WO2006004076A1 - Matière bio physiologiquement active - Google Patents

Matière bio physiologiquement active Download PDF

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Publication number
WO2006004076A1
WO2006004076A1 PCT/JP2005/012292 JP2005012292W WO2006004076A1 WO 2006004076 A1 WO2006004076 A1 WO 2006004076A1 JP 2005012292 W JP2005012292 W JP 2005012292W WO 2006004076 A1 WO2006004076 A1 WO 2006004076A1
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WIPO (PCT)
Prior art keywords
silk
protein
bioactive
biomaterial
silkworm
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PCT/JP2005/012292
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English (en)
Japanese (ja)
Inventor
Rika Hino
Masahiro Tomita
Katsutoshi Yoshizato
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Hiroshima Industrial Promotion Organization
Biointegrence Inc.
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Publication of WO2006004076A1 publication Critical patent/WO2006004076A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention of this application relates to a biomaterial having a given physiological activity. More specifically, the invention of this application includes a transgenic silkworm that produces a silk protein containing a fusion protein of silk hive mouth-in and a physiologically active substance, and a silk protein produced by the silkworm or the fusion protein as a main component. It relates to bioactive biomaterials.
  • Biological tissues are composed of cells and extracellular matrix, and cells carry out various physiological phenomena including adhesion, proliferation and differentiation using the extracellular matrix as a scaffold.
  • the extracellular matrix In the case of damage to living tissue, if the extracellular matrix is not present, the repair and regeneration of the tissue will not be successful because the damaged area and its surrounding cells will not adhere, proliferate or differentiate rapidly. . Therefore, when a living tissue is largely deficient, an extracellular matrix must be provided from the outside to the target tissue that is the deficient site. Therefore, what is needed as a substitute for the extracellular matrix is biomaterial that has an affinity for organisms.
  • Bioactive proteins such as cell growth factors and cytokines that promote cell growth and differentiation play an extremely important role in tissue repair and regeneration.
  • Human basic fibroblast growth factor (bFGF) acts at a low concentration on not only fibroblasts but also a wide range of cells such as vascular endothelial cells, osteoblasts, and nerve cells to promote tissue repair and regeneration. Therefore, an attempt has been made to supply these physiologically active proteins from the outside to the damaged site to repair and accelerate regeneration. However, if the supplied bioactive protein is at a low concentration in the target tissue, tissue repair / regeneration does not proceed quickly.
  • Non-patent Document 1 When an aqueous solution of bFGF is administered to a target tissue, about half of the dose administered in only 4 hours is lost due to diffusion (Non-patent Document 1).
  • Silk hive mouth-in is the main protein that constitutes about 70% of silk thread spouted by silkworms.
  • Silk fiber mouth-in is a natural material and has been used as a material that can be implanted into the body of surgical sutures for many years because it has excellent operability and biocompatibility. .
  • silk fiproin has been liquefied in water and methods for producing films and gels using it have been developed, and the potential for use as new biomaterials is expanding.
  • Non-patent Document 2 a support for cell culture using a silk-fibre mouth-in film
  • Patent Document 3 wound skin materials
  • Patent Document 3 contact lenses
  • Non-patent Document 3 silk fibroin films have been shown to be digested by protease XIV collagenase IA under physiological conditions. It has been suggested that mouth-in has biocompatibility and biodegradability.
  • Non-patent document 4 integrin recognition sequence (RGD) (Non-patent document 4) and ⁇ -galactose residue (Non-patent document 5) specifically recognized by hepatocyte surface
  • RGD integrin recognition sequence
  • Non-patent document 5 ⁇ -galactose residue
  • Non-Patent Document 6 As a material that promotes tissue repair and regeneration by immobilizing bioactive proteins such as cell growth factors and cytokines that promote cell growth and differentiation to biomaterials such as chitosan film through chemical covalent bonds Attempts to use it have been made (Non-Patent Document 6). At this time, purified recombinant protein derived from E. coli or insect cells is used as the physiologically active protein used for immobilization. Therefore, a biomaterial in which a physiologically active protein is immobilized cannot be produced unless complicated steps such as expression, purification, and immobilization of the recombinant protein are performed. In addition, in order for this immobilized bioactive protein to exhibit high biological activity, it is necessary to immobilize a relatively large amount of recombinant protein.
  • the quantitative problem of the physiologically active protein to be immobilized is the biggest problem in producing a biomaterial containing a physiologically active protein. This is because in the process of chemical immobilization, the biological activity of the bioactive protein decreases due to heating or contact with an organic solvent.
  • Transgenic silkworm The applicants of the present invention have invented and have applied for a patent for a transgenic silkworm that produces recombinant human collagen as part of a silk protein (Patent Document 4). In addition, a method for producing a recombinant cytokine using a transgenic silkworm has been devised (Patent Document 5). Thus, silkworms are attracting attention as host animals for producing recombinant proteins.
  • Patent Document 1 Japanese Patent Laid-Open No. 1-243948
  • Patent Document 2 JP-A-11-70160
  • Patent Document 3 Japanese Patent Laid-Open No. 1-52303
  • Patent Document 4 Japanese Patent Laid-Open No. 2004-16144
  • Patent Document 5 Japanese Patent Laid-Open No. 2003-325188
  • Non-Patent Document 1 J. Biomed. Mater. Res. 64A, 177-181 (2003)
  • Non-Patent Document 2 J. Biomed. Mater. Res. 29, 1215-1221 (1995)
  • Non-Patent Document 3 Biomaterials 24, 357-365 (2003)
  • Non-Patent Document 4 J. Biomed. Mater. Res. 54, 139-148 (2001)
  • Non-Patent Document 5 Biomaterials 25, 1 13 1- 1 140 (2004)
  • Non-Patent Document 6 J Biomed. Mater. Res. 64Ai l), 177-181 (2003)
  • biomaterials to which bioactive proteins are immobilized are large as medical materials due to two effects such as “bioactivity” of the protein and “biocompatibility” of the biomaterial. It has potential.
  • bioactive proteins and biomaterials were prepared separately and combined to create them.
  • Another problem is that the activity of the immobilized physiologically active protein is decreasing.
  • the invention of this application has been made in view of the problems of the prior art as described above, and is a novel material that can easily obtain a biomaterial containing a biologically active protein that retains activity and function. The problem is to provide means.
  • This application is obtained from a transgenic silkworm having a fusion polynucleotide encoding a fusion protein of silk fibroin and a physiologically active protein in the genome as a first invention for solving the above-mentioned problems.
  • a bioactive biomaterial mainly composed of a recombinant silk protein containing the fusion protein.
  • this application is a recombinant silk protein obtained from a transgenic silkworm that possesses a fusion polynucleotide encoding a fusion protein of silk fiber mouth-in and a physiologically active protein in the genome. From the above, a bioactive biomaterial from which one or more protein components other than the fusion protein are removed is provided.
  • a transgene which has a fusion polynucleotide encoding a fusion protein of silk fiber mouth-in and a physiologically active protein in its genome, and produces a recombinant silk protein containing the fusion protein.
  • the physiologically active protein is a human fibroblast growth factor.
  • This application provides, as a fourth invention, a silk thread or a protein in the silk line produced by the transgenic silkworm of the third invention.
  • This application provides, as a fifth invention, a bioactive biomaterial solution obtained by treating a silk thread produced by the transgenic silkworm of the third invention with a solubilizing agent.
  • This application provides, as a sixth invention, a method for regenerating the physiological activity of the bioactive biomaterial solution of the fifth invention, wherein the bioactive biomaterial solution is treated with a denaturant, and then in the bioactive biomaterial solution. There is provided a method characterized by reducing the concentration of the denaturant.
  • This application provides, as a seventh invention, a bioactive biomaterial processed product molded using the bioactive biomaterial of the first invention or the second invention.
  • physiologically active protein means a protein having at least one known function that acts on physiological functions of animals and plants including humans.
  • Fusion protein means that the silk fiber mouth-in and the physiologically active protein are physically fused and do not separate by operation in the normal use range.
  • the fusion protein in the present invention also means that the silk fiber mouth-in and the physiologically active protein are bound in a state that retains the function and activity corresponding to each purpose of use.
  • “recombinant silk protein including fusion protein” specifically means a protein component of silk produced by transgene silkworm or a protein in the lumen of silk gland.
  • the term “main component” means, for example, silk thread or silk thread, as long as the function of the bioactive biomaterial of the present invention is substantially carried by the fusion protein contained in the recombinant silk protein. It means that a small amount of reagent or the like used for separating the protein component from the glandular lumen may remain.
  • a bioactive biomaterial containing a fusion protein of silk hive mouth-in and a bioactive protein can be obtained as a recombinant silk protein produced by a transgene silkworm. Therefore, in the conventional method, the operations for securing and immobilizing individual purified recombinant proteins, which were necessary for immobilizing the bioactive protein to silk fibroin, are omitted. Furthermore, since no heating or treatment with an organic solvent is required, the activity of the bioactive protein is not impaired.
  • FIG. 1 shows the expression of a fusion protein between silk hive mouth-in and human bFGF in silkworm silk of Transgenic silkworm.
  • Figure A shows the result of Kumashi staining of SDS polyacrylamide gel developed from silk-five mouth-in samples prepared from silkworms of wild-type silkworm and transgenic silkworm.
  • Figure B shows the results of immunoblot analysis of the nitrocellulose membrane to which the protein developed on SDS polyacrylamide gel was transferred with anti-human bFGF polyclonal antibody.
  • Lane M contains a protein marker
  • Lane W contains a silkworm silk-in sample of a wild-type silkworm
  • Lanes 1 and 2 contain silky silkworms of different transgenic silkworms.
  • FIG. 1 shows the biological activity of silk hive mouth-in solution containing human bFGF prepared from silkworm silk of transgenic silkworm. It is the result of investigating the biological activity of silk fibroin solution containing human bFGF quantitatively using WST-1 method. Below each column of the graph is the concentration of human bFGF in the silk hive mouth-in solution added to the medium.
  • the column shows 100% growth of human umbilical vascular endothelial cells when cultured in normal medium (containing 5 ng / ml human recombinant bFGF), and human umbilical vascular endothelial cells in medium without bFGF.
  • the figure shows the relative growth of the cells when the cells are cultured with the silk fib-in solution added to the medium without bFGF, assuming that the growth of the cells when cultured is 0%.
  • proliferation of umbilical cord vascular endothelial cells by human bFGF in silk-fibre mouth-in solution was similarly performed by adding human recombinant bFGF expressed in E.
  • FIG. 4 is a structural diagram of the vector pL-bFGF prepared in the example.
  • DsRed red fluorescent protein
  • a polynucleotide encoding a fusion protein of silk fibroin and bFGF is incorporated.
  • Transgenic silkworms can be produced by introducing a polynucleotide encoding a fusion protein of a physiologically active protein and silk fib-in into the silkworm chromosome.
  • Silk hive mouth-in is a protein complex in which a hive chain in H chain with a molecular weight of 350 kDa and a hive in L chain with a molecular weight of 25 kDa are associated, and the polynucleotide encoding them is known (the hive in H chain : GenBank Accession No. AF226688; Five mouth in L chain: GenBank Accession No. M76430).
  • a polynucleotide encoding a biologically active protein is linked to either a polynucleotide encoding a hive mouth-in H chain or a hive mouth-in L chain.
  • the ligation position of the polynucleotide encoding the biologically active protein may be 5 ′ side or 3 ′ J of the polynucleotide encoding the five mouth in H chain or fiproin L chain.
  • the polynucleotides may be directly linked or indirectly linked via a linker.
  • the polynucleotide encoding the hive mouth-in H chain or the hive mouth-in L chain may be a sequence encoding the full length of each protein or a partial sequence.
  • Bioactive proteins fused with silk hive mouth-in refer to a group of proteins that exhibit various physiological activities on cells.
  • FGF fibroblast growth factor
  • MBP bone morphogenetic factor
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • TGF insulin-like growth factor
  • IGF insulin-like growth factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • NNF nerve growth factor
  • interleukins This refers to cytosines including IDs, erythropoietin, colony stimulating factor (CSF), tumor necrosis factor (TNF), etc.
  • FGF Fibroblast growth factor
  • FGF-3 Fibroblast growth factor-3
  • X14445, FGF-4 J02986, FGF-5; M37825, FGF-6; X63454, FGF-7; M60828, FGF-8 AH006649, FGF-9; D 14838, FGF-10; AF411527, FGF-11; AY049782, FGF-12; U66197, FGF-13; U66198, FGF-14; U66200, FGF-16; AB009391, FGF-17; AB009249, FGF-18; AF075292, FGF-19; AFl 10400, FGF-20; AB044277, FGF-21; AY359086, FGF-22; AB021925, FGF-23; AF263537, etc. Osteogenic factors
  • BMP (Bone morphogenetic protein) -1; M22488, BMP-2; M22489, BMP-3; M22491, BMP-4; U43842, BMP-5; M60314, BMP-6; M60315, BMP-7; X51801, BMP-8B M97016, BMP-9; AF188285, BMP-10; AF101441, BMP-11; AFl 00907, etc.
  • EGF Epidermal growth factor
  • EGF Epidermal Growth Factor
  • X04571 X04571, etc.
  • PDGF Plate-derived growth factor
  • PDGF-A PDGF (Platelet-derived growth factor)
  • PDGF-B PDGF-B
  • TGF Transforming growth factor
  • TGF Transforming growth factor
  • IGF Insulin-like growth factor
  • IGF Insulin-like growth factor
  • HGF Hepatocyte growth factor
  • HGF Hepatocyte growth factor
  • VEGF Vascular endothelial growth factor
  • VEGF Vascular endothelial growth factor
  • M32977 VEGF-B
  • U52819 VEGF- C
  • X94216 VEGF-D
  • AJ000185 PIGF (Placenta growth factor); X54936, etc.
  • NGF Nema growth factor
  • X52599BDNF Brain-derived neurotrophic factor
  • Interleukin (IL) class class 1
  • IL Interleukin-1 alpha; M28983, IL-1 beta; M 15330, IL-2; U25676, I 3; M14743, IL-4; M13982, IL-5; X04688, IL-6; M29150, L- 7; J04156, IL-8; M26383, IL-9; AF361105, IL-10; M57627, IL-11; M57765, IL-12A; M65291, IL-12B; M65290, IL-13; L06801, IL-15; U14407, IL-16; M90391, IL-17; U32659, IL-18; AY044641, IL-19; AY040367, IL-20; AF212311, IL-21; AF254069, IL-22; AF279437, etc. )
  • EPO Errythropoietin
  • Ml 1319 Ml 1319, etc.
  • GM Granulocyte macrophage
  • M 1220 G (Granulocyte) -CSF
  • G G (Granulocyte) -CSF
  • M 17706 M (Macrophage) -CSF
  • M27087 etc.
  • Tumor necrosis factor (TNF) TNF
  • the polynucleotide and the polynucleotide encoding the bioactive protein are linked by genetic engineering techniques.
  • the polynucleotide encoding the fusion protein may be a fib mouth in H chain, L chain or P25. It needs to be linked downstream of the silk protein gene-derived promo. In order to increase the transcriptional activity of these promoters, an enhancer may be further linked.
  • the enhancer may be derived from a silk protein gene or from a protein other than a silk protein gene. Moreover, it may be derived from silkworms, or may be derived from other animals or viruses. In this way, a fusion protein expression cassette is constructed. This expression cassette is then inserted into a vector to create a transgenic silkworm. Any vector may be used as long as it can insert a foreign gene into the silkworm chromosome. For example, a vector prepared based on a DNA-type transposon can be used. is there. So far, DNA-type transposons that have been shown to have gene transfer activity into the silkworm chromosome include piggyBac and Mariner> Minos.
  • the vector using piggyBac is actually transgeneic silkworm by Tamura et al. (Nat. Biotechnol. 18, 81-84 (2000)) Nyota (Nat. Biotechnol. 21, 52-56 (2003)) et al. Is used to create Use a vector created based on DiggyBac In this case, for example, a method similar to the method of Tamura et al. (Nat. Biotechnol. 18, 81-84, 2000) may be used.
  • a pair of inverted repetitive sequences of piggyBac are incorporated into an appropriate plasmid vector, and a polynucleotide encoding a fusion protein and a promoter are inserted into a region sandwiched between the inverted repetitive sequences in this vector.
  • This plasmid vector is microinjected into silkworm eggs together with a piggyBac transposase expression vector (helper plasmid).
  • helper plasmid is a recombinant plasmid vector lacking one or both of the piggyBac inverted repeats and essentially incorporating only the piggyBac transposase gene region.
  • the promoter for expressing the transposase may be the endogenous transposase promoter, or the silkworm / actin promoter or the Drosophila HSP70 promoter. May be used.
  • the marker gene can be incorporated at the same time in the vector in which the polynucleotide to be inserted is incorporated.
  • a promoter sequence such as a silkworm-actin promoter or a Drosophila HSP70 promoter is incorporated upstream of the marker gene, and the marker gene is expressed by its action. As described above, F0 larvae hatched from silkworm eggs microinjected with Vector 1 are raised.
  • This F0 silkworm is crossed with a sibling or wild-type silkworm, and a transgenic silkworm is screened from the next-generation (F1 generation) silkworm.
  • the marker gene is integrated in the vector, screening is performed using the phenotype.
  • screening can be performed by irradiating F1 generation silkworm eggs and larvae with excitation light and detecting the fluorescence emitted by the fluorescent protein.
  • PCR can be screened by Southern blotting using genomic DNA extracted from larvae and silkworm pupae.
  • transgenic silkworm the polynucleotide encoding the fusion protein is stably integrated into the chromosome and is not lost even in its offspring.
  • Transgenic silkworms that have a polynucleotide encoding a fusion protein of silk fiproin and a bioactive protein synthesize a recombinant fusion protein in the silk gland at the age of five. Since this recombinant fusion protein has a part of the amino acid sequence of silk fiber mouth-in, it forms a complex with the endogenous silk protein, and the silk gland lumen from the silk gland cells together with the endogenous silk protein. Secreted into the body.
  • Liquid silk fibroin containing recombinant fusion protein secreted into the lumen becomes silk thread when spited, and silkworms form cocoons with the silk thread.
  • the bioactive biomaterial (recombinant silk protein) of the present invention is recovered from the liquid silk protein accumulated in the silk gland lumen, it can be obtained, for example, by the following method. Cut the body of Transgenic silkworm the day before spitting silk thread, take out the silk gland, and cut and collect only the rear silk gland. The collected posterior silk gland is cut into pieces of about 5 mm and soaked in distilled water for about 4 hours. Thereafter, tissue pieces of the posterior silk gland are removed by tweezers or removed by light centrifugation.
  • a recombinant silk protein (bioactive biomaterial) containing a fusion protein of a bioactive protein and a silk fiber mouth can be prepared as an aqueous solution.
  • the silk thread spun by Transgenic Silkworm itself becomes a bioactive biomaterial.
  • the silk thread of Transgenic silkworm can be made into a bioactive biomaterial solution (for example, an aqueous solution) by treating the silk thread with a solubilizing agent, for example.
  • chaotropic salts such as 9 M lithium thiocyanate and lithium bromide
  • (Ratio 1: 8: 2) (Nissan 6 1 2, 91 -94 (1998)) can be used.
  • the silk thread is solubilized in these solubilizers by soaking for 10 to 48 hours, preferably 18 to 36 hours, particularly preferably about 24 hours.
  • this aqueous solution can be prepared as a bioactive biomaterial aqueous solution by dialysis with distilled water or the like.
  • bioactive biomaterial solution In the process of isolating silk fiber lumen protein (bioactive biomaterial solution) or in the process of preparing bioactive biomaterial solution from silk thread, the bioactive protein is denatured and its biological activity is lost or reduced.
  • a bioactive biomaterial solution is treated with a denaturing agent, and then the biological activity of the bioactive protein is regenerated by reducing the concentration of the denaturing agent in the bioactive biomaterial solution, thereby A physiologically active biomaterial aqueous solution containing a physiologically active protein can also be obtained.
  • a silk protein solution from a liquid silk protein or silk thread is dissolved in a buffer solution containing a denaturing agent (for example, high-concentration urea-guanidine hydrochloride, etc.) to make a completely denatured silk protein solution.
  • a denaturing agent for example, high-concentration urea-guanidine hydrochloride, etc.
  • dialysis and dilution are performed stepwise to denature the silk protein solution. Decrease the concentration of the agent little by little. That is, the final concentration of the modifier is 5% or less, preferably 3% or less, particularly preferably 1% or less.
  • the biologically active protein in the silk protein solution is refolded from a completely denatured state to the correct body structure, so that the biological activity of the biologically active protein is regenerated.
  • the above method is an example for regenerating a physiologically active protein, and the optimum conditions for efficiently regenerating a physiologically active protein vary depending on the type of the physiologically active protein. Therefore, in order to obtain a bioactive biomaterial solution containing an active recombinant fusion protein, it is important to correctly and efficiently regenerate various bioactive proteins under optimal conditions. For example, the regeneration buffer used for dialysis and dilution needs to be carefully examined in terms of pH, temperature, ionic strength, presence of reducing agent, etc., depending on the type of bioactive protein.
  • one or more protein components other than the fusion protein may be removed from the recombinant silk protein obtained as described above or a solution thereof.
  • silk thread produced by silkworm mainly contains fib mouth in, P25 and sericin.
  • the bioactive biomaterial of the present invention is a silk thread or silk wire tamper containing a physiologically active protein. Proteins other than fib mouth-in (P25 and Z or sericin) may be removed from the protein.
  • sericin is known to have antigenicity to the human body, it is preferable to remove sericin when the bioactive biomaterial is applied directly to the human body.
  • aqueous solution such as 0.3-0.5% aqueous sodium bicarbonate or soapy water, or an 8 M urea aqueous solution.
  • a method of heating at 80 at a protein denaturant aqueous solution or a method of digesting with an enzyme such as trypsin can be used.
  • the silky bioactive biomaterial obtained in this way can be used as a powder material, for example, as a fibrous material or by pulverization using a homogenizer or the like.
  • a liquid bioactive biomaterial obtained from the silk gland lumen or an aqueous bioactive biomaterial prepared from silk can be processed into a film, gel, or sponge.
  • a bioactive film can be produced by pouring a bioactive biomaterial solution onto a flat plate and allowing it to air dry. In order to produce a stronger film, the film may be air-dried and then immersed in 90% (v / v) methanol / distilled water.
  • films mainly composed of silk protein especially have adhesion and proliferation properties to fibroblasts and the like.
  • biomaterial films are highly functional films with added physiological activities such as the proliferation and differentiation induction of cells with various physiologically active proteins.
  • a bioactive biomaterial powder having an active bioactive protein function is prepared by desalting a bioactive biomaterial aqueous solution by dialysis against pure water and lyophilization. be able to.
  • the pH of the silk fiber mouth-in aqueous solution is made weakly acidic using a citrate buffer solution or the like, so that Biomaterial gels can also be made.
  • biologically active biomaterials it is not limited to these forms and methods, and various methods such as solution, sheet, gel, granule, and sponge can be used depending on the application. It can be processed into a shape.
  • Example 1 The following is an example of a method for producing a bioactive biomaterial containing active human basic fibroblast growth factor (bFGF) using transgene silkworm silk thread. The invention will be described in detail, but the invention of this application is not limited to this embodiment.
  • bFGF basic fibroblast growth factor
  • pL-bFGF a vector that expresses a fusion protein of silk hive-in and human bFGF.
  • the insert DNA contained in the pL-bFGF vector is composed of a silkworm silk fib-in L chain promoter, a silkworm silk fib in L chain cDNA, a human bFGF cDNA, and a silkworm silk fiproin L chain poly A addition signal sequence.
  • the silk-fibre-in L chain promoter is a pLE vector (Nat. Biotechnol. 2 1, 52-56 (2003)), and is a primer with a restriction enzyme EcoRI recognition sequence at the 5 'end.
  • the both ends of the obtained DNA fragment were cleaved with Pstl and BamHI and inserted between the Pstl site and BamHI site of pBluescriptIISK +
  • the cDNA encoding human bFGF is The DNA fragment containing the sequence corresponding to nucleotide number 495-935 (GeneBank database accession number NM-002006) was obtained by PCR using human fibroblast cDNA as a saddle type.
  • a cDNA encoding the fusion protein of bFGF and bFGF was prepared, and from this vector, the Pstl cytoplasm present at the 5 'end of the silk-fibre mouth-in L chain cDNA and the 3' end of human bFGF were present.
  • the silk protein in L chain and bFGF fusion protein cDNA was excised, and the above-mentioned silk fiber in L chain promoter was incorporated. Of over and inserted between the Pstl site and EcoRI site at the 3 'end of the silk Fuiburoi down L chain promoter.
  • Silkworm silk of this plasmid vector Using the EcoRI site located at the 5 'end of the hive mouth in light chain promotion and the 3' end of human bFGF, an insert DNA fragment consisting of the fibroin light chain promoter and the fusion protein cDNA was excised and pBac [3xP3- DsRed / pA] (Nat. Biotechnol. 21, 52-56 (2003)) was inserted into the EcoRI site upstream of the fiproin light chain poly A-added signal sequence. In this way, a plasmid vector for microinjection (pL-bFGF) was prepared (see FIG. 4).
  • the pL-bFGF vector prepared in Example 1 was purified by the cesium chloride density gradient method, and this vector and helper plasmid pHA3PIG (Nat. Biotechnol. 18, 81-84). , 2000), and after ethanol precipitation, injection buffer solution (0.5 mM phosphate buffer pH 7.0, 5 niM KC1) was added so that the concentrations of pL-bFGF and pHA3PIG were 200 pg / ml respectively. ). A small amount of this DNA solution was injected at a volume of about 15-20 nl per egg into silkworm eggs at the preblastoderm stage 2-8 hours after laying. A total of 2423 eggs were microinjected.
  • the silk thread produced by the transgenic silkworm obtained in Example 3 was pulverized using a homogenizer.
  • the ground silk thread is suspended in a trypsin solution (0.5 mg / ml aqueous solution of trypsin dissolved in 50 mM Tris-HCl buffer pH 7.6) at a concentration of 5 mg / ml, and the silk thread is shaken.
  • the silk sericin contained in the rice was erased. Thereafter, the silk fiber mouth-in was recovered by centrifugation, and the digested silk sericin was removed.
  • the collected silk fiber mouth-in is washed thoroughly with a neutral buffer and then suspended in a mixed solution of 9 M lithium thiocyanate and 5% ⁇ -mercaptoethanol at a concentration of 5 mg / ml at 4 ° C. Dissolved by shaking. Then, the silk fiproin solution was recovered by removing the undissolved silk hive mouth in by centrifugation. This silk hive mouth-in solution was mixed with SDS-sample buffer, and the proteins contained in the solution were spread by SDS polyacrylamide gel electrophoresis and stained with Kumashi ( Figure 1A). The protein developed on the gel was transferred to a nitrocellulose membrane (PROTRAN) by a conventional method.
  • PROTRAN nitrocellulose membrane
  • This nitrocellulose membrane was treated with a blocking solution (5% Skim milk / 50 mM Tris-HCl buffer pH 7.5, 150 mM NaCl) at 4 for 16 hours, and then TBS (50 mM Tris-HCl buffer pH 7.5, 150 mM). It was reacted with an anti-human bFGF polyclonal antibody (R & D Systems, AF-233-NA) diluted 200-fold with NaCl) solution at room temperature for about 2 hours. Next, it was reacted with a peroxidase-labeled anti-goat IgG polyclonal antibody (VECTOR, PI-9500) diluted 3000 times with a blocking solution at room temperature for about 1 hour.
  • a blocking solution 5% Skim milk / 50 mM Tris-HCl buffer pH 7.5, 150 mM NaCl
  • TBS 50 mM Tris-HCl buffer pH 7.5, 150 mM
  • 2x103 human umbilical vein endothelial cells were seeded in one well (diameter 6.4 mm) of a 96-well plate (Falcon) for tissue culture, and normal medium (2% wild fetal serum, 10 ng / ml EGF, lpg Vascular endothelial cell basal medium (Humedia-EB2 (KURABO)) containing 4 ml / ml hydrocortisone, 50 pg / ml gentamicin, 50 ng / ml amphotericin, lOpg / ml heparin, and 5 ng / ml human recombinant bFGF) for 4 hours Cultivated.
  • normal medium 2% wild fetal serum, 10 ng / ml EGF, lpg Vascular endothelial cell basal medium (Humedia-EB2 (KURABO)
  • the degree of growth per bFGF amount was equivalent to that of human recombinant bFGF expressed in E. coli used as a positive control. As described above, it was confirmed that the silk hive mouth-in solution containing bFGF prepared from silk has high biological activity.
  • silk fibroin solution prepared from silkworm silk of wild-type silkworm and silk-fibre mouth-in solution in which human recombinant bFGF expressed in Escherichia coli was mixed with silk-type mouth silk-in solution of wild-type silkworm were also used.
  • silk-fibre-in-film was prepared and seeded with human umbilical vein endothelial cells.
  • the silk-fibre mouth-in material containing bFGF is useful as a functional biomaterial with added biological activities such as cell proliferation and differentiation.
  • the invention of this application provides a bioactive biomaterial containing an active bioactive protein.
  • This bioactive biomaterial By processing this bioactive biomaterial into films, sheets, gels, sponges, fibers, etc., new biomaterials that can be used in industrial fields such as medical care are provided.

Abstract

Il est destiné à fournir un nouveau moyen par lequel un biomatériau contenant une protéine physiologiquement active tenant son activité et sa fonction et peut être peut obtenu convenablement. Ce biomatériau physiologiquement actif peut s’obtenir à partir d’un ver à soie portant dans son génome un polynucléotide fondu codant une protéine fondue de soie fibroïne avec la protéine physiologiquement active et qui contient une protéine de soie recombiné contenant la protéine fondue ci-dessus décrite comme principal composant.
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