KR100489731B1 - Pharmaceutical compositions containing domains of fibronectin and βig-h3 for wound healing, cell adhesion, migration and proliferation - Google Patents

Abstract

The present invention relates to a recombinant protein having a superior therapeutic effect including a partial domain of fibronectin and βig-h3, and a pharmaceutical composition comprising the recombinant protein. More specifically, the 9th and 10th type III domains of fibronectin and The present invention relates to a recombinant protein comprising a 4 th fas-1 domain of βig-h3 and a pharmaceutical composition for wound treatment containing the recombinant protein as an active ingredient. The pharmaceutical composition for wound treatment of the present invention has an excellent effect on the treatment of wounds by the action of re-epithelialization, angiogenesis, fibroblast proliferation and collagen formation, and can reduce the number of wound treatment as well as chronic wounds. It can be used for healing, and also useful for the purpose of promoting adhesion, migration and proliferation of cells.

Description

Pharmaceutical compositions containing domains of fibronectin and βig-h3 for wound healing, cell adhesion, migration and proliferation}

The present invention relates to a pharmaceutical composition for promoting wound healing, cell adhesion, migration and proliferation, and more particularly, to a recombinant protein comprising a partial domain of fibronectin and βig-h3, and a pharmaceutical composition comprising the recombinant protein. will be.

In recent years, the study of wound healing has been further subdivided into the fields of cell biology and molecular biology, and the clinical application of wound healing promotion is becoming more widespread, but there are still many questions about microcellular and molecular biology mechanisms. To date, wound healing is a tissue response to injury and is a process of tissue repair, including chemotaxis, differentiation and replication of cells, synthesis of matrix proteins, and Complex biological processes, including angiogenesis, reconstruction of wounds, etc. (Steed, DL et al ., Clin. Plast. Surg ., 1998 , 25, 397). The growth factor is one of the representative substances that appear early in the wound healing process and regulate subsequent processes. They have a strong effect throughout the wound healing process and regulate cell growth, differentiation and metabolism, Since the environment is also controlled, the development of therapeutics using the same is steadily progressing.

Current researches for wound treatment include platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), βig-h3, integrin and fibronectin.

βig-h3 is a cell adhesion molecule derived from TGF-β and has four repeat domains named fas-1. βig-h3 has a function of attaching various cells including human corneal epithelial (HCE) cells, fibroblasts, and the like, and can mediate cell attachment only with each fas-1 domain. As in HCE cells, most epithelial cells show fas-1 domains 2 and 4 and all four fas-1 domains in fibroblasts. Thus, fas-1 domains 2 and 4 have a function of mediating cell adhesion of both epithelial and connective tissue cells.

Cell surface adhesion receptors, called integrins, play an important role in cell adhesion.Integrins interact with extracellular matrix to regulate cell growth, differentiation, death, and development, and to Respond. One of the best known extracellular substrates that interact with integrins to regulate cell function is fibronectin. Fibronectin is a relatively large protein with a molecular weight of about 220 kDa and has several motifs, including the RGD motif, which interact with various integrins to regulate cell function (Hynes RO, Arterioscler Thromb Vasc Biol ., 1998 , 18, 1363-1370). ). Among the motifs that interact with the integrins of fibronectin, the most important cell adhesion motif, RGD, is in the type III 10th domain, and the PHSRN motif, which is known to function as a cell adhesion with RGD, is in the type III 9th domain (Richard P. Grant, The Journal of Biological Chemistry , 1997 , 272 (10), 6159-6166).

As described above, both fibronectin and βig-h3 are expected to have a wound healing effect. In the past, the inventors of the present invention prepared a protein obtained by repeating four times the fas-1 domain of βig-h3 four times and found that the recombinant protein has an effect on wound healing (patent application 2000- 25663).

Accordingly, the present inventors have combined the 9th and 10th type III domains containing PHSRN and RGD, the main motifs of fibronectin, and the 4th fas-1 domain of βig-h3, in order to produce a recombinant protein having a superior wound healing effect. The recombinant protein was prepared, and the present invention was completed by revealing that the recombinant protein is superior to fibronectin and βig-h3 in terms of cell adhesion, proliferation, migration, and proliferation and wound healing effects.

Disclosure of Invention An object of the present invention is to provide a recombinant protein having excellent wound healing effect in which fibronectin and βig-h3 partial domains are combined, and to provide a pharmaceutical composition for wound treatment containing the recombinant protein as an active ingredient.

In order to achieve the above object, the present invention provides a recombinant protein comprising a 9 and 10 type III domain of fibronectin and a 4 fas-1 domain of βig-h3.

The present invention also provides a gene encoding the recombinant protein and an expression vector including the gene.

The present invention also provides a pharmaceutical composition for wound treatment containing the recombinant protein as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant protein comprising the 9 and 10 type III domains of fibronectin and the 4 fas-1 domain of βig-h3.

In the present invention, in order to produce a recombinant protein having a very good wound healing effect, the recombinant protein combining the 9 and 10 type III domains containing PHSRN and RGD, which are the main fibronectin motifs, and the 4 fas-1 domain of βig-h3. Was prepared. In the present invention, the 9 and 10 type III domains of fibronectin comprise an amino acid sequence represented by SEQ ID NO: 2 , and the fas-1 domain 4 of βig-h3 comprises an amino acid sequence represented by SEQ ID NO: 4 .

The recombinant protein of the present invention is preferably a recombinant protein comprising the amino acid sequence described in SEQ ID NO: 2 and the amino acid sequence described in SEQ ID NO: 4 , and more preferably comprises the amino acid sequence described in SEQ ID NO: 6 .

The group treated with the recombinant protein of the present invention was assigned to fibronectin, 115 protein corresponding to No. 10 type III domain of fibronectin, 4 fas-1 domain of βig-h3 and βig-h3, which are known to be effective for conventional wound treatment. Comparing the cell attachment, spreading, cell proliferation and cell migration with the corresponding βig-h3D-IV group, fibroblasts showed high adhesion activity similar to fibronectin, and showed better adhesion activity than other proteins. , HaCaT cells showed better cell adhesion and cell spreading activity than fibronectin as well as other proteins (see FIG. 3 ). The results were similar in human skin epithelial cells, CHO, lung fibroblasts and mouse fibroblasts (see FIG. 4 ). In addition, the proliferative effect of fibroblasts showed the same effect as fibronectin or better than other proteins (see FIG. 5 ), and the cell migration activity was found to be the best (see FIG. 6 ).

Recombinant proteins comprising the 9 and 10 type III domains of the fibronectin of the present invention and the 4 fas-1 domain of βig-h3 exhibit excellent effects on cell adhesion, spread, cell proliferation and cell migration. Recombinant protein may be usefully used for wound healing.

The present invention also provides a gene encoding a recombinant protein excellent in the wound healing effect and an expression vector containing the gene.

The gene of the present invention may be a nucleotide sequence described in SEQ ID NO: 1 encoding an amino acid sequence described in SEQ ID NO: 2 and a nucleotide sequence described in SEQ ID NO: 3 encoding an amino acid sequence described in SEQ ID NO: 4 , SEQ ID NO: It is preferable that it is a gene having a nucleotide sequence described as 5 .

The present invention also provides an expression vector comprising the gene encoding the recombinant protein and expressing the recombinant protein of the present invention.

The expression vector of the present invention is preferably an expression vector containing a nucleotide sequence set forth in SEQ ID NO: 5 and expressing a recombinant protein described in SEQ ID NO: 6 . In a preferred embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 5 was inserted into a pET expression vector (Novagen, USA), which was named "pET T-CAM".

The present invention provides a pharmaceutical composition for wound treatment containing a recombinant protein comprising the 9th and 10th type III domains of fibronectin and the 4th fas-1 domain of βig-h3 as an active ingredient.

In the present invention, the 9 and 10 type III domains of fibronectin may have an amino acid sequence represented by SEQ ID NO: 2 , and the fas-1 domain 4 of βig-h3 may be an amino acid sequence represented by SEQ ID NO: 4 . The recombinant protein of the present invention is preferably a recombinant protein comprising the amino acid sequence described in SEQ ID NO: 2 and the amino acid sequence described in SEQ ID NO: 4 , and more preferably comprises the amino acid sequence described in SEQ ID NO: 6 .

The recombinant protein of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the recombinant protein of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used, or Formulated using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, water, or the like in the recombinant protein of the present invention. Prepared with a mixture of sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . For parenteral administration, it may be administered as an external drug, cream, gel, ointment, spray, spray, suspension, etc., and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. Or using excipients. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized preparations. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, ethyl oleate and the like can be used.

In particular, the pharmaceutical composition containing the recombinant protein of the present invention as an active ingredient may be prepared as a ointment or chitosan mixed composition.

The ointment may be prepared by dissolving the recombinant protein in phosphate buffer and then mixing the ointment with a final concentration of 10 to 5,000 μg / ml. As a result of visually confirming that the ointment was applied to the rabbit causing the wound, the group treated with the recombinant protein of the present invention contained 115 protein corresponding to No. 10 and Type III domain of fibronectin, which is known to be effective for conventional wound treatment. It was confirmed that the wound contraction rate and wound healing rate were higher than those of the group treated with βig-h3D-IV (D-IV) corresponding to the fourth fas-1 domain of βig-h3 (see FIGS . 8 and 10 ). Further, histological analysis result performed, in the group treated with the recombinant protein of the present invention multi-core sphere and the infiltration of inflammatory cells around the lymphocytes such as obtained for the reduced result as compared to the group treated with other proteins (Figure 11 and Table reference 1), it was observed excellent angiogenesis (see Fig. 12 and Table 2), proliferation of fibroblasts were significantly increased (see Table 3). In addition, the group treated with the recombinant protein of the present invention was observed that collagen formation was increased and the collagen morphology was better organized than other groups (see FIG. 13 and Table 4 ), and the degree of completion of epithelial regeneration was excellent ( FIG. 14 ).

When the recombinant protein of the present invention is used in wound healing, an excellent effect can be obtained in comparison with existing known proteins in promoting wound healing. On the other hand, fibronectin is difficult to separate from plasma, while the recombinant protein of the present invention can be produced in large quantities in Escherichia coli through genetic engineering, which is much superior in its production or convenience of use. Therefore, the pharmaceutical composition containing the recombinant protein of the present invention as an active ingredient can be usefully used for the treatment of wounds.

In addition, in the present invention, in order to enhance the wound healing effect using the recombinant protein of the present invention, chitosan mixed composition including chitosan as an active ingredient may be prepared and used. The chitosan mixed composition is prepared by dissolving a water-soluble chitosan (Poly (1 → 4) 2-amino-2-deoxy-β-D-glucan) in sterile distilled water to make a 1% solution, and then adding gentamycin to the present invention. Recombinant protein was added at a concentration of 10 to 5,000 ㎍ / ㎖, and then frozen at -70 ℃ and lyophilized for about 12 hours in a freeze drier (Il-Shin, Korea) to prepare a disk-like patch form can do.

To date, many biomaterials have been developed for the purpose of healing wounds. The chitosan used in the present invention is a chitin, a natural polymer that is abundant on the earth, such as shells, insects, mollusks, fungi, yeasts, and mushrooms. It is a material obtained by deacetylation of raw materials such as anionic agents for the treatment of pollutants, agricultural materials, food additives, anticoagulants, antithrombotic agents, hemostatic agents, cosmetic materials, textiles, paper, films, sponges, raw materials such as analytical reagents, It has been used as a material for chromatography (Muzzarelli, R., 1st ed, New York, Marcel Dekker, p179, 1994; US Patent 2,040,880; US Patent 2,040,879; Hirano, S., Biotechnol. Annu. Rev., 1996 , 2 , 237). There have been several experimental and clinical reports since Hoffmeister et al. (Hoffmeister, FS et al. , Surg ., 1964 , 56, 1129,) have known the effects of wound healing on chitosan, but the exact mechanisms have not been determined. Clinical application is expected to be a good wound healing agent due to microbiality, biocompatibility, absorption of exudate, etc. (Kratz, G. et al ., Scand. J. Plast. Reconstr. Hand. Surg., 1998 , 32, 381; Hoekstra, A. et al ., Biomaterials, 1998 , 19, 1467).

In the present invention, it is possible to obtain the effect of reducing the number of treatments and promoting wound healing by using the complex containing the chitosan as a base and the recombinant protein of the present invention. Chitosan is slowly dissolved in the wound, and the protein is slowly secreted by using the property so that the efficacy can be maintained for a long time than one application.

The pharmaceutical composition of the present invention may vary the content of the active ingredient included according to the extent and area of the wound, but in general, the recombinant protein of the present invention is 10 ㎍ to 10 mg per 1 cm 2 of wound area at a single administration. At an effective dose, chitosan can be administered repeatedly several times a day at an effective dose comprising at least 0.1% on the formulation. On the other hand, the pharmaceutical composition containing the recombinant protein of the present invention did not show acute toxicity.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of a Vector Containing Type III Domains 9 and 10 of Fibronectin

We have constructed vectors comprising domains of type III 9 and 10 of fibronectin. To amplify the 552 bp segment of the 1513 specifically from amino acid 1330 of fibronectin cDNA, was carried out PCR (Polymerase Chain Reation) using a reverse primer which is described by the forward primer and SEQ ID NO: 8 which is described in SEQ ID NO: 7. PCR was performed by adding 100 ng of template DNA, 30 pM of primer, 8 μl of 2.5mM dNTP, 10 units of pfu polymerase (Stratagene) and 10 × buffer and reacting for 5 minutes at 94 ° C. with a PCR machine. The reaction was repeated 25 times for 2 minutes at 72 ° C. for 3 minutes. 20 μl of the amplified product was added with 1 μl of restriction enzymes Nde I and Nsi I (TaKaRa) and 3 μl of 10 × buffer, respectively, and reacted at 37 ° C. for 1 hour. The restriction digested product was electrophoresed on agarose gel. After extracting the 552 bp fragment from the gel subjected to the electrophoresis, 17 µl of DNA, 10 µl of buffer, 2.5 µl, 2 µl of Klennow and 2 µl of 2.5 mM dNTP, to make the end of the fragment blunt. 25 µl of each was added and reacted for 15 minutes at 37 ° C to treat Klenow to prepare insert fragments. In addition, 1 μl of pET29b (+) vector (Novagen, USA) DNA was added to 2 μl of EcoRV (TaKaRa) and 10 × buffers, and a vector was prepared by restriction enzyme treatment at 37 ° C. with a total volume of 20 μl for 1 hour. 2 μl of T4 ligase (Gibco BRL) and 4 μl of 5 × buffer were added to 30 ng of the inserted fragment and 100 ng of the prepared fragment so that the total volume was 20 μl. The sections were ligated. 10 μl of the above ligation was added to 200 μl of E. coli strains BL21 (DE3), which is a type of Escherichia coli, and placed on ice for 30 minutes, and then thermally shocked at 42 ° C. for 90 seconds and 800 μl of LB-kanamycin 37 The cells were transformed by reacting at 1 ° C. for 1 hour, and the transformed cells were inoculated on LB-kanamycin medium plate. About 10 of the colonies formed on the inoculated medium plate were randomly selected and incubated overnight in LB-kanamycin medium, followed by minipreparation of the plasmids.

2 μl of the plasmid DNA was added to 1 μl of BamH I (TaKaRa) and 2 μl of 10 × buffer to a total volume of 20 μl, followed by reaction at 37 ° C. for restriction enzyme treatment. The restriction enzyme-treated cleavage product was electrophoresed to determine whether the correct size of the inserted DNA exists. These clones were each cultured in LB-kanamycin medium so as to have an absorbance of about 0.5 to 0.6 at 600 nm, and then IPTG was added to a final concentration of 1 mM and incubated at 37 ° C. for 3 hours. 1 ml of each colony was centrifuged for 5 minutes, the supernatant was removed, and 100 μl of 1 × SDS-PAGE gel application buffer was added to the cell precipitate and treated at 100 ° C. for 5 minutes. Samples obtained as described above were subjected to 15% SDS-PAGE and coomassie (coomassie) staining to select clones showing a protein band of about 29 kDa. The present inventors named the vector including the type III 9 and 10 domains of the fibronectin prepared as described above, and named the protein expressed in the pET 115 as "115".

Example 2 Preparation of Expression Vector Comprising No. 4 fas-1 Domain of βig-h3

The present inventors have previously reported about a recombinant protein comprising the 4th fas-1 domain of βig-h3 (Patent Application No. 2000-25663; Accession No. KCTC 18009P), and prepared a recombinant protein by the same method. . Briefly describing the method for producing the recombinant protein is as follows. In order to amplify the portion of amino acids 498 to 637 corresponding to the fourth fas-1 domain of βig-h3 cDNA, PCR was performed using the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10 . . PCR was performed by adding 100 ng of template DNA, 30 pM of primers, 8 μl of 2.5mM dNTP, 10 units of pfu polymerase (stratagene), 10 × buffer and reacting for 5 minutes at 94 ° C. with a PCR machine. The reaction was carried out 25 times for 2 minutes at 40 ° C. and 3 minutes at 72 ° C. At this time, in order to facilitate subcloning, it was designed to be cleaved with EcoR V and Xho I at both ends of the primer. Into 1 μl of the PCR reaction solution, EcoR V and Xho I, 2 μl of 10 × buffer were added to make the total volume 20 μl, followed by restriction enzyme treatment at 37 ° C. for 1 hour to prepare insert fragments. In addition, pET29b (+) (Novagen, USA) DNA was digested with the above restriction enzyme and extracted from the gel to prepare a vector. Thus prepared 30 ng and 100 ng of the vector was added 2 μl of T4 ligase (Gibco BRL) and 5 μl buffer 4 μl to make a total volume of 20 μl and reacted overnight at 16 ° C. for ligation. 10 µl of the ligation to 200 µl of E. coli strains BL21 (DE3) of Escherichia coli , and put on the ice for 30 minutes, transformed by heat shock at 42 ℃ for 90 seconds and transformed into 800 µl of LB-kanamycin 37 The reaction was carried out at 1 ° C. for 1 hour and then inoculated onto LB-kanamycin medium plate. About 10 of the colonies formed were randomly selected and incubated overnight in LB-kanamycin medium, followed by small-scale plasmid purification.

The plasmid DNA was digested with restriction enzyme Sph I, and electrophoresis was performed to determine whether clones of the correct size were present in LB-kanamycin medium at 600 nm with absorbances of about 0.5 to 0.6, respectively. It was added to a concentration of 1 mM and incubated at 37 ° C. for 3 hours. 1 ml of each colony was centrifuged for 5 minutes to remove the supernatant, and 100 μl of 1 × SDS-PAGE gel application buffer was added to the cell precipitate and treated at 100 ° C. for 5 minutes. Samples obtained above were subjected to 15% SDS-PAGE and Coomassie staining to select clones with a protein band of about 20 kDa. The present inventors named the vector including the 4th fas-1 domain of βig-h3 prepared as above as "pET βig-h3 D-IV", and expressed the protein expressed in the pET βig-h3 D-IV vector. It was named "βig-h3 D-IV".

Example 3 Preparation of T-CAM Expression Vector

The present inventors attempted to prepare a recombinant protein combining the type III 9 and 10 domains of fibronectin and the 4 fas-1 domain of βig-h3.

Specifically, 552 bp fragments corresponding to amino acids 1330 to 1513 of fibronectin cDNA 100 ng, the forward primer described in SEQ ID NO: 7 and the reverse primer described in SEQ ID NO: 8 30 pM, 2.5 mM dNTP 8 μl, pfu polymerase (stratagene, respectively) 10 units, 10 × buffer was added and reacted with a PCR machine at 94 ° C. for 5 minutes, amplified by performing 25 reactions for 1 minute at 94 ° C., 2 minutes at 40 ° C., and 3 minutes at 72 ° C. NlI, NsiI (TaKaRa), and 10µl buffer 2µl were added to 1µl of the amplified DNA to a total volume of 20µl and treated with restriction enzyme for 1 hour at 37 ° C. The cleaved DNA fragment was extracted from the gel after electrophoresis. Into the extracted DNA, 17 μl of 10 × buffer 2.5 μl, 2 μl of Klenow and 2 μl of 2.5 mM dNTP were added to make the total volume 25 μl and reacted at 37 ° C. for 15 minutes. (blunting) to prepare the insert section. In addition, 1 μl of pET βig-h3 D-IV prepared in Example 2 was added EcoR V (TaKaRa), 2 μl of 10 × buffers to a total volume of 20 μl, and the restriction enzyme treatment was performed at 37 ° C. for 1 hour. Vectors were prepared.

2 μl of T4 DNA ligase (Gibco BRL) and 4 μl of 5 × buffer were added to 100 ng of the prepared vector and 30 ng of the inserted fragment to make the total volume 20 μl and reacted overnight at 16 ° C. for the insertion fragment and the vector. After the connection, 10 μl of the ligated strain in Escherichia coli BL21 (E. coli strains BL21, DE3) was put in 200 μl, placed on ice for 30 minutes, and subjected to thermal shock at 42 ° C. for 90 seconds to add 800 μl of LB-kanamycin. The cells were transformed by reaction at 37 ° C. for 1 hour and inoculated in LB-kanamycin medium. About 10 of the colonies formed were randomly selected and incubated overnight in LB-kanamycin medium, followed by small-scale plasmid purification.

The plasmid DNA was limited to restriction enzyme Xho I and electrophoresis confirmed that the correct size of the inserted DNA was present. These clones were incubated in LB-kanamycin medium at 600 nm with absorbances of about 0.5 to 0.6, respectively, and then IPTG was added to a final concentration of 1 mM and incubated at 37 ° C. for 3 hours. 1 ml of each colony was centrifuged for 5 minutes, the supernatant was removed, and 100 μl of 1 × SDS-PAGE gel application buffer was added to the cell precipitate and treated at 100 ° C. for 5 minutes. Samples obtained above were subjected to 15% SDS-PAGE and Coomassie staining to select clones with a protein band of about 37 kDa. The inventors named the vector expressing a recombinant protein in which the type III 9 and 10 domains of the fibronectin prepared as described above and the 4 fas-1 domain of βig-h3 were referred to as "pET T-CAM". Recombinant proteins in which the type III 9 and 10 domains of fibronectin expressed in pET T-CAM and the 4 fas-1 domain of βig-h3 were bound to were named "T-CAM" ( FIG. 1 ).

Example 4 Expression and Purification of T-CAM Recombinant Protein

The present inventors expressed and purified the recombinant protein T-CAM in which the type III 9 and 10 domains of fibronectin expressed in the pET T-CAM prepared in Example 3 and the fas-1 domain of βig-h3 are bound. It was.

Specifically, protein purification was incubated in LB culture medium containing kanamycin 50 ㎍ / ㎖ 537 nm optical density of about 0.5 ~ 0.6 at 37 ℃. After expressing with 1 mM IPTG (Isopropyl β-D-thiogalactopyran oside, Sigma) for 3 hours at 37 ℃, E. coli were collected and dissolved in a lysis buffer and pulverized by an ultrasonic grinder. Then, after reacting with Ni-NTA resin (Qiagen) for 2 hours, the mixture was put in a column, and 5 times the binding volume of the resin (20 mM Tris-Cl, 500 mM NaCl, 5 mM immidazole) was added thereto. After washing with the same volume of wash buffer (20 mM Tris-Cl, 500 mM NaCl, 20 mM imidazole), the T-CAM protein bound to the column was 300 mM in elution buffer containing imidazole (20 mM Tris-Cl). Cl, 500 mM NaCl, 300 mM imidazole). The elution buffer was the same as the wash buffer except for the concentration of imidazole, and the amount of the elution buffer was 5 times the column volume. The eluted fraction was to be less than half the column volume. Thereafter, 15% SDS-PAGE was performed to determine the fraction in which the protein was present and used in combination. 1 and 2 are schematic diagrams of each protein and an electrophoretic photograph of each protein purified separately.

Example 5 Cell Culture

HaCaT (human keratinocyte cell line) was cultured in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 4.5 g / L glucose, 10% Fetal Bovine Serum (FBS), antibiotic penicillin G, streptomycin, and normal Normal human fibroblasts were cultured in DMEM medium supplemented with 4.5 g / L glucose, 10% Fetal Calf Serum (FCS), antibiotic penicillin G and streptomycin, and passages 3-6. Was used. NHEK (Normal human keratinocyte, normal human keratinocytes) was cultured in keratinocyte culture kit (KMK, Sigma) at 37 ℃ 5%, CO ₂ incubator, all experiments were used passage number 3 to 5 times. In addition, CHO (Chineses hamster ovary) cells were cultured in α-MEM (Minimum Essential Medium Alpha Medium) medium containing 10% FBS, antibiotic penicillin G, streptomycin, and MRC5 (Lung fibroblast, lung fiber). Blasts) cells were cultured in RPMI 1640 (RPMI medium 1640) medium supplemented with 10% FBS, penicillin G, streptomycin, and NIH3T3 (mouse fibroblast, mouse fibroblasts) with 4.5 g / L glucose, 10% FBS, penicillin G, streptomycin was incubated in DMEM medium.

Example 6 Cell Attachment and Spread Analysis

The inventors of the present invention, the T-CAM recombinant protein prepared in Example 3, fibronectin, βig-h3, βig-h3 D-IV prepared in Example 2 and 115 protein prepared in Example 1 is a skin epithelial cell ( The effects on the adhesion and spread of keratinocytes, HaCaT cells and fibroblasts, which are present in the dermis, were compared. Specifically, the cell adhesion assay (Cell adhesion assay) was put 100 ㎕ of each protein solution (10 ㎍ / ㎖) in 96-well ELISA plate (Costar) and reacted at 4 ℃ overnight. The proteins used were purified human plasma fibronectin (pFN), T-CAM, βig-h3 WT (normal βig-h3), βig-h3 D-IV, 115 proteins and negative control. Bovine serum albumin (BSA) (Sigma) was used.

Each protein-coated plate was washed twice with phosphate buffer salins (PBS) and then 100 μl of a 2% bovine serum albumin solution was reacted for 2 hours to block the surface of the non-protein coated plate. . Fibroblasts were placed in each well of 2.5 × 10 ⁴ cells and incubated at 37 ℃ for 25 minutes, HaCaT was incubated for 2 hours to put 3 × 10 ⁴ cells in each well. 3 x 10 ⁴ cells were also added to each well. CHO cells were cultured for 1 hour, MRC5 cells for 30 minutes, NHEK cells for 1 hour 30 minutes, and NIH3T3 cells for 40 minutes. After the cells were attached, each well was washed twice with 1 x phosphate buffer, followed by 50 containing 3.75 mM p-nitrophenyl-N-acetyl β-D-glycosamidase substrate and 25% Triton X-100. 60 μl of mM citrate buffer (pH 5.0) was added and reacted at 37 ° C. for 1 hour. Enzyme activity was stopped with 90 μl of 50 mM glycine containing 5 mM EDTA and the absorbance was measured at 405 nm using a Model 550 microplate reader (Bio-Rad Laboratories, Inc., USA). To measure cell area, cells were further incubated for two hours in 96-well plates, then fixed with 8% glutaraldehyde (Sigma) and 0.25% crystal violet (Crystal) containing 20% methanol (w / v). Violet, Sigma) solution. The area of cells was then measured using Image-Pro plus software (Media Cybernetics, Silver Spring MD). All experiments were repeated three times.

As a result, T-CAM showed high adhesion activity similar to fibronectin in fibroblasts, and 115 protein also showed high adhesion activity. Cell spread also showed a similar tendency to adhesion activity. In HaCaT cells, T-CAM had higher cell adhesion activity than fibronectin and 115 protein and also showed excellent cell spreading ( FIG. 3 ). C and D of FIG. 3 are attached for 2 hours in HaCaT cells and 20 minutes in fibroblasts. It was washed with phosphate buffer and fixed with 8% glutaaldehyde and then stained with crystal violet is shown in A and B. E and F are also calculated by Image-Proplus software.

This action is more pronounced in human primary cultured skin epithelial cells. As shown in Figure 4 T-CAM in NHEK (human skin epithelial cells) (C) showed superior cell adhesion activity than other proteins. T-CAM showed equal or better cell adhesion activity than fibronectin in CHO (hamster ovary cells) (A), MRC5 (pulmonary fibroblasts) (B), and NIH3T3 (mouse fibroblasts) (D) cells other than NHEK. ( FIG. 4 ).

Example 7 Cell Proliferation Assay

The present inventors and the fibroblasts by the T-CAM recombinant protein prepared in Example 3, fibronectin, βig-h3, βig-h3 D-IV prepared in Example 2 and 115 protein prepared in Example 1 and Cell proliferation of keratinocytes was compared.

Specifically, 500 μl of each protein at a concentration of 10 μg / ml in a 24-well plate was reacted at 4 ° C. overnight, washed twice with 1 × phosphate buffer, and added with 2% bovine serum albumin solution for 1 hour at room temperature. I was. Then, fibroblasts were put into 5 × 10 ³ cells, HaCaT was put into 2 wells × ⁴ × ⁴ cells in each well, incubated in serum-free medium for 24 hours, and then after 24, 48, 72, 96 hours Numbers were measured with a hemocytometer.

As a result, fibroblast proliferation effect of T-CAM was similar to fibronectin and superior to other proteins. In HaCaT cells, cell proliferation of T-CAM showed better cell proliferation than other proteins including fibronectin ( FIG. 5 ).

Example 8 Cell Migration Analysis

The present inventors the fibroblasts by the T-CAM recombinant protein prepared in Example 3, fibronectin, βig-h3, βig-h3 D-IV prepared in Example 2 and 115 protein prepared in Example 1 and Cell migration of keratinocytes was compared.

Specifically, the cell migration experiment was used a transwell plate (pore size 8 ㎛, Costar, Cambridge, MA). The bottom surface of the transwell was reacted overnight at 4 ° C. with 500 μl of each protein solution (10 μg / ml). Protein-coated transwells were washed twice with 1 x phosphate buffer and then reacted with 2% bovine serum albumin for 1 hour. 1 × 10 ⁴ fibroblasts and 1 × 10 ⁵ HaCaT were placed on the upper surface of the transwell and incubated for 2 hours (fibroblast) and 24 hours (HaCaT), respectively. After incubation, washed twice with 1 x phosphate buffer, fixed with 8% glutaraldehyde (Sigma), and stained with 0.25% crystal violet (Sigma) solution containing 20% methanol (w / v). Cell numbers were measured at nine randomly selected sites under HPF (Microscopic high power fields, x 200).

As shown in FIG. 6 , as a result of confirming cell migration after 2 hours in fibroblasts, all of the proteins except BSA showed good cell migration. In particular, the activities of T-CAM and fibronectin were excellent. In addition, after 24 hours of cell migration in HaCaT, the cell migration activity of T-CAM was found to be superior to other proteins.

Example 9 Wound Healing Analysis

The present inventors have wound healing effect by the T-CAM recombinant protein prepared in Example 3, fibronectin, βig-h3, βig-h3 D-IV prepared in Example 2 and 115 protein prepared in Example 1 Was compared. To this end, first, a water-soluble ointment (sama base) was prepared. The meat-based ointment is 38 mg of light lead per gram, 116 mg of stearyl alcohol, 38 mg of polyethylene glycol, 192 mg of concentrated glycerin, 23 mg of ethanol, sodium lauryl sulfate, 0.87 mg of paraoxybenzoic acid, 0.12 mg of paraoxybenzoic acid and It was prepared including purified water.

To compare the effect of wound healing, four New Zealand white rabbits were made of four round skin epidermal wounds with a diameter of 3 cm and divided into four groups according to the ointment contents applied to each wound. Group A uses ointment base only, Group B uses ointment containing fibronectin 115 protein at a concentration of 500 μg / ml, and Group C uses ointment containing βig-h3 D-IV at a concentration of 500 μg / ml, Group D was tested for 3 weeks by applying 2 g of ointment each mixed with 500 μg / ㎖ T-CAM a day.

<9-1> Visual Evaluation

35 rabbits out of 50 were drawn to the wound size on sterile OHP film at 0, 6, 12 and 18 days after visual evaluation for visual evaluation. Media Cybernetics, Silver Spring MD) was used to determine the wound area, wound shrinkage, wound epithelialization rate and wound healing rate according to wound healing as shown in Equation 1 below. The measured values were examined for significance using ANOVA test and Scheffe's test.

Wound Shrinkage = 100 × (<Wo-Wi> / Wo)

Wound epithelialization rate = 100 × (<Wi-Ui> / Wo)

Wound Healing Rate = 100 × (<Wo-Ui> / Wo)

Wo: Wound area on day 0 after procedure

Ui: Area of granulation tissue undetected on the date of measurement

Wi: Area of wound on measurement day

As a result, the wound area gradually decreased immediately after wound formation. The reduction of wound area was the fastest at 6 to 12 days, and the area reduction of the 115 protein, βig-h3-D-IV and T-CAM groups was faster than that of the control group treated with only ointment. Area reduction was the fastest in the -CAM treated group ( FIG. 7 ).

T-CAM showed higher wound shrinkage and wound healing rate than other groups. 115 days after treatment, 115 protein and T-CAM were significantly higher than the control group (p <0.05), and T-CAM was higher at 12 days. On the 18th day of the CAM group, the 115 protein, βig-h3-D-IV and T-CAM treated groups showed statistically significant (p <0.05) better results than the control group (*: p <0.05, **: p <0.01) ( FIG. 8 and FIG. 10 ) compared to control.

Wound epithelialization rate was higher than that of the other groups 115 days 6 days after the procedure, but overall did not show a significant difference between each protein treatment group and the control group ( Fig. 9 ).

<9-2> Histological Evaluation

Fifteen rabbits out of 50 were sacrificed on 3, 6, 10, 14 and 21 days after the procedure for histological evaluation, biopsied including the open window, and fixed in 10% neutral formalin solution and embedded in paraffin. Tissue sections were then made and stained with hematoxylin and eosin to examine the extent of inflammatory response, angiogenesis, distribution of fibroblasts, and collagen formation under the microscope.

<9-2-1> Infiltration of Inflammatory Cells such as Multinuclear Cells and Lymphocytes

Inflammatory cell infiltration was observed in all experimental groups from day 3, and on day 3 after the procedure, inflammatory cell infiltration was the highest in the control and 115 proteins, and inflammatory cell invasion was observed continuously for 3 weeks. In T-CAM, the inflammatory response was the lowest in the first 6 days compared to the other groups, and 115 protein was found to decrease after 14 days of infiltration of inflammatory cells ( FIG. 11 , Table 1 ).

3 days 6 days 10 days 14 days 21st Control +++ ++++ ++++ +++ +++ 115 Protein +++ +++ +++ ++ ++ βig-h3-D-IV ++ +++ +++ +++ +++ T-CAM ++ ++ +++ +++ +++

Inflammatory cell invasion degree:

-; None, +; Light, ++; (mild),

+++; Moderate, ++++; Severe

<9-2-2> Findings about Angiogenesis

Angiogenesis was observed in the control group and βig-h3-D-IV from day 6, but T-CAM was able to observe neovascularization as early as day 3 and showed the most active angiogenesis. Could see. The most active angiogenesis was observed at 2 weeks in all groups ( FIG. 12 , Table 2 ).

3 days 6 days 10 days 14 days 21st Control - + ++ ++ ++ 115 Protein + +/- ++ +++ ++ βig-h3-D-IV - + ++ +++ ++ T-CAM +/- ++ ++++ ++++ +++

Vascular Growth:

-; None, +; Light, ++; (mild),

+++; Moderate, ++++; Severe

<9-2-3> Findings for Fibroblast Proliferation

Fibroblasts, like angiogenesis, were able to observe fibroblasts from day 3 faster than other groups in T-CAM and showed the most increased findings by day 10. There was an increase in the number of the groups up to 2 weeks after the procedure, but there was no significant difference between the groups after 14 days after the procedure ( Table 3 ).

3 days 6 days 10 days 14 days 21st Control - + ++ +++ +++ 115 Protein + ++ +++ ++ ++ βig-h3-D-IV - +/- +++ +++ +++ T-CAM +/- ++ ++++ +++ +++

Fibroblast proliferation degree:

-; None, +; Light, ++; (mild),

+++; Moderate, ++++; Severe

<9-2-4> Findings for Collagen Production

Collagen formation was increased in T-CAM during the first 6 days, and after 10 days there was no significant difference between the groups. In the T-CAM group was observed that the collagen is well organized than other groups ( Fig. 13 , Table 4 ).

3 days 6 days 10 days 14 days 21st Control +/- + ++ ++ +++ 115 Protein + + ++ +++ +++ βig-h3-D-IV +/- + ++ ++ +++ T-CAM + ++ ++ +++ +++

Collagen production level:

-; None, +; Light, ++; (mild),

+++; Moderate, ++++; Severe

<9-2-5> Histological Findings of Epidermal Skin

In the T-CAM treatment group, the epithelial layer was larger in thickness than the other groups and the rete ridge (epidelial projection) was well formed, and thus, the degree of epithelial regeneration was superior to other groups ( FIG. 14 ). .

As described above, the recombinant protein including the 9 and 10 type III domains of the fibronectin of the present invention and the 4 fas-1 domain of βig-h3 has an easy interaction with integrins, which leads to cell adhesion, spread, and Excellent effect on proliferation and cell migration. In addition, when the recombinant protein is used as a ointment for wound treatment, the wound shrinkage rate and wound healing rate are excellent, angiogenesis, fibroblast proliferation and collagen production are increased, and epithelial regeneration is excellent. Therefore, when the pharmaceutical composition containing the recombinant protein of the present invention as an active ingredient is used for the treatment of wounds, the number of wound treatments can be reduced, and it can be useful for the treatment of chronic wounds.

Figure 1 shows a schematic diagram of the T-CAM protein of the present invention.

Figure 2 is a photograph of the electrophoresis by separating and purifying the T-CAM protein of the present invention.

Figure 3 shows cell adhesion and spread in human dermal fibroblasts by BSA, fibronectin (pFN), βig-h3 (WT), βig-h3 D-IV (D-IV), 115 proteins and T-CAM (A, C, E) and keratinocytes (Keratinocytes) in HaCaT cell adhesion and spread (B, D, F) is a comparison and photographs.

Figure 4 is a graph showing cell adhesion by BSA, fibronectin (pFN), βig-h3 (WT), βig-h3 D-IV (D-IV), 115 protein and T-CAM in different cell types.

A: CHO cells, B: MRC5 cells,

C: NHEK cells, D: NIH3T3 cells

FIG. 5 shows BSA, fibronectin (pFN), βig-h3 (WT), βig-h3 D-IV (D-IV), 115 protein and T-CAM in fibroblast (A) and HaCaT (B) cells. Graph showing proliferation.

Figure 6 shows BSA, fibronectin (pFN), βig-h3 (WT), βig-h3 D-IV (D-IV), 115 protein and T-CAM of fibroblasts (A, C) and HaCaT (B, D). It shows cell migration by.

FIG. 7 is a photograph showing the results of experiments on the wound healing effect of T-CAM, a control (adult application only), βig-h3 D-IV (D-IV), and 115 proteins in rabbits.

FIG. 8 is a graph showing wound contraction rate by a control (applied only with ointment), 115 protein, βig-h3 D-IV (D-IV), and T-CAM.

Figure 9 is a graph showing the wound epithelialization rate by the control (applied only with the ointment), 115 protein, βig-h3D-IV (D-IV) and T-CAM. The significance of each group was analyzed by ANOVA test and Scheffe's test. *: P <0.05 compared to the control

FIG. 10 is a graph showing wound healing rate by a control (applied only with ointment), 115 protein, βig-h3D-IV (D-IV), and T-CAM.

FIG. 11 is a biopsy photograph of inflammatory reactions such as multinucleated cells and lymphocytes by a control (coated with a drug only), 115 protein, βig-h3D-IV (D-IV), and T-CAM.

12 is a tissue photograph observing angiogenesis by a control (applied only with the ointment), 115 protein, βig-h3D-IV (D-IV) and T-CAM.

FIG. 13 is a photograph of tissues observed collagen production by a control (applied only with ointment), 115 protein, βig-h3D-IV (D-IV) and T-CAM.

FIG. 14 is a tissue photograph showing re-epithelialization by a control (applied only with ointment), 115 protein, βig-h3D-IV (D-IV) and T-CAM.

<110> KOREA (KYUNGPOOK NATIONAL UNIVERSITY) <120> Pharmaceutical compositions containing domains of fibronectin and βig-h3 for wound healing, cell adhesion, migration and proliferation <130> 2p-06-76B <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 552 <212> DNA <213> Homo sapiens <400> 1 ggtcttgatt ccccaactgg cattgacttt tctgatatta ctgccaactc ttttactgtg 60 cactggattg ctcctcgagc caccatcact ggctacagga tccgccatca tcccgagcac 120 ttcagtggga gacctcgaga agatcgggtg ccccactctc ggaattccat caccctcacc 180 aacctcactc caggcacaga gtatgtggtc agcatcgttg ctcttaatgg cagagaggaa 240 agtcccttat tgattggcca acaatcaaca gtttctgatg ttccgaggga cctggaagtt 300 gttgctgcga cccccaccag cctactgatc agctgggatg ctcctgctgt cacagtgaga 360 tattacagga tcacttacgg agaaacagga ggaaatagcc ctgtccagga gttcactgtg 420 cctgggagca agtctacagc taccatcagc ggccttaaac ctggagttga ttataccatc 480 actgtgtatg ctgtcactgg ccgtggagac agccccgcaa gcagcaagcc aatttccatt 540 aattaccgaa ca 552 <210> 2 <211> 184 <212> PRT <213> Homo sapiens <400> 2 Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn 1 5 10 15 Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr 20 25 30 Arg Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp 35 40 45 Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro 50 55 60 Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu 65 70 75 80 Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr Val Ser Asp Val Pro Arg 85 90 95 Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp 100 105 110 Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu 115 120 125 Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys 130 135 140 Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile 145 150 155 160 Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys 165 170 175 Pro Ile Ser Ile Asn Tyr Arg Thr 180 <210> 3 <211> 420 <212> DNA <213> Homo sapiens <400> 3 ctgacccccc caatggggac tgtcatggat gtcctgaagg gagacaatcg ctttagcatg 60 ctggtagctg ccatccagtc tgcaggactg acggagaccc tcaaccggga aggagtctac 120 acagtctttg ctcccacaaa tgaagccttc cgagccctgc caccaagaga acggagcaga 180 ctcttgggag atgccaagga acttgccaac atcctgaaat accacattgg tgatgaaatc 240 ctggttagcg gaggcatcgg ggccctggtg cggctaaagt ctctccaagg tgacaagctg 300 gaagtcagct tgaaaaacaa tgtggtgagt gtcaacaagg agcctgttgc cgagcctgac 360 atcatggcca caaatggcgt ggtccatgtc atcaccaatg ttctgcagcc tccagccaac 420 420 <210> 4 <211> 140 <212> PRT <213> Homo sapiens <400> 4 Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn 1 5 10 15 Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu 20 25 30 Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu 35 40 45 Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp 50 55 60 Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile 65 70 75 80 Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln 85 90 95 Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn 100 105 110 Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val 115 120 125 His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn 130 135 140 <210> 5 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> T-CAM <400> 5 ggtcttgatt ccccaactgg cattgacttt tctgatatta ctgccaactc ttttactgtg 60 cactggattg ctcctcgagc caccatcact ggctacagga tccgccatca tcccgagcac 120 ttcagtggga gacctcgaga agatcgggtg ccccactctc ggaattccat caccctcacc 180 aacctcactc caggcacaga gtatgtggtc agcatcgttg ctcttaatgg cagagaggaa 240 agtcccttat tgattggcca acaatcaaca gtttctgatg ttccgaggga cctggaagtt 300 gttgctgcga cccccaccag cctactgatc agctgggatg ctcctgctgt cacagtgaga 360 tattacagga tcacttacgg agaaacagga ggaaatagcc ctgtccagga gttcactgtg 420 cctgggagca agtctacagc taccatcagc ggccttaaac ctggagttga ttataccatc 480 actgtgtatg ctgtcactgg ccgtggagac agccccgcaa gcagcaagcc aatttccatt 540 aattaccgaa cactgacccc cccaatgggg actgtcatgg atgtcctgaa gggagacaat 600 cgctttagca tgctggtagc tgccatccag tctgcaggac tgacggagac cctcaaccgg 660 gaaggagtct acacagtctt tgctcccaca aatgaagcct tccgagccct gccaccaaga 720 gaacggagca gactcttggg agatgccaag gaacttgcca acatcctgaa ataccacatt 780 ggtgatgaaa tcctggttag cggaggcatc ggggccctgg tgcggctaaa gtctctccaa 840 ggtgacaagc tggaagtcag cttgaaaaac aatgtggtga gtgtcaacaa ggagcctgtt 900 gccgagcctg acatcatggc cacaaatggc gtggtccatg tcatcaccaa tgttctgcag 960 cctccagcca ac 972 <210> 6 <211> 324 <212> PRT <213> Artificial Sequence <220> <223> T-CAM <400> 6 Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn 1 5 10 15 Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr 20 25 30 Arg Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp 35 40 45 Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro 50 55 60 Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu 65 70 75 80 Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr Val Ser Asp Val Pro Arg 85 90 95 Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp 100 105 110 Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu 115 120 125 Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys 130 135 140 Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile 145 150 155 160 Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys 165 170 175 Pro Ile Ser Ile Asn Tyr Arg Thr Leu Thr Pro Pro Met Gly Thr Val 180 185 190 Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met Leu Val Ala Ala 195 200 205 Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn Arg Glu Gly Val Tyr 210 215 220 Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg Ala Leu Pro Pro Arg 225 230 235 240 Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu Leu Ala Asn Ile Leu 245 250 255 Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser Gly Gly Ile Gly Ala 260 265 270 Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu Glu Val Ser Leu 275 280 285 Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Val Ala Glu Pro Asp 290 295 300 Ile Met Ala Thr Asn Gly Val Val His Val Ile Thr Asn Val Leu Gln 305 310 315 320 Pro Pro Ala Asn <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 7 attcgatatc ggtgttcggt aatta 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 8 agacagatat ccggtcttga ttccc 25 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 9 atggagatat cgctgacccc ccca 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer 4 <400> 10 tcctgctcga ggttggctgg aggc 24

Claims (10)

A recombinant protein in which the 9 and 10 type III domains of fibronectin described in SEQ ID NO: 2 and the 4 fas-1 domain of βig-h3 described in SEQ ID NO: 4 are fused together. delete delete 2. The recombinant protein of claim 1, wherein said recombinant protein is set forth in SEQ ID NO: 6 . The gene set forth in SEQ ID NO: 5 encoding the recombinant protein of claim 1. delete An expression vector comprising the gene of claim 5 and expressing the recombinant protein of claim 1. 8. The expression vector of claim 7, wherein the expression vector is pET T-CAM. A pharmaceutical composition for treating wounds comprising the recombinant protein of claim 1 as an active ingredient. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is an external preparation, ointment, cream, spray or spray.