Bioadhesive composition containing mussel adhesive protein and preparation method thereof

Abstract

The present invention relates to a bioadhesive composition and a preparation method thereof. The bioadhesive composition comprises: mussel adhesion protein; and at least one of polyacrylic acid and polymethacrylic acid. The mussel adhesion protein is connected to at least one of the polyacrylic acid and polymethacrylic acid through an amide bond. The bioadhesive composition has a three-dimensional network structure.

Description

Technical Field

[0001] The present invention relates to a bioadhesive composition containing mussel adhesive protein and a preparation method thereof, and more particularly, to a bioadhesive composition containing mussel adhesive protein that can be applied to biomedicine and a preparation method thereof. Background Art

[0002] Bioadhesives refer to substances that have adhesive properties in various biological samples such as cell membranes, cell walls, lipids, proteins, deoxyribonucleic acid (DNA), growth factors, cells, tissues, etc. They can be used in various biomedical fields such as tissue adhesives, hemostatic agents, tissue engineering scaffolds, drug delivery carriers, tissue augmentation agents, wound treatment agents or anti-adhesion agents.

[0003] Such bioadhesives need strong bonding and cross-linking capabilities and should maintain their functions in the organism for a long time. Currently commercialized or practical bioadhesives include cyanoacrylate instant adhesives, fibrin glue, gelatin glue, and polyurethane adhesives.

[0004] However, in the case of bioadhesives using synthetic polymers, they show very weak adhesive force in the presence of aqueous solution in vivo, and in the case of cyanoacrylate-based bioadhesives, it is pointed out that they have great limitations such as causing side effects such as immune response in the human body.

[0005] Furthermore, in the case of fibrin glue series bioadhesives, although side effects such as immune response in the human body are rare, their use is limited due to their high price and low adhesion. In the case of gelatin bioadhesives, formalin or glutaraldehyde used as cross-linking agents also undergo cross-linking reactions with proteins in the body, thus causing tissue toxicity. In the case of polyurethane bioadhesives, aromatic diisocyanates used as synthetic raw materials have the problem of causing biological toxicity.

[0006] In contrast, mussels, which are marine organisms, can firmly attach themselves to wet solid surfaces such as rocks in the sea by producing and secreting adhesive proteins, without being affected by wave impact or the buoyancy effect of seawater. Mussel adhesive proteins are known to be strong natural adhesives, and compared with chemical synthetic adhesives, they have tensile strength about twice that of most epoxy resins and flexibility that allows bending.

[0007] Furthermore, mussel adhesive proteins have the ability to adhere to a variety of surfaces including plastic, glass, metal, polytetrafluoroethylene and biological substances, and can also adhere to wet surfaces for several minutes, which is a problem that has not yet been solved in the development of chemical adhesives.

[0008] Furthermore, it is known that mussel adhesive proteins do not attack human cells or cause immune responses, so they are likely to be used in medical fields such as bonding living tissues during surgery or bonding broken teeth. The mussel adhesive proteins can also be used to bond cells to the surface. Cell surface bonding technology is one of the most important technologies required in the field of cell culture and tissue engineering, and is very important in promoting cell proliferation and differentiation.

[0009] Therefore, there is a need for a variety of biomedical applications utilizing mussel adhesive proteins. Summary of the invention

[0010] Technical issues

[0011] The problem to be solved by the present invention is to provide a bioadhesive composition comprising mussel adhesive protein, which can be used as a bioadhesive tape or film and can adjust the degradation time.

[0012] Another problem to be solved by the present invention is to provide a method for preparing the above bioadhesive composition.

[0013] Technical Solution

[0014] In order to solve the above problems, the present invention provides a bioadhesive composition, comprising: mussel adhesive protein; and at least one of polyacrylic acid and polymethacrylic acid. The mussel adhesive protein and at least one of polyacrylic acid and polymethacrylic acid are connected through an amide bond, and the bioadhesive composition has a three-dimensional network structure.

[0015] The above-mentioned mussel adhesion protein can be composed of one or more amino acid sequences selected from the group consisting of sequence 1, sequence 2, sequence 3, sequence 4, sequence 5, sequence 6, sequence 7, sequence 8, sequence 9, sequence 10, sequence 11, sequence 12, sequence 13, sequence 14 and sequence 15.

[0016] The mussel adhesion protein may be a mussel adhesion protein in which tyrosine residues are converted into catechol compounds, a mussel adhesion protein in which catechol derivatives are introduced on the surface of the mussel adhesion protein, or a mussel adhesion protein comprising both of the above.

[0017] The catechol compound may be one or more selected from the group consisting of DOPA (3,4-dihydroxyphenylalanine, DOPA), Dopa o-quinone (Dopa o-quinone), 6-hydroxyDOPA (2,4,5-trihydroxyphenylalanine, TOPA), 6-hydroxydopaquinone (Topa quinone) and derivatives thereof.

[0018] The bioadhesive composition may be a first component in which the mussel adhesive protein and polyacrylic acid are bonded via an amide bond, or a second component in which the mussel adhesive protein and polymethacrylic acid are bonded via an amide bond, or a mixture of the first component and the second component. The mass ratio of the mussel adhesive protein to the polyacrylic acid or polymethacrylic acid contained in the first component or the second component may be 1:1 to 1:5.

[0019] The mass ratio of the mussel adhesive protein to the polyacrylic acid or polymethacrylic acid in the first component or the second component may be 1:2.5 to 1:3.5.

[0020] The first component and the second component may be mixed at a mass ratio of 3:1 to 1:3.

[0021] The bioadhesive composition can exhibit non-adhesiveness and non-degradability in a dry state, and exhibit adhesiveness and biodegradability in a wet state.

[0022] The bioadhesive composition may be a film, a single-sided or double-sided tape or a hydrogel.

[0023] Furthermore, the present invention provides a method for preparing a bioadhesive composition, comprising: step (a), preparing a mixed solution formed by mixing a solution containing mussel adhesive protein with a solution containing at least one of acrylic acid N-hydroxysuccinic acid imide ester and methacrylic anhydride; step (b), dissolving a photoinitiator in the mixed solution; step (c), adding at least one of an acrylic acid monomer and a methacrylic acid monomer to the mixed solution; and step (d), solidifying the mixed solution;

[0024] Specifically, the present invention provides a method for preparing a bioadhesive composition, comprising: step (a-1), preparing a first mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing N-hydroxysuccinic acid imide ester of acrylic acid, and a second mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing methacrylic anhydride; step (b-1), dissolving a photoinitiator in the first mixed solution and the second mixed solution, respectively; step (c-1), adding an acrylic acid monomer to the first mixed solution and adding a methacrylic acid monomer to the second mixed solution; and step (d-1), mixing the first mixed solution and the second mixed solution and curing them.

[0025] As another specific example, the present invention provides a method for preparing a bioadhesive composition, comprising: step (a-2), preparing a mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing acrylic acid N-hydroxysuccinic acid imide ester; step (b-2), dissolving a photoinitiator in the above-mentioned mixed solution; step (c-2), adding an acrylic acid monomer; and step (d-2), curing.

[0026] As another specific example, the present invention provides a method for preparing a bioadhesive composition, comprising: step (a-3), preparing a mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing methacrylic anhydride; (b-3), dissolving a photoinitiator in the mixed solution; step (c-3), adding methacrylic acid monomer; and step (d-3), curing.

[0027] The photoinitiator may be α-ketoglutaric acid.

[0028] Based on the total weight of the mixed solution, the mussel adhesive protein may be contained in an amount of 7 weight percent to 15 weight percent.

[0029] Based on the total weight of the mixed solution, the mixed solution may contain 0.6 weight percent to 2.0 weight percent of at least one of the above-mentioned N-hydroxysuccinimidyl acrylate and methacrylic anhydride.

[0030] Based on the total weight of the mixed solution, the photoinitiator may be included in an amount of 0.1 weight percent to 0.4 weight percent.

[0031] The step (d-1) may be a step of mixing the first mixed solution and the second mixed solution at a mass ratio of 3:1 to 1:3.

[0032] In the above step (d-2), the mass ratio of the mussel adhesive protein to the acrylic acid monomer may be 1:1 to 1:5.

[0033] Effects of the Invention

[0034] The bioadhesive composition of the present invention forms a three-dimensional network structure in which mussel adhesive protein and poly(meth)acrylic acid are connected by amide bonds, exhibits non-adhesiveness and non-degradability in a dry state, and has excellent bioadhesive ability in a wet state, and can be used as a bioadhesive tape or film that can be biodegraded under the action of biological enzymes.

[0035] Furthermore, the bioadhesive composition of the present invention can adjust the degradation time by adjusting the mixing ratio of two mussel adhesive proteins after cross-linking them with poly(meth)acrylic acid respectively, without leaching toxic (meth)acrylic acid monomers, so the biological stability is very outstanding. Since it has no cytotoxicity or very low cytotoxicity, it can be used in a variety of biomedical applications requiring biological adhesion.

[0036] Furthermore, the bioadhesive composition of the present invention can be used as a double-sided tape or film, can be used as a tissue adhesive based on biomaterials, and can be used to adhere time-limited circuits for in vivo signal transduction and drug administration, etc. After adhesion, organ normalization and periodic drug administration can be achieved without the need for additional surgery.

[0037] The effects of the present invention are not limited to the above-mentioned effects, and it should be understood that they also include all the effects that can be inferred from the inventive structure described in the detailed description of the present invention or the scope of protection of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The diagram shows a first component in which a mussel adhesive protein and polyacrylic acid are bonded via an amide bond according to Example 1 of the present invention.

[0039] Figure 2 The figure shows a second component in which the mussel adhesive protein and polymethacrylic acid are bonded via an amide bond according to Example 2 of the present invention.

[0040] Figure 3A The MALDI mass spectrum of the existing mussel adhesion protein (FP-151) is shown. Figure 3B The MALDI mass spectrum of the methacrylated mussel adhesion protein prepared in Example 2 is shown.

[0041] Figure 4A The Fourier transform infrared spectroscopy (FT-IR) data of the existing mussel adhesion protein (FP-151) are shown. Figure 4B The Fourier transform infrared spectroscopy data of the methacrylated mussel adhesion protein prepared in Example 2 are shown. Figure 4C This is the result of confirming whether the composition of Example 7 was synthesized by Fourier transform infrared spectroscopy.

[0042] Figure 5To confirm the results of the degradation data of the bioadhesive compositions prepared according to Examples 1 to 3 of the present invention.

[0043] Figure 6 To confirm the results of the adhesive force of the bioadhesive compositions prepared according to Examples 1 to 3 of the present invention.

[0044] 7A to 7D To evaluate the cytotoxicity of the bioadhesive composition of the present invention, Fig. 7A The CCK-8 test results of Example 3 at a wavelength of 450 nm using a microplate spectrophotometer are shown in a bar graph. Figure 7B Live-dead assay was performed to confirm the results of cell survival in Example 3 (living cells are green, dead cells are red). Figure 7C The CCK-8 test results of Example 7 at a wavelength of 450 nm using a microplate spectrophotometer are shown in a bar graph. Fig.7D A live / dead test was performed to confirm the results of cell survival in Example 7 (live cells are green, dead cells are red).

[0045] Fig. 8A To confirm the images of the dry state of the bioadhesive compositions prepared according to Examples 1 to 5 of the present invention, Figure 8B To confirm the images of the dry state of the compositions prepared according to Examples 6 to 9, Figure 8C The images are for comparison and show the dried states of the compositions prepared according to Comparative Example 1, Comparative Example 2, and Example 7.

[0046] Fig. 9 The results are those of evaluating the biodegradability of the bioadhesive composition of the present invention in the absence and presence of biological enzymes.

[0047] Fig.10 The results are as follows: the adhesive ability in water was confirmed in pig skin using the bioadhesive composition of Example 7 of the present invention, the composition of Comparative Example 2, and a commercially available adhesive.

[0048] Fig.11A and Fig. 11B This is the tensile test result of the bioadhesive composition of Example 2 of the present invention. Fig.11A The tensile test results of the dry tape are shown in Figure 2. Fig. 11B The figure shows the tensile test result of the tape in wet state.

[0049] Fig. 12A and Fig. 12BTo confirm the results of the tensile test of the bioadhesive composition of Example 7 of the present invention by repeated contraction / expansion tests in porcine bladder, Fig. 12A The results for commercially available anti-adhesion films are Fig. 12B These are the results for the bioadhesive composition of Example 7. DETAILED DESCRIPTION

[0050] It should be noted that in the following description, only parts necessary for understanding the embodiments of the present invention will be described, and other parts will be omitted within the scope not to obscure the gist of the present invention.

[0051] The terms or words used in the present specification and the scope of protection of the invention described below should not be interpreted in a limited manner to the usual meaning or dictionary meaning, but should be interpreted as the meaning and concept that conforms to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term in order to explain his invention in the most preferred way. Therefore, the embodiments described in this specification and the structures shown in the drawings are only preferred embodiments of the present invention and do not represent all the technical ideas of the present invention. Therefore, it should be understood that there may be a variety of equivalents and modifications that can replace them when filing this application.

[0052] Hereinafter, the present invention will be described in detail.

[0053] The present invention provides a bioadhesive composition, comprising: mussel adhesion protein; and at least one of polyacrylic acid and polymethacrylic acid. The mussel adhesion protein and at least one of polyacrylic acid and polymethacrylic acid are connected through an amide bond, and the bioadhesive composition has a three-dimensional network structure.

[0054] The mussel adhesion protein is a protein derived from the byssus of mussels, preferably, a mussel adhesion protein derived from Mytilus edulis, Mytilus galloprovincialis or Mytilus coruscus or a variant thereof, but is not limited thereto.

[0055] The mussel adhesive protein of the present invention may include Mefp (Mytilus edulis foot protein)-1, Mgfp (Mytilus galloprovincialis foot protein)-1, Mcfp (Mytilus coruscus foot protein)-1, Mefp-2, Mefp-3, Mgfp-3 and Mgfp-5 or variants thereof, respectively derived from the above-mentioned mussel species, preferably, a protein selected from the group consisting of fp (foot protein)-1 (sequence 1), fp-2 (sequence 4), fp-3 (sequence 5), fp-4 (sequence 6), fp-5 (sequence 7) and fp-6 (sequence 8), or a fusion protein connecting two or more proteins, or a variant of the above-mentioned protein, but is not limited thereto.

[0056] Furthermore, the mussel adhesion protein of the present invention includes all mussel adhesion proteins described in International Publication No. WO2006 / 107183 or WO2005 / 092920. Preferably, the mussel adhesion protein may include a fusion protein selected from the group consisting of fp-151 (sequence 9), fp-131 (sequence 10), fp-353 (sequence 11), fp-153 (sequence 12) and fp-351 (sequence 13), but is not limited thereto.

[0057] Furthermore, the mussel adhesive protein of the present invention may include a polypeptide that is continuously linked to the decapeptide (sequence 2) repeated about 80 times in fp-1 for 1 to 12 times or more. Preferably, it may be a fp-1 variant polypeptide (sequence 3) that is continuously linked to the decapeptide of sequence 2 for 12 times, but is not limited thereto.

[0058] Furthermore, the mussel adhesion protein of the present invention may be a variant of fp-151 (sequence 15), but is not limited thereto. Compared with sequence 9, the protein sequence of sequence 15 is a sequence in which a linker sequence and the like are removed. Specifically, it is a fusion protein sequence in which the Mgfp-5 sequence represented by sequence 16 is fused between the fp-1 variant sequence represented by sequence 14. More specifically, the mussel adhesion protein of the present invention may be composed of one or more amino acid sequences selected from the group consisting of sequence 1, sequence 2, sequence 3, sequence 4, sequence 5, sequence 6, sequence 7, sequence 8, sequence 9, sequence 10, sequence 11, sequence 12, sequence 13, sequence 14, sequence 15, and sequence 16.

[0059] The mussel adhesive protein of the present invention can also be modified within the scope of the conservative amino acid sequence that can maintain the characteristics of the mussel adhesive protein mentioned above. That is, the scope of the present invention can include an amino acid sequence having a sequence identity of 70% or more with the amino acid sequence of the above sequence that exhibits substantially the same effect, preferably, an amino acid sequence having a sequence identity of 80% or more, and more preferably, an amino acid sequence having a sequence identity of 90% or more, that is, 95%, 96%, 97%, 98%, 99% or more.

[0060] The mussel adhesion protein may be a mussel adhesion protein in which tyrosine residues are converted into catechol compounds, a mussel adhesion protein in which catechol derivatives are introduced on the surface of the mussel adhesion protein, or a mussel adhesion protein comprising both of the above.

[0061] Preferably, the mussel adhesive protein of the present invention is a mussel adhesive protein in which tyrosine residues are converted into catechol compounds, preferably, 10% to 100% of the total tyrosine residues are converted into catechol compounds. In the total amino acid sequence of most mussel adhesive proteins, the proportion of tyrosine can be about 1% to 50%. The tyrosine in the mussel adhesive protein can be converted into DOPA (DOPA) as a catechol compound by adding OH groups through a hydration process.

[0062] However, the tyrosine residues of the mussel adhesive protein produced in E. coli are not converted, and therefore, preferably, a modification reaction is performed to convert tyrosine into DOPA by an additional enzyme and chemical treatment method. The modification method for converting the tyrosine residues contained in the mussel adhesive protein into DOPA can use a method known in the technical field to which the present invention belongs, and is not particularly limited.

[0063] The catechol compound is a compound containing a dihydroxyl group, and refers to a compound that imparts adhesion to the mussel adhesive protein through cross-linking, and specifically, may be one or more selected from the group consisting of dopa, dopa-o-quinone, 6-hydroxydopa, 6-hydroxydopaquinone, and derivatives thereof.

[0064] In the present invention, preferably, the variants of the mussel adhesive protein may be an adhesive protein containing an additional sequence at the carboxyl terminus or amino terminus of the mussel adhesive protein, or a part of the amino acids are converted into other amino acids, while maintaining the adhesive force of the mussel adhesive protein. More preferably, the mussel adhesive protein may be a mussel adhesive protein in which a polypeptide consisting of 3 to 25 amino acids including arginine-glycine-aspartic acid (RGD) is connected to the carboxyl terminus or amino terminus of the mussel adhesive protein, or a mussel adhesive protein in which 1% to 100%, preferably 5% to 100%, of the total number of tyrosine residues forming the mussel adhesive protein is converted into 3,4-dihydroxyphenyl-L-alanine (DOPA).

[0065] Preferably, the mussel adhesion protein can be inserted into a common vector prepared for the purpose of expressing external genes in an expressible manner and mass-produced by genetic engineering methods. The above-mentioned vector can be appropriately selected or newly prepared according to the type and characteristics of the host cell used to produce the protein. The method of transforming the above-mentioned vector in the host cell and the method of producing recombinant protein from the transformant can be easily implemented by the usual method. The methods of selecting, preparing, transforming and expressing the recombinant protein of the above-mentioned vector can be easily implemented by a person of ordinary skill in the art to which the present invention belongs, and some variations of the usual methods are also included in the present invention.

[0066] The bioadhesive composition of the present invention comprises: mussel adhesion protein; and at least one of polyacrylic acid and polymethacrylic acid, specifically, it can be a first component in which the mussel adhesion protein and polyacrylic acid are bonded via an amide bond, or a second component in which the mussel adhesion protein and polymethacrylic acid are bonded via an amide bond, or a mixture of the first component and the second component.

[0067] The mass ratio of the mussel adhesion protein to the polyacrylic acid or polymethacrylic acid contained in the first component or the second component may be 1:1 to 1:5, preferably 1:2.5 to 1:3.5, more preferably 1:3. The physical properties of the prepared composition can be adjusted by adjusting the content ratio (mass ratio) of the mussel adhesion protein to the polyacrylic acid or polymethacrylic acid.

[0068] If the content of polyacrylic acid or polymethacrylic acid in the bioadhesive composition of the present invention is less than the above range compared to the mussel adhesive protein, residual protein crystals will remain in the composition, and when preparing films and tapes, the flexibility of the film will be reduced, for example, it will break or break, and the expensive protein will be discarded due to unreacted, thereby reducing economic efficiency. On the other hand, if the content of polyacrylic acid or polymethacrylic acid is greater than the above range, the water adhesion of the dopamine residue of the mussel adhesive protein will be hindered due to the excessively high proportion of polyacrylic acid or polymethacrylic acid in the composition, thereby reducing the adhesive force.

[0069] On the contrary, when polyacrylic acid or polymethacrylic acid is contained in an amount within the above range, no unreacted protein is present in the composition, so that the composition can be prepared into a film or tape having excellent flexibility and tensile strength, and the aqueous adhesion of the prepared composition through the dopamine residues of the mussel adhesive protein can be maximized, so that a composition having excellent biodegradability and no cytotoxicity or very low cytotoxicity can be prepared.

[0070] The bioadhesive composition of the present invention may include the first component or the second component alone. For example, the mass ratio of the first component to the second component may be 10:0 or 0:10.

[0071] Furthermore, the bioadhesive composition of the present invention may be a mixture of the first component and the second component. When the first component and the second component are mixed, the mass ratio of the first component to the second component may be 9:1 to 1:9, preferably 3:1 to 1:3, and more preferably 1:1. When the first component and the second component are mixed, the bioadhesive composition of the present invention can adjust the degradation time when bonding in vivo by adjusting the mixing ratio (mass ratio) of the first component and the second component to prepare a film and a tape. Furthermore, if the mussel adhesive protein is maximized within the above mixing ratio and is not crystallized, the protein can be saved.

[0072] The bioadhesive composition of the present invention is a bioadhesive composition of a three-dimensional network structure in which the mussel adhesive protein of the present invention is bonded to at least one of polyacrylic acid and polymethacrylic acid through an amide bond. It does not show adhesive force in a dry state and is non-degradable, but can show adhesiveness and biodegradability in a moist (wet) state. In a moist state, the adhesive force is improved by the combined action of the van der Waals force between the mussel adhesive protein and the polyacryloyl or polymethacryloyl group in the composition, the hydrophilic attraction, and the catechol residues of the mussel adhesive protein, such as the adhesive force in water of dopamine residues.

[0073] Furthermore, the bioadhesive composition of the present invention is a bioadhesive composition of a three-dimensional network structure in which the mussel adhesive protein of the present invention is bonded to at least one of polyacrylic acid and polymethacrylic acid through an amide bond, and can be biodegraded in vivo by biological enzymes. The above-mentioned bioadhesive composition of the present invention does not separate or dissolve acrylic acid monomers from the composition even when biodegraded, and therefore has no cytotoxicity and outstanding stability.

[0074] The bioadhesive composition of the present invention can be in the form of a film, a single-sided or double-sided tape or a hydrogel, but is not limited thereto, and can be effectively applied to biomedical applications requiring biological adhesion. For example, it can be used as a tissue adhesive based on biomaterials, and can be used as a double-sided tape for fixing a device installed in a living body for in vivo signal transduction and drug administration.

[0075] Furthermore, the present invention provides a method for preparing a bioadhesive composition, comprising: step (a), preparing a mixed solution formed by mixing a solution containing mussel adhesive protein with a solution containing at least one of acrylic acid N-hydroxysuccinic acid imide ester and methacrylic anhydride; step (b), dissolving a photoinitiator in the mixed solution; step (c), adding at least one of an acrylic acid monomer and a methacrylic acid monomer to the mixed solution; and step (d), curing the mixed solution.

[0076] Specifically, the preparation method of the bioadhesive composition of the present invention may include: step (a-1), preparing a first mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing acrylic acid N-hydroxysuccinic acid imidate, and a second mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing methacrylic anhydride; step (b-1), dissolving a photoinitiator in the first mixed solution and the second mixed solution, respectively; step (c-1), adding an acrylic acid monomer to the first mixed solution and adding a methacrylic acid monomer to the second mixed solution; and step (d-1), mixing the first mixed solution and the second mixed solution and curing them.

[0077] As another specific example, the present invention provides a method for preparing a bioadhesive composition, comprising: step (a-2), preparing a mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing acrylic acid N-hydroxysuccinic acid imide ester; step (b-2), dissolving a photoinitiator in the above-mentioned mixed solution; step (c-2), adding an acrylic acid monomer; and step (d-2), curing.

[0078] As another specific example, the present invention provides a method for preparing a bioadhesive composition, comprising: step (a-3), preparing a mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing methacrylic anhydride; (b-3), dissolving a photoinitiator in the mixed solution; step (c-3), adding methacrylic acid monomer; and step (d-3), curing.

[0079] In the present invention, the mussel adhesion protein is defined as above.

[0080] In the present invention, the N-hydroxysuccinimidyl acrylate may be a compound represented by the following Chemical Formula 1, and the methacrylic anhydride may be a compound represented by the following Chemical Formula 2.

[0081] Chemical formula 1

[0082]

[0083] Chemical formula 2

[0084]

[0085] In the present invention, the photoinitiator may be α-ketoglutaric acid, but is not limited thereto, and any photoinitiator may be used as long as it can form polyacrylic acid or polymethacrylic acid.

[0086] In the present invention, α-ketoglutaric acid may be a compound represented by the following Chemical Formula 3.

[0087] Chemical formula 3

[0088]

[0089] According to the present invention, the acrylic monomer may be a compound represented by the following Chemical Formula 4, and the methacrylic monomer may be a compound represented by the following Chemical Formula 5.

[0090] Chemical formula 4

[0091]

[0092] Chemical formula 5

[0093]

[0094] According to the present invention, the curing in the above step (d), step (d-1), step (d-2), and step (d-3) may be photocuring, preferably, irradiation with light of 200 nm to 300 nm, for example, irradiation with light of 254 nm. Through the above light irradiation and the photoinitiator, polyacrylic acid is formed from acrylic acid monomers, or polymethacrylic acid is formed from methacrylic acid monomers, and an amide bond is formed between the mussel adhesive protein and the polyacrylic acid or polymethacrylic acid.

[0095] like Figure 1 and Figure 2 As shown, the mussel adhesive protein of the present invention forms a three-dimensional network structure with at least one of polyacrylic acid and polymethacrylic acid through amide bonds.

[0096] Specifically, the above Figure 1 The first component of the mussel adhesive protein and polyacrylic acid bonded by amide bonds is shown in the figure. Figure 2 The figure shows the mussel adhesive protein and the second component of polymethacrylic acid bound by an amide bond.

[0097] According to the present invention, based on the total weight of the mixed solution, the mussel adhesion protein may be included in an amount of 7 to 15 weight percent, and the mass ratio of the mussel adhesion protein to the acrylic acid monomer or the methacrylic acid monomer may be 1:1 to 1:5, preferably, 1:2.5 to 1:3.5, and more preferably, 1:3.

[0098] When the mussel adhesive protein is contained in the above range based on the total weight of the mixed solution, the mixed solution has an excellent bioadhesive effect. Moreover, when the ratio of the mussel adhesive protein to the acrylic acid monomer or the methacrylic acid monomer is within the above range, the mixed solution exhibits excellent mechanical properties in both dry and wet states, and for example, a bioadhesive film or double-sided tape having outstanding tensile strength and excellent bioadhesive ability can be prepared.

[0099] In the present invention, based on the total weight of the mixed solution, the photoinitiator may be included in an amount of 0.1 to 0.4 weight percent, and more preferably, the photoinitiator may be included in an amount of 0.2 to 0.3 weight percent.

[0100] When the photoinitiator is contained in the above-mentioned range as a catalyst during the reaction based on the total weight of the mixed solution, the reaction between the mussel adhesive protein and N-hydroxysuccinimidyl acrylate or methacrylic anhydride can be facilitated.

[0101] In the present invention, based on the total weight of the mixed solution, at least one of acrylate N-hydroxysuccinimidyl ester and methacrylic anhydride may be included in an amount of 0.6 to 2.0 weight percent, and more preferably, at least one of acrylate N-hydroxysuccinimidyl ester and methacrylic anhydride may be included in an amount of 0.8 to 1.5 weight percent. If the content of at least one of acrylate N-hydroxysuccinimidyl ester and methacrylic anhydride is less than the above range, the pores may become larger due to the insufficient density of the three-dimensional network structure, thereby possibly preparing a film or tape with reduced tensile force. If the content is greater than the above range, the elasticity may be reduced and the adhesive force may be reduced due to the excessive density of the three-dimensional network structure. On the contrary, when at least one of acrylate N-hydroxysuccinimidyl ester and methacrylic anhydride is included in the above range, the tensile force is excellent, and the adhesive force is particularly improved, so it is preferred.

[0102] In the present invention, the first mixed solution or the second mixed solution may be prepared and used separately. In this case, the mass ratio of the first mixed solution to the second mixed solution may be 10:0 or 0:10.

[0103] Furthermore, in the present invention, the mass ratio of the first mixed solution to the second mixed solution may be 9:1 to 1:9, preferably 3:1 to 1:3, and more preferably 1:1. In this case, the bioadhesive composition of the present invention can adjust the degradation time during adhesion in vivo by adjusting the mixing ratio (mass ratio) of the first mixed solution to the second mixed solution to prepare a film and a tape. Furthermore, if the mussel adhesive protein is maximized within the above mixing ratio and is not crystallized, the protein can be saved.

[0104] The method for preparing the bioadhesive composition of the present invention may further include a drying step after solidifying the mixed solution in the above-mentioned step (d), or after mixing the first mixed solution and the second mixed solution and solidifying them in step (d-1), step (d-2) or step (d-3).

[0105] The bioadhesive composition of the present invention is non-adhesive and non-degradable in a dry state, and exhibits adhesiveness in a wet state. In addition, it can exhibit biodegradability under wet conditions containing biological enzymes, such as under wet conditions in the presence of collagenase. However, the above-mentioned biodegradation is the biodegradation of the part that binds to the mussel adhesive protein, and does not excrete toxic acrylic acid monomers or methacrylic acid monomers, so it has no cytotoxicity and maintains biocompatibility.

[0106] The bioadhesive composition of the present invention is a promising adhesive material and can be prepared into a film, a single-sided or double-sided tape, or a hydrogel, but is not limited thereto.

[0107] Furthermore, the bioadhesive composition can be effectively used as an adhesive for wearable electronic products, artificial skin, supercapacitor robots, energy storage materials, bioelectrodes, implantable current detection biosensors, electrical stimulation drug sustained release devices, neural prostheses, and the like.

[0108] The above description uses an example to illustrate the content of the technical idea of ​​the present invention. Ordinary technicians in the technical field to which the present invention belongs can make various modifications and deformations without departing from the essential characteristics of the present invention. Therefore, the embodiments described in the present invention are only used to illustrate the technical idea of ​​the present invention, but not to limit the technical idea of ​​the present invention. The scope of the technical idea of ​​the present invention is not limited to the above embodiments. The protection scope of the present invention should be interpreted by the scope of protection of the invention claims, and all technical ideas within the same scope should be interpreted as included in the scope of rights of the present invention.

[0109] Preparation Example

[0110] Preparation Example 1: Preparation of mussel adhesion protein

[0111] The mussel adhesive protein of sequence 9 (fp-151) was prepared by the following method: a decapeptide consisting of 10 amino acids repeated about 80 times in the mussel adhesive protein fp-1 existing in nature was synthesized into an fp-1 variant consisting of 6 decapeptides in a manner that can be expressed in Escherichia coli, and the gene of Mgfp-5 (Genbank No. AAS00463 or AY521220) was added between two fp-1 variants, and then produced in Escherichia coli (DSHwang et al., Biomaterials 28, 3560-3568, 2007). Then, the tyrosine of the mussel adhesive protein (fp-151) of the sequence 9 was converted into DOPA by an in vitro enzyme reaction using mushroom tyrosinase to prepare a mussel adhesive protein into which a catechol DOPA residue was introduced.

[0112] Preparation Example 2: Preparation of mussel adhesion protein

[0113] The mussel adhesion protein of sequence 9 (fp-151) was prepared by the following method: a decapeptide consisting of 10 amino acids repeated about 80 times in the mussel adhesion protein fp-1 existing in nature was synthesized into an fp-1 variant consisting of 6 decapeptides in a manner that can be expressed in Escherichia coli, and the Mgfp-5 gene (Genbank No. AAS00463 or AY521220) was added between two fp-1 variants, and then produced in Escherichia coli (DSHwang et.al., Biomaterials 28, 3560-3568, 2007).

[0114] Example

[0115] Example 1. Preparation of the first component

[0116] After dissolving 10 weight percent of the mussel adhesive protein introduced with catechol-DOPA residues prepared in the above-mentioned Preparation Example 1, 1 weight percent of acrylic acid N-hydroxysuccinic acid imide ester, and 0.2 weight percent of α-ketoglutaric acid as a photoinitiator in deionized water, 20 weight percent of acrylic acid was added and mixed to prepare a first mixed solution. The first mixed solution was filtered using a 0.4 μm sterilized syringe filter and poured into a quartz mold. Then, an ultraviolet (UV) lamp was used to cure for 30 minutes at a wavelength of 254 nm and a power of 10 W.

[0117] Example 2. Preparation of the second component

[0118] After dissolving 0.1 weight percent of the mussel adhesive protein prepared according to the above Preparation Example 2 in a buffer solution (phosphate buffer solution (PBS) at pH 7.5), 2 ml of methacrylic anhydride was added per 1 g of the mussel adhesive protein, and the mixture was stirred at 120 rpm in a water bath at 40° C. for 1 hour. The mussel adhesive protein was placed in a No. 4 dialysis bag (Spectra / Por 4 dialysis membrane Standard RC Tubing) and dialyzed in DW in a water bath at 40° C. for one day. After the mussel adhesive protein into which methacryloyl groups were introduced was freeze-dried, the tyrosine of the mussel adhesive protein was converted into DOPA to prepare a mussel adhesive protein into which catechol DOPA residues were introduced. After dissolving 10 weight percent of the mussel adhesive protein introduced with catechol-DOPA residues and 0.2 weight percent of α-ketoglutaric acid as a photoinitiator in deionized water, 30 weight percent of methacrylic acid was added and mixed to prepare a second mixed solution. The second mixed solution was filtered using a 0.4 μm sterilized syringe filter and poured into a quartz mold. Then, an ultraviolet lamp was used to cure the solution at a wavelength of 254 nm and a power of 10 W for 1 hour.

[0119] Example 3. Mixing the first component and the second component (1:1 mass ratio)

[0120] After filtering the mixed solution of the first mixed solution prepared according to the above Example 1 and the second mixed solution prepared according to the above Example 2 at a mass ratio of 1:1 using a 0.4 μm sterilized syringe filter, the mixed solution was poured into a quartz mold and then cured for 30 minutes using an ultraviolet lamp at a wavelength of 254 nm and a power of 10 W.

[0121] Example 4. Mixing the first component and the second component (1:3 mass ratio)

[0122] After filtering the mixed solution of the first mixed solution prepared according to the above Example 1 and the second mixed solution prepared according to the above Example 2 at a mass ratio of 1:3 using a 0.4 μm sterilized syringe filter, the mixed solution was poured into a quartz mold and then cured for 30 minutes using an ultraviolet lamp at a wavelength of 254 nm and a power of 10 W.

[0123] Example 5. Mixing the first component and the second component (3:1 mass ratio)

[0124] After filtering the mixed solution of the first mixed solution prepared according to the above Example 1 and the second mixed solution prepared according to the above Example 2 at a mass ratio of 3:1 using a 0.4 μm sterilized syringe filter, the mixed solution was poured into a quartz mold and then cured for 30 minutes using an ultraviolet lamp at a wavelength of 254 nm and a power of 10 W.

[0125] Example 6. Preparation of the first component (mussel adhesion protein: acrylic acid = 1:2 mass ratio)

[0126] After dissolving 10 weight percent of the mussel adhesive protein introduced with catechol-dopa residues prepared according to Preparation Example 1, 1 weight percent of acrylic acid N-hydroxysuccinic acid imide ester, and 0.2 weight percent of α-ketoglutaric acid as a photoinitiator in deionized water, 20 weight percent of acrylic acid was added and mixed. The mixed solution was filtered using a 0.4 μm sterilized syringe filter and poured into a quartz mold. Then, an ultraviolet lamp was used to cure at a wavelength of 254 nm and a power of 10 W for 1 hour and completely dried. The image of the composition in a dry state is shown in FIG. Figure 8B shown.

[0127] Example 7. Preparation of the first component (mussel adhesive protein: acrylic acid = 1:3 mass ratio)

[0128] The method of Example 6 was used to prepare a composition of mussel adhesive protein and polyacrylic acid crosslinked except that the concentration of acrylic acid was 30 weight percent. Figure 8B and Figure 8C The prepared composition was confirmed by Fourier transform infrared spectroscopy (FT-IR), and the results were as follows Figure 4C As shown. At 3000cm -1 The Dopa peak is shown at 1710 cm -1 The C=O stretching of polyacrylic acid was confirmed at 1219 cm -1 Symmetrical CNC telescoping was confirmed at 1307cm -1 Asymmetric SNC stretching was confirmed in the PCR product, confirming that the composition was synthesized normally.

[0129] Example 8. Preparation of the first component (Mussel adhesion protein: acrylic acid = 1:4 mass ratio)

[0130] The method of Example 6 was used to prepare a composition of mussel adhesive protein and polyacrylic acid crosslinked except that the concentration of acrylic acid was 40 weight percent. Figure 8B shown.

[0131] Example 9. Preparation of the first component (mussel adhesive protein: acrylic acid = 1:5 mass ratio)

[0132] The method of Example 6 was used to prepare a composition of mussel adhesive protein and polyacrylic acid crosslinked except that the concentration of acrylic acid was 50 weight percent. Figure 8B shown.

[0133] Comparative Example

[0134] Comparative Example 1

[0135] Gelatin was added to replace mussel adhesive protein, and the composition was prepared according to the method of Example 1 so that the acrylic acid concentration was 30 weight percent. Figure 8C shown.

[0136] Comparative Example 2. Composition containing non-modified mussel adhesive protein and polyacrylic acid

[0137] Using non-modified mussel adhesive protein without DOPA, a composition was prepared by the method of Example 6 so that the acrylic acid concentration was 30 weight percent. Figure 8C shown.

[0138] like Figure 8B As shown in the figure, when the mass ratio of mussel adhesive protein to polyacrylic acid is 1:2, the mechanical properties are slightly weak in the dry state. On the other hand, when the mass ratio of mussel adhesive protein to polyacrylic acid is greater than 1:4, it is confirmed that the hardness is strong but the elasticity is reduced in the wet state. The experimental results show that the composition of Example 7, in which the mass ratio of mussel adhesive protein to polyacrylic acid is 1:3, is excellent in mechanical properties, tensile force and elastic force.

[0139] On the other hand, by comparing the results of Comparative Examples 1 and 2 with Example 7, it was confirmed that the composition of Example 7 had outstanding mechanical stability in a dry state and excellent tensile strength in a wet state compared with Comparative Examples 1 and 2.

[0140] Test example

[0141] Experimental Example 1. MALDI mass spectrometry analysis

[0142] The methacryloyl group was introduced into the existing mussel adhesive protein (fp-151) and the mussel adhesive protein prepared in Example 2 to crystallize the methacrylated mussel adhesive protein and then put it into a maldi plate to check the molecular weight. The results are as follows: Figure 3A and Figure 3B shown.

[0143] Figure 3A The MALDI mass spectrum of the existing mussel adhesion protein (FP-151) is shown. Figure 3B The MALDI mass spectrum of the methacrylated mussel adhesion protein prepared in Example 2 is shown.

[0144] Reference Figure 3A and Figure 3B , the average molecular weight of the large peak increased from 22.6 kDa to 27.5 kDa, thus confirming that the mussel adhesion protein was methacryloylated.

[0145] Test Example 2. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0146] The methacryloyl group was introduced into the existing mussel adhesion protein (fp-151) and the mussel adhesion protein prepared in Example 2 to crystallize the methacrylated mussel adhesion protein, and then the Fourier transform infrared spectrum was measured. The result is shown in FIG4 .

[0147] Referring to Figure 4, when gelatin is changed to gelatin methacrylate, by -1 The observed peak change confirmed that the mussel adhesive protein of Example 2 was methacrylated.

[0148] Test Example 3. Degradation Test

[0149] The adhesive composition prepared according to Examples 1 to 3 was prepared to have a size of 1 cm in length and width and 0.5 mm in height.

[0150] The adhesive composition prepared according to Examples 1 to 3 was placed in 10 ml of a phosphate buffer solution containing 0.01% collagenase and 0.01% lysozyme, and placed in an incubator at 37 degrees for up to 2 weeks before being recovered. The remaining amount was confirmed in percentage (%) by comparing the dry weight before being placed in the phosphate buffer solution with the weight after freeze-drying after recovery. The results are as follows: Figure 5 shown.

[0151] Test Example 4. Adhesion Test

[0152] Prepare adhesive compositions prepared according to Examples 1 to 3 and cyanoacrylic acid and fibrin glue as control groups. Prepare adhesive compositions prepared according to Examples 1 to 3 with a length, width, and height of 1 cm and 0.5 mm, respectively, using 300 μl of cyanoacrylic acid and collagen glue. Paste pig skin on an acrylic plate with a length and width of 1 cm. After pasting the adhesive between the pig skin and placing it in a phosphate buffer solution for 4 hours, the shear stress and interfacial stress are tested. The results are as follows: Figure 6 shown.

[0153] Reference Figure 6 It can be confirmed that although the adhesive force of the adhesive composition of the present invention is lower than that of cyanoacrylic acid which is difficult to use on the human body, it exhibits outstanding adhesive force compared with commercially available fibrin glue.

[0154] Test Example 5. Cytotoxicity Test

[0155] 5×10 per well 4 The cell density was 24-well tissue culture plates inoculated with HaCaT cells and cultured overnight. The cells were treated with 2% of the composition solutions of Example 3 and Example 7 dissolved in Dulbecco Modified Eagle Medium (DMEM) high glucose solution. After culturing the cells for 1 day, 5% CCK-8 (Dojindo Laboratories, Tokyo, Japan) reagent dissolved in Dulbecco Modified Eagle Medium was added to each well. After culturing the cells for 3 hours, the absorbance at a wavelength of 450 nm was measured using a microplate spectrophotometer. In addition, under the same conditions, a live / dead test was performed to evaluate cytotoxicity (surviving cells are green and dead cells are red). The results are as follows: 7A to 7D shown.

[0156] Fig. 7A The CCK-8 test results of Example 3 at a wavelength of 450 nm using a microplate spectrophotometer are shown in a bar graph. Figure 7B To perform a live / dead test to confirm the results of cell survival in Example 3 (live cells are green, dead cells are red), Figure 7C The CCK-8 test results of Example 7 at a wavelength of 450 nm using a microplate spectrophotometer are shown in a bar graph. Fig.7D A live / dead test was performed to confirm the results of cell survival in Example 7 (live cells are green, dead cells are red).

[0157] like Fig. 7A As shown in Figure 2, in the CCK-8 test, the cell survival rate showed more than 70%. Figure 7B As shown, almost no red color was observed. The above results confirmed that the bioadhesive composition of the present invention has very low cytotoxicity. In addition, such a result can be considered that no leaching of acrylic acid monomer occurred.

[0158] like Figure 7C As shown in Figure 2, in the CCK-8 test, the cell survival rate showed more than 90%. Fig.7DAs shown, almost no red color was observed. The above results confirmed that the bioadhesive composition of the present invention has very low cytotoxicity. In addition, such a result can be considered that no leaching of acrylic acid monomer occurred.

[0159] Test Example 6. Confirmation of Adhesive Formation

[0160] It was confirmed that the bioadhesive compositions prepared according to Examples 1 to 5 could form adhesives with various concentration changes of the first component and the second component (first component: second component = 10:0, 3:1, 1:1, 1:3, 0:10), as shown in FIG. 8 .

[0161] Test Example 7. Evaluation of in vitro biodegradability

[0162] The bioadhesive composition of Example 7 was prepared as a film sample of about 10 mm×10 mm in size with the same weight. In order to evaluate biodegradability and prevent the growth of other microorganisms in vitro, two culture media were prepared, one without collagenase and one with 0.005% (w / v) collagenase added to Dulbecco's Phosphate-Buffered Saline (DPBS) with 0.01% (w / v) sodium azide. Before placing the prepared samples in each culture medium, they were sterilized in 75% ethanol for 15 minutes and washed three times with Dulbecco's phosphate buffered saline (DPBS) before use. 15 ml of culture medium without collagenase or with 5% collagenase was added to a conical tube, and then the bioadhesive composition sample was placed in the tube and shaken at 60 rpm at 37°C to evaluate the biodegradability at different times. The evaluation was performed after the bioadhesive composition sample was taken out of the culture medium, completely washed with deionized water and freeze-dried. The weight loss was determined by taking the dry mass of the freeze-dried sample before the experiment as the standard and the mass percentage of the freeze-dried sample after the experiment at each time interval. The results are shown in Fig. 9 shown.

[0163] The bioadhesive composition of the present invention tends to be slowly biodegraded in a Dulbecco's phosphate buffer solution medium, and tends to be biodegraded by 50% or more within 4 hours in the presence of collagenase as a biological enzyme. This means that the bioadhesive medium of the present invention is easily biodegradable in an environment in which the same enzyme as the in vivo environment exists.

[0164] Test Example 8. Adhesion test in water

[0165] In order to confirm the adhesion of the film in water, a tensile test was performed using Instron. Before the test, the pig skin and the tape prepared by the bioadhesive composition of Example 7 were placed in a Dulbecco's phosphate buffer solution with 0.01% (w / v) sodium azide added, and kept for more than 10 minutes to prevent decomposition, dehydration and contamination. The test was performed as follows: the wet pig skin was cut into 10mm×10mm sizes and pasted on an acrylic rod. The tape of Example 7 cut into 10mm×10mm sizes was placed between the pig skin, and the surface was cleaned with a phosphate buffer solution. The tape of Example 7 was attached to the pig skin by compressing it at a pressure of 1kPa for 5 seconds. For the mechanical test of the bonded sample, in order to ensure the parallel expansion of the adhesive tape in a humid environment, it was carried out for 6 hours after the initial pressure. Two commercially available adhesives (fibrin glue, gelatin) and the composition of Comparative Example 2 were used as the control group, and their shear stress and interfacial stress were confirmed according to different materials. All tests were performed at a specified peel speed of 50 mm / min, and the interfacial toughness was determined as twice the stagnation force (in the case of 180° peel test) or the stagnation force (in the case of 90° peel test).

[0166] like Fig.10 As shown, the bioadhesive composition of Example 7 of the present invention has superior adhesive strength in water compared to fibrin glue and gelatin which are commercially available adhesives. In addition, it was confirmed that surface modification with a catechol compound further improved the adhesive strength.

[0167] Test Example 9. Tensile Test

[0168] Using Instron, a 1 cm x 1 cm tape of Example 7 was fixed on the instrument and pulled up and down continuously at a speed of 50 mm / min. The tape was checked in both a completely dry state and a wet state until the moment the tape was torn. The wet tape was taken out after the completely dry tape was completely immersed in Dulbecco's phosphate buffer solution for 10 seconds. Fig.11A The tensile test results of the dry tape are shown in Figure 2. Fig. 11B The figure shows the tensile test result of the tape in wet state.

[0169] Reference Fig.11A and 11B In a dry state, the stretch was 0.4583 mm, which was 4.6%. In contrast, in a wet state, the stretch was 5.32 mm, which was 53.2%.

[0170] Test Example 10. Tensile test in pig bladder

[0171] After attaching a film of the bioadhesive composition of Example 7 of the present invention and a commercially available anti-adhesion film as a control group to an excised pig bladder, 250 ml of phosphate buffer solution was repeatedly injected into the pig bladder 9 times at an injection rate of 2.5 ml / s in a water tank filled with 1× phosphate buffer solution to observe changes in the performance of the attached films.

[0172] Fig. 12A This is an image of a pig bladder with an anti-adhesion film attached. Fig. 12B is an image of a porcine bladder to which the film of Example 7 is attached. Fig. 12A It was observed in the repeated bladder expansion / contraction test that the anti-adhesion film did not stretch to the size of the bladder, resulting in a portion of the attachment site falling off the bladder. Fig. 12B It was observed that the film of Example 7 also showed a tendency to expand / contract in the repeated bladder expansion / contraction test, showing that it remained attached to the surface of the porcine bladder with good efficiency.

[0173] The above description is made with reference to the preferred embodiments of the present invention, but it can be understood that skilled practitioners or ordinary technicians in the technical field to which the present invention belongs can make various modifications and changes to the present invention without departing from the scope of the concept and technical field of the present invention as described in the attached invention claims.

[0174] Therefore, the technical scope of the present invention should not be limited to the contents described in detail in the specification, but should be determined by the scope of protection claimed for the invention. <110> Pohang University of Science and Technology Industry-Academic Cooperation Group <120> Bioadhesive composition containing mussel adhesive protein and preparation method thereof <130> POSTECH1-90P <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 800 <212> PRT <213> Artificial sequence <220> <223> fp-1 <400> 1 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 165 170 175 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 180 185 190 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 195 200 205 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 210 215 220 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Tyr Lys 225 230 235 240 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 245 250 255 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 260 265 270 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 275 280 285 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 290 295 300 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Tyr Lys 305 310 315 320 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 325 330 335 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 340 345 350 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 355 360 365 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 370 375 380 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 385 390 395 400 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 405 410 415 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 420 425 430 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 435 440 445 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 450 455 460 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 465 470 475 480 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 485 490 495 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 500 505 510 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 515 520 525 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 530 535 540 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 545 550 555 560 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 565 570 575 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 580 585 590 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 595 600 605 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 610 615 620 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 625 630 635 640 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 645 650 655 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 660 665 670 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 675 680 685 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 690 695 700 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 705 710 715 720 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 725 730 735 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 740 745 750 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 755 760 765 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 770 775 780 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 785 790 795 800 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <220> <223> fp-1 variant <400> 2 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 1 5 10 <210> 3 <211> 120 <212> PRT <213> Artificial sequence <220> <223> fp-1 variant <400> 3 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys 115 120 <210> 4 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 4 Leu Phe Ser Phe Phe Leu Leu Leu Thr Cys Thr Gln Leu Cys Leu Gly 1 5 10 15 Thr Asn Arg Pro Asp Tyr Asn Asp Asp Glu Glu Asp Asp Tyr Lys Pro 20 25 30 Pro Val Tyr Lys Pro Ser Pro Ser Lys Tyr Arg Pro Val Asn Pro Cys 35 40 45 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Arg Gly Gly 50 55 60 Ser Tyr Lys Cys Phe Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn 65 70 75 80 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys 85 90 95 Val Pro Val Gly Lys Thr Phe Lys Cys Val Cys Arg Asn Gly Asn Phe 100 105 110 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys Asn 115 120 125 Asn Gly Lys Cys Ser Pro Leu Gly Lys Thr Gly Tyr Lys Cys Thr Cys 130 135 140 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro 145 150 155 160 Asn Pro Cys Lys Asn Lys Gly Arg Cys Phe Pro Asp Gly Lys Thr Gly 165 170 175 Tyr Lys Cys Arg Cys Val Asp Gly Tyr Ser Gly Pro Thr Cys Gln Glu 180 185 190 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Thr Cys Ser Ala 195 200 205 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Arg Pro Gly Tyr Phe Gly 210 215 220 Pro Glu Cys Glu Arg Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly 225 230 235 240 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly Tyr Arg Cys Arg Cys Lys 245 250 255 Gly Gly Tyr Ser Gly Pro Thr Cys Lys Val Asn Val Cys Lys Pro Thr 260 265 270 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn 275 280 285 Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu Asn Val 290 295 300 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Asp Asn 305 310 315 320 Ser Asp Asp Gly Phe Lys Cys Arg Cys Val Gly Gly Tyr Lys Gly Pro 325 330 335 Thr Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn 340 345 350 Gly Gly Lys Cys Asn Tyr Asn Gly Lys Ile Tyr Thr Cys Lys Cys Ala 355 360 365 Tyr Gly Trp Arg Gly Arg His Cys Thr Asp Lys Ala Tyr Lys Pro Asn 370 375 380 Pro Cys Val Val Ser Lys Pro Cys Lys Asn Arg Gly Lys Cys Ile Trp 385 390 395 400 Asn Gly Lys Ala Tyr Arg Cys Lys Cys Ala Tyr Gly Tyr Gly Gly Arg 405 410 415 His Cys Thr Lys Lys Ser Tyr Lys Lys Asn Pro Cys Ala Ser Arg Pro 420 425 430 Cys Lys Asn Arg Gly Lys Cys Thr Asp Lys Gly Asn Gly Tyr Val Cys 435 440 445 Lys Cys Ala Arg Gly Tyr Ser Gly Arg Tyr Cys Ser Leu Lys Ser Pro 450 455 460 Pro Ser Tyr Asp Asp Asp Glu Tyr 465 470 <210> 5 <211> 50 <212> PRT <213> Artificial sequence <220> <223> fp-3 <400> 5 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Gly Ser 50 <210> 6 <211> 750 <212> PRT <213> Artificial sequence <220> <223> fp-4 <400> 6 Tyr Gly Arg Arg Tyr Gly Glu Pro Ser Gly Tyr Ala Asn Ile Gly His 1 5 10 15 Arg Arg Tyr Tyr Glu Arg Ala Ile Ser Phe His Arg His Ser His Val 20 25 30 His Gly His His Leu Leu His Arg His Val His Arg His Ser Val Leu 35 40 45 His Gly His Val His Met His Arg Val Ser His Arg Ile Met His Arg 50 55 60 His Arg Val Leu His Gly His Val His Arg His Arg Val Leu His Asn 65 70 75 80 His Val His Arg His Ser Val Leu His Gly His Val His Arg His Arg 85 90 95 Val Leu His Arg His Val His Arg His Asn Val Leu His Gly His Val 100 105 110 His Arg His Arg Val Leu His Lys His Val His Asn His Arg Val Leu 115 120 125 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Arg 130 135 140 His Gln Val Leu His Lys His Val His Asn His Arg Val Leu His Lys 145 150 155 160 His Leu His Lys His Gln Val Leu His Gly His Val His Thr His Arg 165 170 175 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Leu 180 185 190 His Lys His Gln Val Leu His Gly His Ile His Thr His Arg Val Leu 195 200 205 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Thr 210 215 220 His Arg Val Leu His Lys His Val His Lys His Arg Val Leu His Lys 225 230 235 240 His Leu His Lys His Gln Val Leu His Gly His Val His Met His Arg 245 250 255 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Val 260 265 270 His Lys His His Val Val His Lys His Val His Ser His Arg Val Leu 275 280 285 His Lys His Val His Lys His Arg Val Glu His Gln His Val His Lys 290 295 300 His His Val Leu His Arg His Val His Ser His His Val Val His Ser 305 310 315 320 His Val His Lys His Arg Val Val His Ser His Val His Lys His Asn 325 330 335 Val Val His Ser His Val His Arg His Gln Ile Leu His Arg His Val 340 345 350 His Arg His Gln Val Val His Arg His Val His Arg His Leu Ile Ala 355 360 365 His Arg His Ile His Ser His Gln Ala Ala Val His Arg His Val His 370 375 380 Thr His Phe Glu Gly Asn Phe Asn Asp Asp Gly Thr Asp Val Asn Leu 385 390 395 400 Arg Ile Arg His Gly Ile Ile Tyr Phe Gly Gly Asn Thr Tyr Arg Leu 405 410 415 Ser Gly Gly Arg Arg Arg Phe Met Thr Leu Trp Gln Glu Cys Leu Glu 420 425 430 Ser Tyr Gly Asp Ser Asp Glu Cys Phe Val Gln Leu Leu Glu Gly Asn 435 440 445 Gln His Leu Phe Thr Val Val Gln Gly His His Ser Thr Ser Phe Arg 450 455 460 Ser Asp Leu Ser Asn Asp Leu His Pro Asp Asn Asn Ile Glu Gln Ile 465 470 475 480 Ala Asn Asp His Val Asn Asp Ile Ala Gln Ser Thr Asp Gly Asp Ile 485 490 495 Asn Asp Phe Ala Asp Thr His Tyr Asn Asp Val Ala Pro Ile Ala Asp 500 505 510 Val His Val Asp Asn Ile Ala Gln Thr Ala Asp Asn His Val Lys Asn 515 520 525 Ile Ala Gln Thr Ala His His His Val Asn Asp Val Ala Gln Ile Ala 530 535 540 Asp Asp His Val Asn Asp Ile Gly Gln Thr Ala Tyr Asp His Val Asn 545 550 555 560 Asn Ile Gly Gln Thr Ala Asp Asp His Val Asn Asp Ile Ala Gln Thr 565 570 575 Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val 580 585 590 Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val Asn Asp Ile Gly Asp 595 600 605 Thr Ala Asn Ser His Ile Val Arg Val Gln Gly Val Ala Lys Asn His 610 615 620 Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His Ile Gln His Leu Lys 625 630 635 640 Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn Asn His Ala Thr Lys 645 650 655 Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln Gln Thr Ile Glu Arg 660 665 670 Glu Ile Gln His Lys Arg His Leu Ser Glu Lys Glu Asp Ile Asn Leu 675 680 685 Gln His Glu Asn Ala Met Lys Ser Lys Val Ser Tyr Asp Gly Pro Val 690 695 700 Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln Gly Ser Tyr Asn Glu 705 710 715 720 Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr Asn Gly Lys Val Ser 725 730 735 Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly Tyr Ala Tyr 740 745 750 <210> 7 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 7 Lys His His His His His His Ser Ser Glu Glu Tyr Lys Gly Gly Tyr 1 5 10 15 Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly 20 25 30 Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys 35 40 45 Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys 50 55 60 Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly 65 70 75 80 Watching Watching <210> 8 <211> 103 <212> PRT <213> artificial sequence <220> <223> fp-6 <400> 8 Ile Ala Ala Leu Cys Gly Ile Val Lys Ser Ile Asp Ser Asp Asp Ser 1 5 10 15 Asp Tyr Asp Tyr Lys Gly Arg Gly Tyr Cys Thr Asn Lys Gly Cys Arg 20 25 30 Ser Gly Tyr Asn Tyr Phe Gly Asn Lys Gly Tyr Cys Lys Tyr Gly Glu 35 40 45 Lys Ser Tyr Thr Tyr Asn Cys Asn Ser Tyr Ala Gly Cys Cys Leu Pro 50 55 60 Arg Asn Pro Tyr Gly Lys Leu Lys Tyr Tyr Cys Thr Asn Lys Tyr Gly 65 70 75 80 Cys Pro Asn Asn Tyr Tyr Phe Tyr Asn Asn Lys Gly Tyr Tyr Tyr Leu 85 90 95 Glu His His His His His His 100 <210> 9 <211> 199 <212> PRT <213> artificial sequence <220> <223> fp-151 <400> 9 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Light Tyr Tyr Gly Light Ala Light Light Tyr Tyr Tyr Light Tyr Light Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro 130 135 140 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 145 150 155 160 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 165 170 175 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 180 185 190 Tyr Pro Pro Thr Tyr Lys Leu 195 <210> 10 <211> 171 <212> PRT <213> artificial sequence <220> <223> fp-131 <400> 10 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala Asp 50 55 60 Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn 65 70 75 80 Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn 85 90 95 Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu 165 170 <210> 11 <211> 175 <212> PRT <213> artificial sequence <220> <223> fp-353 <400> 11 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys 85 90 95 Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala 115 120 125 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly 130 135 140 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn 145 150 155 160 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 165 170 175 <210> 12 <211> 187 <212> PRT <213> artificial sequence <220> <223> fp-153 <400> 12 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Light Tyr Tyr Gly Light Ala Light Light Tyr Tyr Tyr Light Tyr Light Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala Asp Tyr Tyr Gly 130 135 140 Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr Asn Arg 145 150 155 160 Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly Trp Lys 165 170 175 Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 180 185 <210> 13 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 13 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr 85 90 95 Tyr Light Asn Ser Gly Light Tyr Light Tyr Leu Light Light Ala Arg Light Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala 115 120 125 Light Pro Ser Tyr Pro Pro Thr Tyr Light Ala Light Pro Ser Tyr Pro Pro 130 135 140 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 145 150 155 160 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 165 170 175 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 180 185 <210> 14 <211> 60 <212> PRT <213> artificial sequence <220> <223> fp-1 change <400> 14 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 50 55 60 <210> 15 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> fp-151 Variant <400> 15 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu 50 55 60 Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly 65 70 75 80 Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr 85 90 95 Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys 100 105 110 Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys 115 120 125 Lys Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr 130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Light 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 180 185 190 Pro Thr Bull Light 195 <210> 16 <211> 76 <212> PRT <213> artificial sequence <220> <223> mgfp-5 <400> 16 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 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 Light Gly Light Tyr Tyr Gly Light Ala Light Light Tyr Tyr Tyr Light Tyr Light 35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg 50 55 60 Light Gly Tyr Light Light Tyr Tyr Gly Gly Gly Ser Ser 65 70 75

Claims

1. A bioadhesive composition, characterized in that: Include: mussel adhesion proteins; and At least one of polyacrylic acid and polymethacrylic acid, The mussel adhesive protein is connected to at least one of the polyacrylic acid and polymethacrylic acid via an amide bond. Wherein, the above-mentioned bioadhesive composition has a three-dimensional network structure. The bioadhesive composition is a mixture of a first component of the mussel adhesive protein and polyacrylic acid bonded by an amide bond and a second component of the mussel adhesive protein and polymethacrylic acid bonded by an amide bond in a mass ratio of 3:1 to 1:

3. The first component comprises mussel adhesive protein and polyacrylic acid in a mass ratio of 1:1 to 1:5, and The second component comprises mussel adhesion protein and polymethacrylic acid in a mass ratio of 1:1 to 1:

5.

2. The bioadhesive composition according to claim 1, characterized in that The above-mentioned mussel adhesion protein consists of one or more amino acid sequences selected from the group consisting of sequence 1, sequence 2, sequence 3, sequence 4, sequence 5, sequence 6, sequence 7, sequence 8, sequence 9, sequence 10, sequence 11, sequence 12, sequence 13, sequence 14 and sequence 15.

3. The bioadhesive composition according to claim 1, characterized in that The mussel adhesion protein is a mussel adhesion protein in which tyrosine residues are converted into catechol compounds, a mussel adhesion protein in which catechol derivatives are introduced on the surface of the mussel adhesion protein, or a mussel adhesion protein including the above two.

4. The bioadhesive composition according to claim 3, characterized in that: The catechol compound is one or more selected from the group consisting of DOPA, DOPA-O-quinone, 6-hydroxyDOPA, 6-hydroxyDOPAquinone and derivatives thereof.

5. The bioadhesive composition according to claim 1, characterized in that: The mass ratio of the mussel adhesive protein to the polyacrylic acid or polymethacrylic acid contained in the first component or the second component is 1:2.5 to 1:3.

5.

6. The bioadhesive composition according to claim 1, characterized in that: The bioadhesive composition exhibits non-adhesiveness and non-degradability in a dry state, and exhibits adhesiveness and biodegradability in a wet state.

7. The bioadhesive composition according to claim 1, characterized in that: The bioadhesive composition is a film, a single-sided or double-sided tape, or a hydrogel.

8. A method for preparing a bioadhesive composition, characterized in that: include: Step (a), preparing a first mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing acrylate N-hydroxysuccinic acid imide ester, and preparing a second mixed solution formed by mixing a solution containing mussel adhesion protein and a solution containing methacrylic anhydride; Step (b), dissolving a photoinitiator in each of the first mixed solution and the second mixed solution; Step (c), adding acrylic acid monomer to the first mixed solution, and adding methacrylic acid monomer to the second mixed solution; and Step (d), mixing the first mixed solution and the second mixed solution in a mass ratio of 3:1 to 1:3, and then curing; The first mixed solution comprises mussel adhesive protein and acrylic acid monomer in a mass ratio of 1:1 to 1:5, and Wherein, the second mixed solution contains mussel adhesion protein and methacrylic acid monomer in a mass ratio of 1:1 to 1:

5.

9. The method for preparing the bioadhesive composition according to claim 8, characterized in that: The photoinitiator was α-ketoglutaric acid.

10. The method for preparing the bioadhesive composition according to claim 8, characterized in that: Based on the total weight of the mixed solution, the mixed solution contains 7 weight percent to 15 weight percent of mussel adhesive protein.

11. The method for preparing the bioadhesive composition according to claim 8, characterized in that: Based on the total weight of the mixed solution, the mixed solution contains 0.6 weight percent to 2.0 weight percent of at least one of acrylic acid N-hydroxysuccinic acid imide ester and methacrylic anhydride.

12. The method for preparing the bioadhesive composition according to claim 8, characterized in that: Based on the total weight of the mixed solution, the mixed solution contains 0.1 weight percent to 0.4 weight percent of the photoinitiator.

13. The method for preparing the bioadhesive composition according to claim 8, characterized in that: The bioadhesive composition exhibits non-adhesiveness and non-degradability in a dry state, and exhibits adhesiveness and biodegradability in a wet state.

14. The method for preparing the bioadhesive composition according to claim 8, characterized in that: The mussel adhesion protein is a mussel adhesion protein in which tyrosine residues are converted into catechol compounds, and the catechol compounds are selected from at least one of the group consisting of dopa, dopa-o-quinone, 6-hydroxydopa, 6-hydroxydopaquinone and derivatives thereof.

15. The method for preparing the bioadhesive composition according to claim 8, characterized in that: The above-mentioned mussel adhesion protein consists of one or more amino acid sequences selected from the group consisting of sequence 1, sequence 2, sequence 3, sequence 4, sequence 5, sequence 6, sequence 7, sequence 8, sequence 9, sequence 10, sequence 11, sequence 12, sequence 13, sequence 14 and sequence 15.

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