacillus Subtilis), the self-coagulation binding proteins are typically derived from mussel (latin name：Mytilidae).It is specific in the present invention one In embodiment, the BslA fusion proteins include that BslA protein fragments and self-coagulation bond egg successively from N-terminal to C-terminal White tiles section.The BslA fusion proteins are typically to be modified through tyrosinase (tyrosinase), described to be modified through tyrosinase The tyrosine moieties being often referred in BslA fusion proteins are modified as DOPA, so as to further increase its tribute to viscosity It offers.
In composition provided by the present invention, TasA fusion proteins, exocellular polysaccharide mass ratio typically 0.1-10： 60-90 or 1-5：The mass ratio typically 0.1-10 of 80-90, TasA fusion protein, BslA fusion proteins：0.1-10, or 1-5：1-5.The preparation method of the composition should be known to those skilled in the art, for example, can pass through The methods of chemical synthesis, microculture prepare, purify above-mentioned fusion protein and/or exocellular polysaccharide, and they are mixed to obtain Obtain above-mentioned composition.
Another aspect of the present invention provides a kind of biomembrane, and the biomembrane may include TasA fusions egg as described above In vain.In the biomembrane, the content of TasA fusion proteins can be 0.1-10wt% or 1-5wt%.
Can also include exocellular polysaccharide as described above in biomembrane provided by the present invention.It is extracellular in the biomembrane The content of polysaccharide is 60-90wt% or 80-90wt%.
Can also include BslA fusion proteins as described above in biomembrane provided by the present invention.In the biomembrane, The content of BslA fusion proteins can be 0.1-10wt% or 1-5wt%.
In biomembrane provided by the present invention, the biomembrane is typically to derive from bacillus (latin name： Bacillus), it can be specifically such as bacillus subtilis (latin name：Bacillus subtilis), bacillus megaterium (latin name：Bacillus megatherium), Bacillus cereus (latin name：Bacillus cereus), i.e., the described biology Film can usually be generated by above-mentioned mushroom, and bacterial strain can express secretion related fusion and polysaccharide, as described above to generate Biomembrane.For example, since bacillus subtilis strain belongs to gram-positive bacteria, the ability with stronger exocytosis albumen will Fusion binding proteins are expressed and are secreted in expressing bacterial strain, and the albumen of secretion can be self-assembly of binder fibre extracellular.
Another aspect of the present invention provides a kind of expression system, and the expression system can form biomembrane as described above.
In expression system provided by the present invention, the expression system can be engineering bacteria.It, can be in the expression system TasA fusion proteins, exocellular polysaccharide and BslA fusion proteins as described above are expressed simultaneously in same engineering bacteria (group), for example, Engineering bacteria can contain whole in the construct or genome that can express the TasA fusion proteins and BslA fusion proteins simultaneously The polynucleotides of the coding TasA fusion proteins and BslA fusion proteins that have external source are closed, the engineering bacteria can be gemma bar Bacterium (for example, bacillus subtilis) etc..In the expression system, can also respectively it be expressed in multiple and different engineering bacterias (group) TasA fusion proteins, exocellular polysaccharide and BslA fusion proteins as described above, for example, at least part of engineering bacteria can be with table Up to TasA fusion proteins as described above, for another example at least part of engineering bacteria, which can contain, can express the TasA fusions The polynucleotides of the coding TasA fusion proteins of external source are integrated in the construct or genome of albumen；For another example at least The partial engineering bacteria can express exocellular polysaccharide as described above, for another example the engineering bacteria can be bacillus (example Such as, bacillus subtilis) etc.；For another example at least part of engineering bacteria can express BslA fusion proteins as described above, For another example at least part of engineering bacteria contain it is whole in the construct that can express the BslA fusion proteins or genome Close the polynucleotides for the coding BslA fusion proteins for having external source.Suitable engineering bacteria, structure may be selected in those skilled in the art Body etc. is built, and the expression system is built using suitable method, for example, the engineering bacteria usually can be bacillus, more Can be specifically one or more including but not limited in bacillus subtilis, bacillus megaterium, Bacillus cereus etc. Combination, the construct can be include being not limited in pHT01 carriers, pHT43 carriers, pHT254 carriers, pMAD carriers etc. One or more combinations.
In expression system provided by the present invention, at least part of engineering bacteria can also express tyrosinase, for example, The coding junket that at least part of engineering bacteria contains the construct that can express tyrosinase or is integrated with external source in genome The polynucleotides of propylhomoserin enzyme.Expressed tyrosinase can be used for modifying BslA fusion proteins, so as to further increase Its contribution to viscosity.
Another aspect of the present invention provides the preparation method of the biomembrane, including：Suitably forming the biomembrane Under the conditions of, cultivate expression system as described above.The condition for forming biomembrane generally depends on the kind of used expression system Class, the condition for being suitably used to form biomembrane should be known to those skilled in the art, for example, may be used The method of solid MSGG or MSGG Liquid Culture biomembrane.
The life that another aspect of the present invention provides expression system as described above or preparation method as described above expression obtains Object film.
Another aspect of the present invention provides the purposes of biomembrane adhesive preparation field under water.The underwater adhesive Agent can be the adhesive for different base material, for example, it may be various applicable inorganic matters and/or organic matter, described Inorganic matter can be including but not limited in unorganic glass, metal, paper material, ceramic material, bone and artificial skelecton etc. One or more combinations, the organic matter can be including but not limited to organic glass, braided wire, branch, plastics, poly- ammonia One or more combinations in textile materials such as the macromolecule polymeric materials such as ester, PDMS, cloth, nylon etc..
Underwater adhesive provided by the present invention based on bacillus subtilis biomembrane is living materials, has component can The advantages such as controllable, material is renewable are secreted in adjusting, sticky ingredient expression, to further building the underwater of intelligent environment response type Jointing material (such as light-operated, quorum sensing, odor sensing regulation and control) is of great significance.Compared with prior art, of the invention Major technique advantage and characteristic includes the following aspects：
1) finally obtained jointing material have live body (living cells) fundamental characteristics, including bio-regeneration, environmental response, The superior functions such as programmable and self-regeneration, these are characterized in what general macromolecule and pure protein jointing material did not had Feature.
2) live body jointing material is the bacillus subtilis biofilm based on genetic modification, and utilizes base on this basis Because modular approach designs the jointing material with different molecular feature, these jointing materials show the spy of different adhesive strengths Sign；It designs and integrates the jointing material for including a variety of bonding characterization of molecules in the genome finally by genetic engineering, obtain excellent The live body jointing material of change.
3) there is stronger environmental resistance, syringeability etc. by the integrated live body jointing material that design and rational obtains Feature, and preferable underwater viscosity is shown to general material substrate.
4) live body jointing material is built based on the bacillus subtilis of genetic modification, can also be introduced on this basis Other functional groups or albumen (including bonding and non-adhering relevant functional group) build multifunctional active jointing material.
5) since biomembrane is directly related with active somatic cell, which in the future may be by using bacterium to extraneous ring Expression regulation of the perception (such as pH, illumination, temperature and smell) or Gene circuits in border to bacterium (feel by such as chemical induction, group Should wait), controllability expression and secretion of the feature capability fusion protein on time and space are realized, to realize intelligent bonding Living materials concept.
6) compared with Escherichia coli, bacillus subtilis belongs to generally accepted safety (Generally Recognized As Safe, GRAS) microorganism, biological safety is more preferable, is expected to have important application potential in biological medicine and other industrial circles.
Illustrate that embodiments of the present invention, those skilled in the art can be by this specification below by way of specific specific example Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from Various modifications or alterations are carried out under the spirit of the present invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text In addition explicitly point out, singulative "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The specific construction method of engineering bacteria used in the present embodiment is as follows：
The main composition of bacillus subtilis biomembrane has：Exocellular polysaccharide, amyloid protein and surface hydrophobicity albumen, point Not by epsA-O gene clusters, tasA genes, the expression of bslA gene codes is the expression of modularized design tasA-R fusion proteins, The bacterial strain TD that structure biomembrane related gene all knocks out.For modularized design EPS and Fe3+Interaction, build biomembrane The bacterial strain DD that eps genes retain.For the expression of modularized design bslA-R ' interfacial viscosity fusion proteins, structure biomembrane bslA The bacterial strain Db of gene knockout.
By gradually knocking out bslA, above-mentioned three kinds of different bacillus subtilises can be obtained in tasA and epsA-O genes Bacterial strain.Specifically, 168 genome sequence of bacillus subtilis (the gene order-checking genebank being sequenced with complete genome Number NC_000964) it is foundation, design upstream specific primer upstream-F and upstream-R and downstream special primer Downstream-F and downstream-R.Using 3610 genome of wild-type B. subtilis as template, PCR expands purpose and strikes Except the upstream and downstream homologous sequence about 1kb of gene in the genome, use pMAD for integrative plasmid carrier, involved plasmid structure It builds, bacterial strain converts, the same patent of strain construction equimolecular biological experimental method (201611156490.X).
Difference has, and for building Db bacterial strains, it is respectively D bslA that upstream and downstream fragment primer needed for bslA is deleted in amplification upstream-F(SEQ ID NO：1),D bslA upstream-R(SEQ ID NO：And D bslA downstream-F 2) (SEQ ID NO：3),D bslA downstream-R(SEQ ID NO：4), integration vector restriction enzyme site used be SmaI and EcoRI.DD bacterial strains are further built on this basis, and it is respectively D tasA that upstream and downstream fragment primer needed for tasA is deleted in amplification upstream-F(SEQ ID NO：5),D tasA upstream-R(SEQ ID NO：And D tasA downstream-F 6) (SEQ ID NO：7),D tasA downstream-R(SEQ ID NO：8), integration vector restriction enzyme site used be SmaI and NcoI.TD bacterial strains are further built on this basis, and it is respectively D eps that upstream and downstream fragment primer needed for epsA-O is deleted in amplification upstream-F(SEQ ID NO：9),D eps upstream-R(SEQ ID NO：And D eps downstream-F 10) (SEQ ID NO：11),D eps downstream-R(SEQ ID NO：12).Integration vector restriction enzyme site used be BamHI and SalI first synthesizes tapA-sipW-tasA-mefp5 gene clusters (SEQ ID in addition, for building integrated bacterial strain T5B3 by company NO：14) and its primer is designed as tasA-mefp5-F (SEQ ID NO：25),tasA-mefp5-R(SEQ ID NO：26), if Count upstream and downstream amplimer tasA-mefp5upstream-F (SEQ ID NO：23),tasA-mefp5upstream-R(SEQ ID NO：And tasA-mefp5downstream-F (SEQ ID NO 24)：27),tasA-mefp5downstream-R(SEQ ID NO：28), integration vector restriction enzyme site used is SmaI and NcoI.(3sp is when genome introduces bslA-3sp on this basis Mfp3spep abbreviations, similarly hereinafter), bslA-3sp-spytag (SEQ ID NO are equally synthesized by company：19) it and designs its primer and is bslA-3sp-F(SEQ ID NO：31),bslA-3sp-R(SEQ ID NO：32), design upstream and downstream amplimer bslA-3sp upstream-F(SEQ ID NO：29),bslA-3sp upstream-R(SEQ ID NO：And bslA-3sp 30) downstream-F(SEQ ID NO：33,bslA-3sp downstream-R(SEQ ID NO：34), integration vector enzyme used Enzyme site is SmaI and NcoI.
Described tapA-sipW-tasA-R, bslA-R ' and tyrosinase related genes all obtained by company is fully synthetic , expression carrier used thereof is pHT01 (mobitec companies), and restriction enzyme site is BamHI and SmaI.Structure contains target gene Expression vector the same patent of specific method (201611156490.X).Particularly, for building control vector pHT01-tapA- SipW-tasA, tapA-sipW-tasA gene order are SEO ID NO.13, and upstream and downstream primer is respectively TO-F (SEQ ID NO：And TO-R (SEQ ID NO 15)：16).For building pHT01-tapA-sipW-tasA-mefp5, upstream and downstream primer difference For TO-F (SEQ ID NO：And TO-mefp5-R (SEQ ID NO 15)：17), for building control vector pHT01-bslA, BslA gene orders are SEO ID NO.18, and upstream and downstream primer is respectively bslA-F (SEQ ID NO：And bslA-R (SEQ 20) ID NO：21).For building pHT01-bslA-3sp-spytag, by the fully synthetic segment bslA-3sp-spytag (SEQ of company ID NO：19), upstream and downstream primer is respectively bslA-F (SEQ ID NO：And bslA-3sp-spytag-R (SEQ ID NO 20)： 22).For building pHT01-tyrosinase, by company fully synthetic segment tyrosinase (SEQ ID NO：36), upstream and downstream Primer is respectively tyrosinase-F (SEQ ID NO：And tyrosinase-R (SEQ ID NO 37)：38).
In addition to said gene Engineering operation, involved LB culture mediums, biofilm medium MSGG, liquid training in the present invention Support the method equating patent (201611156490.X) of biomembrane.It should be pointed out that solid LB or MSGG culture medium is in liquid The agar powder of addition 1.5% is formulated on the basis of body culture medium, and the Biofilm Environment cultivated on solid medium is：Inoculation Stay overnight 37 DEG C of the monoclonal on streak plate in the LB culture mediums of 5mL, 220rpm shaken cultivation 3h, again in 1% ratio 37 DEG C are inoculated in the LB of 5mL, 220rpm shaken cultivations 3h.Thalline were collected by centrifugation by 5000g, is resuspended to OD=with deionized water 0.8-1.0 draws 2.5 μ L points on MSGG solid plates.If containing pHT01 expression vectors in host strain, in seeded process Culture medium contains the chloramphenicol of 5 μ g/mL；For containing the bacterial strain of pHT1-bslA-R ' carriers, the final culture for cultivating biomembrane The IPTG containing 1mmol/L is needed in base；For the bacterial strain containing pHT1-tyrosinase carriers, the final culture for cultivating biomembrane The CuSO of 0.4 μ g/mL is needed in base4；
In addition, the same patent of method for making sample is immunized in transmission electron microscope (TEM) involved in the present invention (201611156490.X)。
Embodiment 1：Modularized design based on amyloid protein TasA and mussel byssus protein adhesive characteristics
This example, because of the bacterial strain TD (Fig. 1 d) knocked out completely, can be dropped to greatest extent using bacillus subtilis biomembrane base It is viscous to improving biomembrane to be also beneficial to significantly more efficient reflection design for influence of the low biomembrane intrinsic viscosity to modularized design The contribution of property.The present invention constructs the pHT01-tapA-sipW-tasA plasmids of heterogenous expression amyloid protein TasA as a contrast (Fig. 2 a), and the pHT01-tapA-sipW-tasA-mefp5 plasmids of amyloid protein and mussel byssus protein can be expressed simultaneously (Fig. 2 a).
The e. coli tg1 (Biovecter, BioVector1058 12-6) that activation is chosen from LB tablets, is inoculated in 5ml LB culture mediums, 37 DEG C of shaken cultivations stay overnight.Bacterium solution is pressed 1:100 ratio inoculation, takes 250 μ l bacterium solutions to be cultivated in the LB of 25ml In base, 37 DEG C of shaken cultivations 2-3 hours to OD 600=0.5 or so.Bacterium solution is transferred in 50ml centrifuge tubes, is placed on ice 10min.At 4 DEG C, 4000r/min centrifuges 10min, abandons supernatant, and pipe inversion 1min to liquid is flow to end.With what is be pre-chilled on ice The CaCl2 solution 10ml of 0.1mol/L gently suspension cells, stand 30min on ice.0-4 DEG C, 4000r/min centrifuges 10min, abandons The CaCl2 solution of the 0.1mol/L of 2ml precoolings is added in supernatant, and gently suspension cell, is placed on ice, obtains competent cell.It takes The above-mentioned competent cells prepared of 200 μ l, are added 1 μ g integrated plasmids of step structure, and chemical transformation is transferred to.Using upgrading Matter is integrated in extraction in the e. coli tg1 that grain kit (Tiangeng, DP103) is obtained according to the method in the discussion of aforementioned competence Grain, hay bacillus bacterial strain to be knocked out is transferred to using Spizizen conversion methods.Spizizen conversion methods are as follows：It is living Change hay bacillus 3610 to be transformed (bacillus collection Bacillus Genetic Stock Center (BGSC), 1L26).Choose the 2xYT culture mediums that fresh monoclonal is inoculated in 3ml, 37 DEG C of overnight shaking cultures.By 1:100 ratios connect strain Kind is in the medium A of 5ml, 37 DEG C of shaken cultivation 3.5h.By 1:10 ratios are inoculated with the bacterium in medium A in medium B In, competent cell is made in 37 DEG C of shaken cultivation 1.5h.Competent cell is made this moment, is added in the competent cell of 500 μ l The integrated plasmid DNA that 1 μ g are extracted from e. coli tg1.37 DEG C of water-baths stand competence 60min.37 DEG C of shaken cultivation impressions State cell 2h, is coated onto on the tablet with chloramphenicol resistance gene, and 37 DEG C of constant incubators stand 8h.The bacterial strain of -80 DEG C of preservations It is seeded to overnight incubation in LB culture mediums, thalline were collected by centrifugation, is resuspended to OD600=1.0 or so with deionized water, by 1% ratio Example is added in 4ml MSGG fluid nutrient mediums, stationary culture two days.Take the biological coating solution cultivated in liquid MSGG culture mediums 2 days 10 μ l are dripped in standing 2-5min on copper mesh (middle mirror tech, BZ10024a), are blotted with filter paper.Take the blocking of 10 μ l Buffer stands 30min, blots on copper mesh.Blocking buffer are formulated：200ml PBS(life Technology, 00051) plus 40 μ l Tween 20 (raw work, A600560-0500) and 0.4g skimmed milk powers (BD in Difco, 232100).Take primary antibody solution (primary antibody uses after diluting 150 times of blocking buffer) drop of 10 μ l in copper mesh On, 2h is stood, is blotted.It takes the PBST of 10 μ l to wash once, blots.The formula of PBST is：PBS+0.1%Tween 20.Take 10 μ l Two corresponding anti-solution (secondary antibody dilute 50 times of PBST then use) drop in 1h on copper mesh, blot.It takes the PBST of 10 μ l to wash once, inhales It is dry.It is washed once, is blotted with the deionization of 10 μ l again.(2.5mg/ml) acetic acid uranium solution of 10 μ l is taken to drip in copper mesh, dyeing 30s is blotted.Biomembrane base can be observed because the bacterial strain TD knocked out completely does not contain any nanofibrous structures, and carry The TD bacterial strains of pHT01-tapA-sipW-tasA with pHT01-tapA-sipW-tasA-mefp5 plasmids have similar amyloid egg White fiber structure, to show that fusion protein TasA-mefp5 can correctly simultaneously shape be secreted in expression in bacillus subtilis strain At fiber.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biomembrane using rheometer (Anton Paar MCR101) Viscoplasticity.Cultured TD, TD/pHT01-tapA-sipW-tasA (are carried into pHT01-tapA-sipW-tasA plasmids TD bacterial strains) and the TD/pHT01-tapA-sipW-tasA-mefp5 (TD of carrying pHT01-tapA-sipW-tasA-mefp5 plasmids Bacterial strain) biomembrane scrapes off from MSSG solid mediums respectively, it is smeared on rheometer rotor cp25-2 uniform.Measure storage Variation (Fig. 4 a) of the modulus (Storage modulus) to strain (γ), wherein measurement parameter frequency of oscillation are fixed as 10rad/ S, strain amplitude 0.01%-100%, it can be seen that the expression of fusion protein TasA-mefp5 effectively raises TD bacterial strains Elasticity and toughness.Variation (Fig. 4 b) of the storage modulu to frequency (ω) is measured simultaneously, wherein measurement parameter strain amplitude is fixed It is 0.1%, frequency of oscillation amplitude is 0.1-100rad/s, it can be seen that the table of fusion protein TasA-mefp5 under high and low frequency Up to the equal elasticity modulus that can effectively raise TD bacterial strains, promote the cross-linked network structure of its inside.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biology using general-purpose mechanics device (Instron) The shear viscosity intensity of film.Cultured biomembrane is scraped from MSSG solid mediums respectively, is smoothened smooth in two panels Between stainless steel iron sheet.Stainless steel and iron chip size is long 5cm, wide 1cm, thick 0.2mm, and it is long 1cm to smoothen area, and wide 1cm is just Square region (Fig. 5 a).Test sample is statically placed in 30 DEG C of constant incubators (DHP-9032, the limited public affairs of the permanent scientific instrument in Shanghai one Department) be incubated 2 hours after carry out extension test.Be arranged general-purpose mechanics device tensile speed be 5mm/min, up-down stretch fixture it Between distance be 5cm.In addition, for the biomembrane of TD/pHT01-tapA-sipW-tasA-mefp5, pass through additional 1mmol/L 2.5 μ L of tyrosinase, the tyrosine being rich in fusion protein TasA-mefp5 is changed into the excellent catechol of viscosity (DOPA), and its shear viscosity (Fig. 5 b) after modification is tested.The result shows that containing fusion protein without tyrosinase processing The biomembrane of TasA-mefp5 has higher viscosity compared to TD, its viscosity gets a greater increase after tyrosinase is handled, and reaches To 63.47 ± 9.75kPa.This show the fusion of mussel byssus protein and amyloid protein TasA really to biomembrane viscosity have compared with Big contribution.
In conclusion the shallow lake that skeleton function will be played in mussel byssus protein and biomembrane with bond properties under excellent water The TasA fusions of powder sample protein fusion, can improve biomembrane viscoplasticity, toughness and shear viscosity so that biofilm structure is more Closely, inherent viscosity and interfacial viscosity are greatly enhanced.
Embodiment 2：Based on EPS and Fe3+The adhesive characteristics modularized design that electrostatic interaction mediates
Main component of the EPS polysaccharide as biomembrane, is typically considered a kind of polyanionic polymer, by introducing Spend metal ion (Fe3+) chelate therewith, then it can further increase its internal crosslinking feature.This example use TD bacterial strains for pair According to using the DD bacterial strains (Fig. 1 c) containing EPS ingredients, by comparing TD and DD biomembranes to Fe3+Differential responses, determine EPS With Fe3+Electrostatic interaction, and measure its influence to biomembrane viscosity.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biomembrane using rheometer (Anton Paar MCR101) Viscoplasticity.Measurement method and parameter are the same as examples detailed above 1.It can be seen that polysaccharide EPS ingredients generate, although not greatly improving life The elasticity of object film, but greatly enhance the toughness (Fig. 6 a) of biomembrane.In addition, the storage modulu of TD and DD is almost in low frequency Unanimously, DD is about twice of TD modulus and in high frequency, shows that EPS is effectively increased the physical crosslinking structure (figure of biomembrane 6b)。
The method that biomembrane is cultivated using above-mentioned solid MSGG, and use Fourier transform infrared spectroscopy (FTIR, PerkinElmer EPS and Fe) is identified3+Interaction.Cultured TD and DD biomembranes are scraped respectively to 50mL centrifuge tubes In, distilled water, which is added, makes biomembrane fully be resuspended, and solution is divided into two parts, and portion is to contain only biomembrane suspension, another The middle 1mol/L ferric chloride solutions that 0.1mL is added.Two parts of samples of TD and DD are placed in freeze drier (LABCONCO) Freeze-drying 24 hours.After freeze-drying, sample is measured, wherein measurement parameter spectral region is 900cm-1—1800cm-1(Fig. 7). As can be seen that Fe is being added in TD and DD3+In 1084cm-1And 979cm-1Place, which absorbs, significant difference, shows EPS and Fe3+Phase interaction Main functional group is phosphate radical PO3 2-；In addition, the infrared absorption spectrum of TD and DD is in 1598cm-1Also there is significant change at place, Illustrate that there are part COO in biological film component-With Fe3+Interaction.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and use scanning electron microscope (SEM, JSM 7800F) Observe EPS and Fe3+Interaction after the varying topography that brings.Cultured DD biomembranes bacterium colony on MSGG culture mediums is taken, is added It after the ferric chloride solution of 1mol/L, is applied on clean aluminium-foil paper, then fixes 1 hour with 5% glutaraldehyde solution (An Naiji), Then carry out serial dehydration with ethyl alcohol (concentration of alcohol is followed successively by 50%, 60%, 70%, 80%, 90%, 100%).It prepares After sample is placed in gold spraying instrument sputtering 30s, observed (Fig. 8, engineer's scale are 1 μm) using SEM.As can be seen that Fe is added3+It Afterwards, distance is clearly more close between biofilm matrix and bacterium, this also shows electrostatic interaction to biomembrane internal junction The dehydrating polycondensation of structure plays a crucial role.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biology using general-purpose mechanics device (Instron) The shear viscosity intensity (Fig. 9) of film.Measurement method and parameter setting are the same as embodiment 1.As can be seen that Fe is added3+Later, biomembrane The shear viscosity of material substantially increases about 4 times, up to 63.8 ± 8.01kPa.
In conclusion the biomembrane containing polysaccharide component, can have a considerable amount of phosphate groups PO3 2-, these poly- the moon The presence of ion serves not only as the toughness that skeleton improves biomembrane, more can be with the apparent electrostatic of generation of metal cation Interaction is eventually exhibited as significantly increasing for biomembrane viscosity to further increase the inherent cohesion of biomembrane.
Embodiment 3：Modularized design based on the sticky small peptide of surface hydrophobicity protein B slA fusions
Biofilm surface hydrophobin of the BslA albumen as bacillus subtilis, having naturally can engineering interfacial adhesion Advantage.The present embodiment imports the plasmid (Fig. 2 d) of pHT01-bslA-3sp-spytag, to obtain based on Db bacterial strains Fusion protein bslA-mefp3s-spytag biological membrane interface expression, while with pHT01-bslA plasmids (Fig. 2 c) be pair According to the transfection method of plasmid is referring to embodiment 1.Pass through the phase interaction of Spytag and mCherry-spycatcher (pacifying with patent) It is successful to be transformed with the functionalization proved to bslA genes, and uses rheometer test, the identification of general-purpose mechanics device extension test Merge influences of the BslA-3sp-spytag to biomembrane viscosity.
The method that biomembrane is cultivated using aforesaid liquid MSGG, culture Db/pHT01-bslA-3sp-spycatcher biologies Film.The biomembrane bacterium solution of 500 μ L is drawn in the EP pipes of 1.5mL, 10000g centrifuges 1min, is added 200 μ L's after abandoning supernatant MCherry-spycatcher solution on rotary shaker (QB-208, woods Bell) combine 40min, 10000g centrifuge 1min, Supernatant is abandoned, the sediment after appropriate centrifugation is chosen, glass slide center is applied to, 20 μ L deionized waters, covered is added dropwise.Control Group is then not added with mCherry-spycatcher, and directly plus 200 μ L deionized waters combine 40min on rotary shaker, remaining operation Ibid.Sample is observed under fluorescence microscope (Leica) (Figure 10, engineer's scale are 20 μm), it can be seen that carry fusion plasmid The bacillus subtilis biomembrane of pHT01-bslA-3sp-spytag can with mCherry-spycatcher in conjunction with and aobvious red It is red, to which surface BslA-3sp-spytag can correctly express secretion in bacillus subtilis.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biomembrane using rheometer (Anton Paar MCR101) Viscoplasticity (Figure 11).Measurement method and parameter are the same as examples detailed above 1.As can be seen that compared to Db biomembranes, amalgamation and expression The biomembrane elasticity of BslA-3sp-spytag greatly improves, and toughness is not also lost too much, but the expression compared to simple BslA, Its elasticity and toughness decrease.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biology using general-purpose mechanics device (Instron) The shear viscosity intensity (Figure 12) of film.Measurement method and parameter setting are the same as embodiment 1.Meanwhile to DB/pHT01-bslA-3sp- The 2.5 μ L of tyrosinase of the additional 1mmol/L of spytag biomembranes, the junket that will be rich in fusion protein BslA-3sp-spytag Propylhomoserin is changed into the excellent catechol (DOPA) of viscosity, and tests its shear viscosity after modification.The result shows that without junket The biomembrane of the BslA-3sp-spytag containing fusion protein of propylhomoserin enzymatic treatment has higher viscosity compared to Db, but than simple expression The Db biomembranes of BslA albumen want low.But its viscosity is greatly improved after tyrosinase is handled, and about reaches 250kPa.This shows that fusion protein BslA-3sp-spytag can be so that bacillus subtilis biomembrane after tyrosinase is modified It is improved with more preferable, more significant viscosity.
Shown in sum up, by the surface hydrophobicity protein B slA of engineered bacillus subtilis, biology can actually be enhanced The adhesive force of film, and biomembrane elasticity and toughness are improved to a certain extent.
Embodiment 4：The structure of integrated adhesive based on bacillus subtilis
On the basis of fully understanding effect and the characteristic of each ingredient of withered grass biomembrane, pass through the modularization in hay bacillus Design different adhesive characteristics, such as amyloid protein, mussel byssus protein and positive and negative charge interaction substance, Ke Yiti The high biomembrane that it is generated viscosity.Therefore, by the way that further all adhesive characteristics are integrated on genome, according to above-mentioned bacterium Strain construction method, obtains final T5B3 bacterial strains and passes through in addition, in order to play the advantage of mussel byssus protein to the maximum extent Tyrosinase heterogenous expression (Figure 13) in bacillus subtilis is built, T5B3/pHT01-tyrosinase bacterial strains is obtained and (carries There are the T5B3 bacterial strains of pHT01-tyrosinase plasmids).And it is final using rheometer test, the identification of general-purpose mechanics device extension test The adhesive force of integrated biology film adhesive.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biomembrane using rheometer (Anton Paar MCR101) Viscoplasticity (Figure 14).Measurement method and parameter are the same as above-described embodiment 1.It can be seen that integrated bacterial strain T5B3/pHT01- The obtained biomembrane modulus of modularized design, illustrates all adhesive characteristics before the storage modulu of Tyrosinase is all higher than It is integrated with conducive to more powerful adhesive is built.But the expression of tyrosinase can weaken biomembrane to a certain extent Toughness, to reduce adhesive reversibility.
The method that biomembrane is cultivated using above-mentioned solid MSGG, and measure biology using general-purpose mechanics device (Instron) The shear viscosity intensity (Figure 15) of film.Measurement method and parameter setting are the same as embodiment 1.It can be seen that integrated T5B3/pHT01- The viscosity of tyrosianse biomembranes is adding Fe3+It greatly improves later, is 255.76 ± 39.24kPa, than modularized design before The biomembrane viscosity with partial cohesive feature be intended to height.
In conclusion by the further integration designed single moduleization, the final stronger biology of viscosity can be obtained Film bacterial strain.In addition, compared to the modularized design of plasmid series, final genome conformity reduces the risk of plasmid loss, The stability of bacterial strain is improved, there is more real application advantage.
Embodiment 5：The performance of integrated adhesive based on bacillus subtilis and application
For building complete integration bacillus subtilis T5B3/pHT01-tyrosinase, this example takes several acts Example shows its performance and operational, and material is all made of the biomembrane that above-mentioned solid MSGG is cultivated in example.
As illustrated in fig 16 a, it is control with wild-type B. subtilis biomembrane (WT), measures integrated biomembrane bonding Agent is through detergent (dodecyl sodium sulfate (SDS), urea (Urea)) viscosity change that treated.As it can be seen that two kinds of detergents pair The viscosity of biofilm has certain influence.In general, control group viscosity fall is up to 55.1%, and integrated life Adhesive strength after object envelope adds Fe3+ to react does more declines only 20.9%.And under 90% relative humidity of highest, wild type The sticky fall of biomembrane is up to 81.7%, and integrated jointing material is only 40.6% (Figure 16 b).As it can be seen that integrated viscous Mixture has preferable environmental resistance, this may can only make albuminous degeneration due to detergent and cannot destroy EPS exocellular polysaccharides In PO3 2-Group, to which the electrostatic interaction for making EPS-Fe3+ be mediated can still play bonding effect, in addition, biological The DOPA cross-linked structures and Fe formed in film3+To knot of the dehydration flocculating result in relation to enabling to biofilm of biofilm Structure is more fine and close, to enable to resist destruction of the high humidity environment to viscosity.
By the streak inoculation again on LB solid mediums by the biomembrane of solid culture, and turn out on this basis New biomembrane grows the biofilm in 5 generations repeatedly, the shear viscosity of biomembrane between more different passages.As it can be seen that working as It is not added with Fe3+When, biomembrane 1-4 is (1 for afterst-2nd-3rd-4th) shear viscosity be held essentially constant, about 150kPa；And with Fe3+Although the viscosity after effect is fluctuated, substantially remain between 200-260kPa (Figure 17).And the 5th generation (5th) training Foster biofilm, adds Fe3+Shear viscosity after effect then drops to 189.24 ± 22.61kPa.It can speculate and be grown in passage In the process, the possibility changed greatly in biofilm composition is EPS, because on the one hand EPS is conducive to maintain the tough of biofilm Property, it on the other hand also can be with Fe3+It generates electrostatic interaction and leads to significantly improving for viscosity.In general, biomembrane bonds Material has preferable recyclability, and cost is reduced for the use of material.
As shown in figure 18 a, the integrated biomembrane of collection is poured into syringe, then it can be by biofilm according to a setting Shape squeezes out, such as alphabetical " B.subtilis " shape (Figure 18 b).As it can be seen that biofilm be it is a kind of it is very soft, plasticity is stronger Gel rubber material, this for material storage and using will offering convenience property, especially when need using adhesive filling or Bonding is when such as crack, hole are difficult to the place being in direct contact.
If Figure 19 a are filling cracks schematic diagram.By the way that is there is the structure in crack applied to various in the adhesive built In, biomembrane can bond crack as soft material by its stronger toughness and viscosity, and maintain preferable viscosity under water. As shown in fig. 19b, suitable biomembrane is scraped on MSGG solid plates, is coated in one end of glass slide, is fixed one thin One end of line, application area are about 1 × 1cm, and the length of fixed filament is about 1cm, and the FeCl of excess 1mM is then added dropwise3Solution, So that it is fully acted on biological film adhesive, extra FeCl is siphoned away with blotting paper3Solution.The other end for stretching filament is carried, glass will be carried Piece is placed in the 500mL beakers equipped with 500mL water, it is seen that glass slide still can keep tacky state with filament in water.Separately Outside, as shown in Figure 19 c, the same appropriate cultured T5B3/pHT01-tyrosinase bacillus subtilises biomembrane of scraping will It is equably applied on the plane of disruption of the bone of two pieces of fractures, and the plane of disruption for being coated with biological film adhesive is then pressed in one It rises, after 2min, carries bone one end, its crack is immersed in the 500mL beakers equipped with 500mL water, it is seen that the bone of two pieces of fractures Head, which is still closely joined together, not will disconnect.
In conclusion the integrated underwater adhesive developed of this patent have preferable environmental resistance, recyclability, Injectable row and relatively strong underwater viscosity to different base, to be conducive to low cost, high efficiency, convenient under various circumstances The use biomembrane live body jointing material.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology can all carry out modifications and changes to above-described embodiment without violating the spirit and scope of the present invention.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should by the present invention claim be covered.
Sequence table
<110>Shanghai Science and Technology Univ.
<120>A kind of live body underwater adhesive material and application thereof
<160> 50
<170> SIPOSequenceListing 1.0
<210> 1
<211> 57
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tagacagatc tatcgatgca tgccatggta cccgggtctt cttgattaaa tgggttg 57
<210> 2
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gcgttaaccg gtcttttaca aacaaaattc cccctaaaaa a 41
<210> 3
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tttttagggg gaattttgtt tgtaaaagac cggttaacgc c 41
<210> 4
<211> 57
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgacgtcga cgcgtctgca gaagcttcta gaattccctt tgtctacatc ccttcgt 57
<210> 5
<211> 59
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ttacacatta actagacaga tctatcgatg catgccatgg aaaccagaaa gcggactta 59
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
taacagcaaa aaaaagagac ggccc 25
<210> 7
<211> 47
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gggccgtctc ttttttttgc tgttaggtaa gctccccttt tattgaa 47
<210> 8
<211> 55
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
tctgcagaag cttctagaat tcgagctccc gggatcaaac ggatacgaaa ggcac 55
<210> 9
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
aaaggatccg caatcctcgg actggcggg 29
<210> 10
<211> 46
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
gagaatcaaa ataaaccttc cgcgtattca tagccttcag ccttcc 46
<210> 11
<211> 46
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
ggaaggctga aggctatgaa tacgcggaag gtttattttg attctc 46
<210> 12
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
aaagtcgact tccgctgcga tgtgcccat 29
<210> 13
<211> 2670
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tcagagttaa atggtattgc ttcactgctt catcttttct tttacggtcc catacttttt 60
gtttgaacag tacctgtgcg agcgggtacc ttttttttgc ttcttttaca gcaatctctt 120
cccatttgga catgtggcgg gcggttacaa gcggtgtttc ttctgcgtga gcggctgtgg 180
tgccaaagac gagaagagat agacaaatca cacattgttt gatcatcatg ctgtcaccct 240
ttctttgttt attattacca aataataatg ggatatgcat ttaaattctc acataacaat 300
cccaaaaatt tctaaaaaat tgaaaaaatg agcaatactg agcaagactt tgtaatatga 360
tgaaaacatt cttttaaacg aacaaaatga gcgatttcgg tgtttttaaa tctataaatc 420
gttgattata ctctatttgt gaagttcttt aaagagaacg attgtcatat caagttacag 480
tgttttacag gaggtaagat atgtttcgat tgtttcacaa tcagcaaaag gcgaagacga 540
aactgaaagt tctgcttatc tttcagcttt cagtcatttt cagtctgact gccgcaatat 600
gcttacaatt ttccgatgat acaagcgctg cttttcatga tattgaaaca tttgatgtct 660
cacttcaaac gtgtaaagac tttcagcata cagataaaaa ctgccattat gataaacgct 720
gggatcaaag tgatttgcac atatcagatc aaacggatac gaaaggcact gtatgctcac 780
ctttcgcctt atttgctgtg ctcgaaaata caggtgagaa acttaagaaa tcaaagtgga 840
agtgggagct tcataagctt gaaaatgccc gcaaaccgtt aaaggatggg aacgtgatcg 900
aaaaaggatt tgtctccaat caaatcggcg attcacttta taaaattgag accaagaaaa 960
aaatgaaacc cggcatttat gcatttaaag tatataaacc ggcaggctac ccggcaaacg 1020
gcagtacatt tgagtggtcg gagcctatga ggcttgcaaa atgcgatgaa aaaccgacag 1080
tccctaaaaa agaaacaaag tcggacgtca aaaaggagaa tgaaacaaca caaaaagata 1140
taccggaaaa aacaatgaaa gaagaaacat ctcaagaagc tgtaaccaaa gaaaaagaaa 1200
ctcaatcaga ccagaaggaa agcggggaag aggatgaaaa aagcaatgaa gctgatcagt 1260
aatattttat acgtgatcat ctttactctt attattgtgc tgacacttgt cgtgatttca 1320
acacgttcat ccgggggaga gccggcagtg tttgggtata cgctgaaatc agttctgtca 1380
ggttcgatgg agccggagtt caatacaggt tccttaatat tggtcaaaga aatcactgat 1440
gtgaaagagc tccaaaaagg tgacgttatt acatttatgc aggatgcaaa tacggcggtc 1500
acccacagaa ttgttgacat aacaaagcaa ggagaccatt tgttatttaa aacaaaaggt 1560
gataataatg cagcagctga ttcagcgcct gtatcggacg aaaatgttcg cgcgcaatac 1620
acaggttttc agcttccata tgccggctat atgcttcatt ttgccagcca gccgattgga 1680
acggctgtat tattgattgt tcccggcgtg atgctgttag tttacgcttt tgtgacgatc 1740
agcagcgcca ttagagaaat tgaaagaaag acaaaagcct tggaaacaga tacaaaggac 1800
agcaccatgt ctacttaact tcagttgtaa acctggcaac aggtttcgat ataaaatcat 1860
tcaataaaag gggagcttac catgggtatg aaaaagaaat tgagtttagg agttgcttct 1920
gcagcactag gattagcttt agttggagga ggaacatggg cagcatttaa cgacattaaa 1980
tcaaaggatg ctacttttgc atcaggtacg cttgatttat ctgctaaaga gaattcagcg 2040
agtgtgaact tatcaaatct aaagccggga gataagttga caaaggattt ccaatttgaa 2100
aataacggat cacttgcgat caaagaagtt ctaatggcgc ttaattatgg agattttaaa 2160
gcaaacggcg gcagcaatac atctccagaa gatttcctca gccagtttga agtgacattg 2220
ttgacagttg gaaaagaggg cggcaatggc tacccgaaaa acattatttt agatgatgcg 2280
aaccttaaag acttgtattt gatgtctgct aaaaatgatg cagcggctgc tgaaaaaatc 2340
aaaaaacaaa ttgaccctaa attcttaaat gcaagcggta aagtcaatgt agcaacaatt 2400
gatggtaaaa ccgctcctga atatgatggt gttccaaaaa caccaactga cttcgatcag 2460
gttcaaatgg aaatccaatt caaggatgat aaaacaaaag atgaaaaagg gcttatggtt 2520
caaaataaat atcaaggcaa ctccattaag cttcaattct cattcgaagc tacacagtgg 2580
aacggcttga caatcaaaaa ggaccatact gataaagatg gttacgtgaa agaaaatgaa 2640
aaagcgcata gcgaggataa aaattaataa 2670
<210> 14
<211> 2943
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
tcagagttaa atggtattgc ttcactgctt catcttttct tttacggtcc catacttttt 60
gtttgaacag tacctgtgcg agcgggtacc ttttttttgc ttcttttaca gcaatctctt 120
cccatttgga catgtggcgg gcggttacaa gcggtgtttc ttctgcgtga gcggctgtgg 180
tgccaaagac gagaagagat agacaaatca cacattgttt gatcatcatg ctgtcaccct 240
ttctttgttt attattacca aataataatg ggatatgcat ttaaattctc acataacaat 300
cccaaaaatt tctaaaaaat tgaaaaaatg agcaatactg agcaagactt tgtaatatga 360
tgaaaacatt cttttaaacg aacaaaatga gcgatttcgg tgtttttaaa tctataaatc 420
gttgattata ctctatttgt gaagttcttt aaagagaacg attgtcatat caagttacag 480
tgttttacag gaggtaagat atgtttcgat tgtttcacaa tcagcaaaag gcgaagacga 540
aactgaaagt tctgcttatc tttcagcttt cagtcatttt cagtctgact gccgcaatat 600
gcttacaatt ttccgatgat acaagcgctg cttttcatga tattgaaaca tttgatgtct 660
cacttcaaac gtgtaaagac tttcagcata cagataaaaa ctgccattat gataaacgct 720
gggatcaaag tgatttgcac atatcagatc aaacggatac gaaaggcact gtatgctcac 780
ctttcgcctt atttgctgtg ctcgaaaata caggtgagaa acttaagaaa tcaaagtgga 840
agtgggagct tcataagctt gaaaatgccc gcaaaccgtt aaaggatggg aacgtgatcg 900
aaaaaggatt tgtctccaat caaatcggcg attcacttta taaaattgag accaagaaaa 960
aaatgaaacc cggcatttat gcatttaaag tatataaacc ggcaggctac ccggcaaacg 1020
gcagtacatt tgagtggtcg gagcctatga ggcttgcaaa atgcgatgaa aaaccgacag 1080
tccctaaaaa agaaacaaag tcggacgtca aaaaggagaa tgaaacaaca caaaaagata 1140
taccggaaaa aacaatgaaa gaagaaacat ctcaagaagc tgtaaccaaa gaaaaagaaa 1200
ctcaatcaga ccagaaggaa agcggggaag aggatgaaaa aagcaatgaa gctgatcagt 1260
aatattttat acgtgatcat ctttactctt attattgtgc tgacacttgt cgtgatttca 1320
acacgttcat ccgggggaga gccggcagtg tttgggtata cgctgaaatc agttctgtca 1380
ggttcgatgg agccggagtt caatacaggt tccttaatat tggtcaaaga aatcactgat 1440
gtgaaagagc tccaaaaagg tgacgttatt acatttatgc aggatgcaaa tacggcggtc 1500
acccacagaa ttgttgacat aacaaagcaa ggagaccatt tgttatttaa aacaaaaggt 1560
gataataatg cagcagctga ttcagcgcct gtatcggacg aaaatgttcg cgcgcaatac 1620
acaggttttc agcttccata tgccggctat atgcttcatt ttgccagcca gccgattgga 1680
acggctgtat tattgattgt tcccggcgtg atgctgttag tttacgcttt tgtgacgatc 1740
agcagcgcca ttagagaaat tgaaagaaag acaaaagcct tggaaacaga tacaaaggac 1800
agcaccatgt ctacttaact tcagttgtaa acctggcaac aggtttcgat ataaaatcat 1860
tcaataaaag gggagcttac catgggtatg aaaaagaaat tgagtttagg agttgcttct 1920
gcagcactag gattagcttt agttggagga ggaacatggg cagcatttaa cgacattaaa 1980
tcaaaggatg ctacttttgc atcaggtacg cttgatttat ctgctaaaga gaattcagcg 2040
agtgtgaact tatcaaatct aaagccggga gataagttga caaaggattt ccaatttgaa 2100
aataacggat cacttgcgat caaagaagtt ctaatggcgc ttaattatgg agattttaaa 2160
gcaaacggcg gcagcaatac atctccagaa gatttcctca gccagtttga agtgacattg 2220
ttgacagttg gaaaagaggg cggcaatggc tacccgaaaa acattatttt agatgatgcg 2280
aaccttaaag acttgtattt gatgtctgct aaaaatgatg cagcggctgc tgaaaaaatc 2340
aaaaaacaaa ttgaccctaa attcttaaat gcaagcggta aagtcaatgt agcaacaatt 2400
gatggtaaaa ccgctcctga atatgatggt gttccaaaaa caccaactga cttcgatcag 2460
gttcaaatgg aaatccaatt caaggatgat aaaacaaaag atgaaaaagg gcttatggtt 2520
caaaataaat atcaaggcaa ctccattaag cttcaattct cattcgaagc tacacagtgg 2580
aacggcttga caatcaaaaa ggaccatact gataaagatg gttacgtgaa agaaaatgaa 2640
aaagcgcata gcgaggataa aaatggagga ggaggctcag gatccagcag cgaagagtat 2700
aagggcggct attatcctgg taatgcgtat cactaccatt caggcggcag ctatcatggc 2760
tcaggctatc atggcggcta taagggcaaa tactacggca aggcgaaaaa atattattat 2820
aaatataaaa atagcggcaa gtataaatat ctgaaaaaag cgcgcaaata ccatcgcaag 2880
ggctataagt attacggcgg cagcagcact agtcaccatc atcaccatca tcattggcca 2940
taa 2943
<210> 15
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
acaattccca attaaaggag g 21
<210> 16
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ctgccccggg gacgtcgact ctagattatt aatttttatc ctcgctatgc 50
<210> 17
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
gacgtcgact ctagaggatc cttatggcca atgatgatgg tga 43
<210> 18
<211> 546
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis)
<400> 18
atgaaacgca aattattatc ttctttggca attagtgcat taagtctcgg gttactcgtt 60
tctgcaccta cagcttcttt cgcggctgaa tctacatcaa ctaaagctca tactgaatcc 120
actatgagaa cacagtctac agcttcattg ttcgcaacaa tcactggcgc cagcaaaacg 180
gaatggtctt tctcagatat cgaattgact taccgtccaa acacgcttct cagccttggc 240
gttatggagt ttacattgcc aagcggattt actgcaaaca cgaaagacac attgaacgga 300
aatgccttgc gtacaacaca gatcctcaat aacgggaaaa cagtaagagt tcctttggca 360
cttgatttgt taggagctgg cgaattcaaa ttaaaactga ataacaaaac acttcctgcc 420
gctggtacat atactttccg tgcggagaat aaatcattaa gcatcggaaa taaattttac 480
gcagaagcca gcattgacgt ggctaagcgc agcactcctc cgactcagcc ttgcggttgc 540
aactaa 546
<210> 19
<211> 675
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
atgaaacgca aattattatc ttctttggca attagtgcat taagtctcgg gttactcgtt 60
tctgcaccta cagcttcttt cgcggctgaa tctacatcaa ctaaagctca tactgaatcc 120
actatgagaa cacagtctac agcttcattg ttcgcaacaa tcactggcgc cagcaaaacg 180
gaatggtctt tctcagatat cgaattgact taccgtccaa acacgcttct cagccttggc 240
gttatggagt ttacattgcc aagcggattt actgcaaaca cgaaagacac attgaacgga 300
aatgccttgc gtacaacaca gatcctcaat aacgggaaaa cagtaagagt tcctttggca 360
cttgatttgt taggagctgg cgaattcaaa ttaaaactga ataacaaaac acttcctgcc 420
gctggtacat atactttccg tgcggagaat aaatcattaa gcatcggaaa taaattttac 480
gcagaagcca gcattgacgt ggctaagcgc agcactcctc cgactcagcc ttgcggttgc 540
aacggaagcg gctacgatgg ctataactgg ccgtatggct acaacggcta ccgctatggc 600
tggaacaaag gctggaatgg ctatggaagc gtcccgacaa tcgtcatggt cgacgcctac 660
aaacgctaca aataa 675
<210> 20
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
tcccattaaa ggaggaagga tccatgaaac gcaaattatt a 41
<210> 21
<211> 69
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
ccgctcatta ggcgggctgc cccgggttaa tgatggtgat ggtgatggtt gcaaccgcaa 60
ggctgagtc 69
<210> 22
<211> 45
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
ttaggcgggc tgccccgggt tatttgtagc gtttgtaggc gtcga 45
<210> 23
<211> 62
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
ttacacatta actagacaga tctatcgatg catgccatgg aaaccagaaa gcggacttaa 60
gc 62
<210> 24
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
taacagcaaa aaaaagagac ggccc 25
<210> 25
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
atgggtatga aaaagaaatt g 21
<210> 26
<211> 56
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
tatgaatact gggccgtctc ttttttttgc tgttattatg gccaatgatg atggtg 56
<210> 27
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
agcaactcct aaactcaatt tc 22
<210> 28
<211> 54
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
tctgcagaag cttctagaat tcgagctccc gggtaaaaca aaaggtgata ataa 54
<210> 29
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
acagatctat cgatgcatgc catggtgcgt gaggagttgc aaatgagcaa 50
<210> 30
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
gataataatt tgcgtttcat aacaaaattc cccctaaaaa 40
<210> 31
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
atgaaacgca aattattatc 20
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
ttaatagcca ttccagcctt 20
<210> 33
<211> 53
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
tggaacaaag gctggaatgg ctattaatgt aaaagaccgg ttaacgccgg tct 53
<210> 34
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
agcttctaga attcgagctc ccgggatctc catgatgttt aatggccagg 50
<210> 35
<211> 1170
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccaa actgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggcg 720
ggtggcggtg gtggtagcgg tggtggcggt agctcgtact accatcacca tcaccatcac 780
gattacgaca tcccaacgac cgaaaacctg tattttcagg gcgccatggt aaccacctta 840
tcaggtttat caggtgagca aggtccgtcc ggtgatatga caactgaaga agatagtgct 900
acccatatta aattctcaaa acgtgatgag gacggccgtg agttagctgg tgcaactatg 960
gagttgcgtg attcatctgg taaaactatt agtacatgga tttcagatgg acatgtgaag 1020
gatttctacc tgtatccagg aaaatataca tttgtcgaaa ccgcagcacc agacggttat 1080
gaggtagcaa ctgctattac ctttacagtt aatgagcaag gtcaggttac tgtaaatggc 1140
gaagcaacta aaggtgacgc tcatacttaa 1170
<210> 36
<211> 840
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
atgacagtcc gtaaaaatca agcatccttg acggcggaag agaaacgtcg ttttgtagcg 60
gcattacttg aattaaaacg taccggccgt tatgacgcgt tcgtgactac ccacaatgca 120
ttcattctgg gggataccga taacggtgaa cgcacaggcc accgctcgcc cagctttctg 180
ccctggcatc gccgcttctt gttggagttt gaacgcgcac tgcaatcggt cgatgcgagc 240
gtcgccctgc cgtattggga ttggtccgcg gatcgctcaa cccgctcttc gctgtgggcg 300
ccagattttt tgggcggcac gggccgtagc cgcgacggcc aggttatgga tggccctttc 360
gcagcctcgg caggtaattg gccgattaat gtccgtgtgg acggccgcac gttcctccgc 420
cgtgcgttag gcgccggcgt gagcgaactg ccaacccgcg ctgaagtgga cagcgtcctg 480
gccatggcta cctacgatat ggcgccctgg aactccggca gcgatggctt tcgcaatcat 540
ttagagggtt ggcgcggtgt caatctgcat aaccgcgtgc acgtttgggt tggtggtcaa 600
atggccacgg gcgttagccc caatgatccg gttttctggt tacaccatgc ctatattgat 660
aaattatggg cggaatggca gcgccgtcat ccgagcagcc cgtatttgcc aggcggaggt 720
acgcccaacg tggtggactt aaatgaaact atgaagccgt ggaatgacac gacccccgca 780
gccctgttag atcatacccg tcactatacc tttgatgtcc atcaccatca ccaccattaa 840
<210> 37
<211> 45
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
ttcccaatta aaggaggaag gatccatgac agtccgtaaa aatca 45
<210> 38
<211> 59
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
tgaaaaaagc ccgctcatta ggcgggctgc cccgggttaa tggtggtgat ggtgatgga 59
<210> 39
<211> 261
<212> PRT
<213>Bacillus subtilis (Bacillus subtilis)
<400> 39
Met Gly Met Lys Lys Lys Leu Ser Leu Gly Val Ala Ser Ala Ala Leu
1 5 10 15
Gly Leu Ala Leu Val Gly Gly Gly Thr Trp Ala Ala Phe Asn Asp Ile
20 25 30
Lys Ser Lys Asp Ala Thr Phe Ala Ser Gly Thr Leu Asp Leu Ser Ala
35 40 45
Lys Glu Asn Ser Ala Ser Val Asn Leu Ser Asn Leu Lys Pro Gly Asp
50 55 60
Lys Leu Thr Lys Asp Phe Gln Phe Glu Asn Asn Gly Ser Leu Ala Ile
65 70 75 80
Lys Glu Val Leu Met Ala Leu Asn Tyr Gly Asp Phe Lys Ala Asn Gly
85 90 95
Gly Ser Asn Thr Ser Pro Glu Asp Phe Leu Ser Gln Phe Glu Val Thr
100 105 110
Leu Leu Thr Val Gly Lys Glu Gly Gly Asn Gly Tyr Pro Lys Asn Ile
115 120 125
Ile Leu Asp Asp Ala Asn Leu Lys Asp Leu Tyr Leu Met Ser Ala Lys
130 135 140
Asn Asp Ala Ala Ala Ala Glu Lys Ile Lys Lys Gln Ile Asp Pro Lys
145 150 155 160
Phe Leu Asn Ala Ser Gly Lys Val Asn Val Ala Thr Ile Asp Gly Lys
165 170 175
Thr Ala Pro Glu Tyr Asp Gly Val Pro Lys Thr Pro Thr Asp Phe Asp
180 185 190
Gln Val Gln Met Glu Ile Gln Phe Lys Asp Asp Lys Thr Lys Asp Glu
195 200 205
Lys Gly Leu Met Val Gln Asn Lys Tyr Gln Gly Asn Ser Ile Lys Leu
210 215 220
Gln Phe Ser Phe Glu Ala Thr Gln Trp Asn Gly Leu Thr Ile Lys Lys
225 230 235 240
Asp His Thr Asp Lys Asp Gly Tyr Val Lys Glu Asn Glu Lys Ala His
245 250 255
Ser Glu Asp Lys Asn
260
<210> 40
<211> 181
<212> PRT
<213>Bacillus subtilis (Bacillus subtilis)
<400> 40
Met Lys Arg Lys Leu Leu Ser Ser Leu Ala Ile Ser Ala Leu Ser Leu
1 5 10 15
Gly Leu Leu Val Ser Ala Pro Thr Ala Ser Phe Ala Ala Glu Ser Thr
20 25 30
Ser Thr Lys Ala His Thr Glu Ser Thr Met Arg Thr Gln Ser Thr Ala
35 40 45
Ser Leu Phe Ala Thr Ile Thr Gly Ala Ser Lys Thr Glu Trp Ser Phe
50 55 60
Ser Asp Ile Glu Leu Thr Tyr Arg Pro Asn Thr Leu Leu Ser Leu Gly
65 70 75 80
Val Met Glu Phe Thr Leu Pro Ser Gly Phe Thr Ala Asn Thr Lys Asp
85 90 95
Thr Leu Asn Gly Asn Ala Leu Arg Thr Thr Gln Ile Leu Asn Asn Gly
100 105 110
Lys Thr Val Arg Val Pro Leu Ala Leu Asp Leu Leu Gly Ala Gly Glu
115 120 125
Phe Lys Leu Lys Leu Asn Asn Lys Thr Leu Pro Ala Ala Gly Thr Tyr
130 135 140
Thr Phe Arg Ala Glu Asn Lys Ser Leu Ser Ile Gly Asn Lys Phe Tyr
145 150 155 160
Ala Glu Ala Ser Ile Asp Val Ala Lys Arg Ser Thr Pro Pro Thr Gln
165 170 175
Pro Cys Gly Cys Asn
180
<210> 41
<211> 10
<212> PRT
<213> Mytilus edulis
<400> 41
Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys
1 5 10
<210> 42
<211> 42
<212> PRT
<213> Mytilus galloprovincialis
<400> 42
Thr Asp Lys Ala Tyr Lys Pro Asn Pro Cys Val Val Ser Lys Pro Cys
1 5 10 15
Lys Asn Arg Gly Lys Cys Ile Trp Asn Gly Lys Ala Tyr Arg Cys Lys
20 25 30
Cys Ala Tyr Gly Tyr Gly Gly Arg His Cys
35 40
<210> 43
<211> 48
<212> PRT
<213> Mytilus edulis
<400> 43
Ala Asp Tyr Tyr Gly Pro Asn Tyr Gly Pro Pro Arg Arg Tyr Gly Gly
1 5 10 15
Gly Asn Tyr Asn Arg Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr
20 25 30
Lys Gly Trp Asn Asn Gly Trp Asn Arg Gly Arg Arg Gly Lys Tyr Trp
35 40 45
<210> 44
<211> 74
<212> PRT
<213> Mytilus edulis
<400> 44
Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ala Tyr His
1 5 10 15
Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr
20 25 30
Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys
35 40 45
Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg
50 55 60
Lys Gly Tyr Lys Tyr Tyr Gly Gly Ser Ser
65 70
<210> 45
<211> 99
<212> PRT
<213> Mytilus californianus
<400> 45
Gly Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser
1 5 10 15
Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser
20 25 30
Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Cys Leu Pro Arg Asn Pro
35 40 45
Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys Pro Asp
50 55 60
Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn Asp Lys
65 70 75 80
Asp Tyr Gly Cys Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Arg
85 90 95
Ser Gly Tyr
<210> 46
<211> 47
<212> PRT
<213> Perna viridis
<400> 46
Gln Leu Thr Cys Phe Pro Thr Ile Asp Cys Gly Phe Asn Ile Asp Gly
1 5 10 15
Cys Gln Ser Phe Cys Arg Asp Arg Asn Cys Ser Pro Tyr Gly Ser Glu
20 25 30
Cys Arg Asn Asn Asn Leu Cys Cys Cys Leu Tyr Cys Arg Phe Gly
35 40 45
<210> 47
<211> 82
<212> PRT
<213> Perna viridis
<400> 47
Val Tyr Tyr Pro Asn Pro Cys Ser Pro Tyr Pro Cys Arg Asn Gly Gly
1 5 10 15
Thr Cys Lys Lys Arg Gly Leu Tyr Ser Tyr Lys Cys Tyr Cys Arg Lys
20 25 30
Gly Tyr Thr Gly Lys Asn Cys Gln Tyr Asn Ala Cys Phe Pro Asn Pro
35 40 45
Cys Leu Asn Gly Gly Thr Cys Gly Tyr Val Tyr Gly Tyr Pro Tyr Tyr
50 55 60
Lys Cys Ser Cys Pro Tyr Gly Tyr Tyr Gly Lys Gln Cys Gln Leu Lys
65 70 75 80
Lys Tyr
<210> 48
<211> 25
<212> PRT
<213> Mytilus californianus
<400> 48
Gly Tyr Asp Gly Tyr Asn Trp Pro Tyr Gly Tyr Asn Gly Tyr Arg Tyr
1 5 10 15
Gly Trp Asn Lys Gly Trp Asn Gly Tyr
20 25
<210> 49
<211> 41
<212> PRT
<213> Mytilus californianus
<400> 49
Gly Tyr Asp Gly Tyr Asn Trp Pro Tyr Gly Tyr Asn Gly Tyr Arg Tyr
1 5 10 15
Gly Trp Asn Lys Gly Trp Asn Gly Tyr Gly Ser Val Pro Thr Ile Val
20 25 30
Met Val Asp Ala Tyr Lys Arg Tyr Lys
35 40
<210> 50
<211> 45
<212> PRT
<213> Mytilus californianus
<400> 50
Gly Tyr Gly Tyr Asp Leu Gly Tyr Asn Ala Pro Trp Pro Tyr Asn Asn
1 5 10 15
Gly Tyr Tyr Gly Tyr Asn Gly Tyr Asn Gly Tyr His Gly Arg Tyr Gly
20 25 30
Trp Asn Lys Gly Trp Asn Asn Gly Pro Trp Gly Gly Tyr
35 40 45

Claims (11)

1. a kind of composition, the composition includes TasA fusion proteins, exocellular polysaccharide, BslA fusion proteins, the TasA Fusion protein includes TasA protein fragments and mussel byssus protein segment, the BslA fusion proteins include BslA protein fragments and Self-coagulation binding proteins segment.
2. composition as described in claim 1, which is characterized in that further include one or more of following technical characteristic：
A1) the TasA protein fragments are：A) amino acid sequence polypeptide fragment as shown in SEQ ID No.39；Or b) amino acid Sequence has the function of 80% or more homology with SEQ ID NO.39 and has the polypeptide fragment of polypeptide fragment a) limited；
A2) the mussel byssus protein be selected from Mytilus galloprovincialis byssus protein, Perna viridis byssus protein, California mussel byssus protein, Mediterranean mussel byssus protein；
A3) the mussel byssus protein is selected from mefp-1, mgfp-2, mefp-3, mefp-5, mcfp-6, pvfp3, pvfp5；
A4) the mussel byssus protein segment is：C) amino acid sequence polypeptide as shown in one of SEQ ID No.41-47 One of segment or d) amino acid sequence and SEQ ID No.41-47 have 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more or 99% or more homology and have the function of c) limit polypeptide fragment it is more Peptide fragment；
A5) the TasA albumen sources are in bacillus subtilis；
A6) the mussel byssus protein derives from mussel；
A7) the TasA fusion proteins include TasA protein fragments and mussel byssus protein segment successively from N-terminal to C-terminal；
A8) in the composition, TasA fusion proteins, exocellular polysaccharide, BslA fusion proteins mass ratio be 1-5：80-90：1- 5。
3. composition as described in claim 1, which is characterized in that further include one or more of following technical characteristic：
B1) exocellular polysaccharide is polyanionic polymer；
B2) exocellular polysaccharide be metal cation chelating exocellular polysaccharide, the metal cation be selected from iron ion, magnesium from One or more combinations in son, calcium ion, manganese ion；
B3) exocellular polysaccharide derives from bacillus.
4. composition as described in claim 1, which is characterized in that further include one or more of following technical characteristic：
C1) BslA protein fragments are：E) amino acid sequence polypeptide fragment as shown in SEQ ID No.40；Or f) amino acid sequence Has the function of 80% or more homology with SEQ ID NO.40 and with the polypeptide fragment of polypeptide e) limited；
C2) the self-coagulation binding proteins are selected from California mussel byssus protein and derivative sticky small peptide；
C3) the self-coagulation binding proteins are selected from mcfp-3s, mcfp3spep, mcfp3spep-spytag；
C4) the self-coagulation binding proteins segment is：G) amino acid sequence is as shown in one of SEQ ID No.48-50 One of polypeptide fragment or h) amino acid sequence and SEQ ID No.48-50 have 80% or more, 85% or more, 90% with Upper, 93% or more, 95% or more, 97% or more or 99% or more homology and has the function of the g) polypeptide fragment of restriction Polypeptide fragment；
C5) the BslA albumen sources are in bacillus subtilis；
C6) the self-coagulation binding proteins derive from mussel；
C7) the BslA fusion proteins include BslA segments and self-coagulation binding proteins segment successively from N-terminal to C-terminal；
C8) the BslA fusion proteins are modified through tyrosinase.
5. a kind of biomembrane, the biomembrane includes the TasA in the composition as described in claim 1-4 any claims Fusion protein, exocellular polysaccharide, BslA fusion proteins.
6. biomembrane as described in claim 1, which is characterized in that further include one or more of following technical characteristic：
D1) in the biomembrane, the content of TasA fusion proteins is 1-5wt%；
D2) in the biomembrane, the content of exocellular polysaccharide is 80-90wt%；
D3) in the biomembrane, the content of BslA fusion proteins is 1-5wt%；
D4) biomembrane derives from bacillus.
7. a kind of expression system, the expression system can form the biomembrane as described in claim 5-6 any claims.
8. expression system as claimed in claim 7, which is characterized in that the expression system includes engineering bacteria, at least part of The engineering bacteria contains the construct that can express the TasA fusion proteins or is integrated with described in the coding of external source in genome The polynucleotides of TasA fusion proteins；
At least part of engineering bacteria contains the construct that can express the BslA fusion proteins or is integrated in genome The polynucleotides of the coding BslA fusion proteins of external source；
At least part of engineering bacteria can generate EPS polysaccharide；
At least part of engineering bacteria contains the construct that can express tyrosine or is integrated with the coding of external source in genome The polynucleotides of tyrosine；
The engineering bacteria is selected from bacillus.
9. the preparation method of the biomembrane as described in claim 5-6 any claims, including：Suitably forming the biology Under conditions of film, the expression system as described in claim 7-8 any claims is cultivated.
10. the expression system as described in claim 7-8 any claims or preparation method as claimed in claim 9 expression The biomembrane of acquisition.
11. composition as described in claim 1-4 any claims, as described in claim 5-6 any claims The purposes of biomembrane adhesive preparation field under water.
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WO2019228291A1 (en) * 2018-05-28 2019-12-05 Shanghaitech University Engineered biofilms
WO2022052745A1 (en) * 2020-09-09 2022-03-17 Shanghaitech University Programming living glue systems to perform autonomous mechanical repairs
CN115181169A (en) * 2022-05-13 2022-10-14 南京工业大学 Method for improving adhesion of recombinant mussel protein

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WO2019228291A1 (en) * 2018-05-28 2019-12-05 Shanghaitech University Engineered biofilms
WO2022052745A1 (en) * 2020-09-09 2022-03-17 Shanghaitech University Programming living glue systems to perform autonomous mechanical repairs
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CN115181169B (en) * 2022-05-13 2024-01-30 南京工业大学 Method for improving adhesion of recombinant mussel protein

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