WO2007108643A1

WO2007108643A1 - New gene and polypeptides of platelet derived growth factor b

Info

Publication number: WO2007108643A1
Application number: PCT/KR2007/001369
Authority: WO
Inventors: Hyun Su Bae; Moon Kyu Kang; Min Kyu Shin; Moo Chang Hong; Hwan Suck Chung; Chong Woon Cho; Sang Moon Lee; Shoukat Parvez; Chang Sook Kim; Hwa Jin Lee; Eun A Lee; Sam Woong Rho; Eun Jung Ko
Original assignee: Purimed Co., Ltd.
Priority date: 2006-03-21
Filing date: 2007-03-21
Publication date: 2007-09-27
Also published as: KR100742313B1

Abstract

Disclosed herein are a novel deer PDGF-B polypeptide and a gene encoding the same. The PDGF-B is superior in wound healing activity to human PDGF-B. Also, a composition comprising the platelet-derived growth factor-B polypeptide as an active ingredient is provided for wound healing and the treatment and prevention of neurodegenerative diseases, ischemic neurological diseases and diseases causing neurological damage, through cell proliferation, development and differentiation.

Description

NEWGENEANDPOLYPEPTIDESOFPLATELETDERIVEDGROWTH

FACTORB

Technical Field

The present invention relates to a novel deer platelet derived growth factor (PDGF)-B polypeptide and a gene encoding the same, which is superior to human PDGF-B polypeptide. Also, the present invention is concerned with a composition comprising the platelet-derived growth factor-B protein as an active ingredient, which is useful in wound healing and in the treatment and prevention of neurodegenerative diseases, ischemic neurological diseases and neurological damage diseases through cell proliferation, development and differentiation.

Background Art

Platelet derived growth factor (PDGF) plays a significant role in cell proliferation, development and differentiation, and wound healing, and has also been linked to several diseases such as arteriosclerosis and malignant diseases. There are five different isoforms of PDGF, three combinations of homo and heterodimers of ligands A and B, that is, PDGF-AA, PDGD-AB and PDGF-BB, and PDGF C and PDGF D, the last two recently discovered by Li and Chen (Xuri Li and UIf Eriksson, Cytokine & Growth Factor reviews (2003) Vol. 14, pp91-93; Jingzhou Chen, Yu Han, Yu Ha, Chunxia Lin, Yisong Zhen, Xiaodong Song, Siyong Teng, Chen Chen, Yu Chen, Yinhui Zhang and Rutai Hui, Biochemical and Biophysical Research Communications (2005) , Vol. 329, pp 976-983) . Growth factors are polypeptide, hormone-like molecules, which interact with specific receptors on the surface of their target cells. The wound healing process is controlled and regulated by growth factors which: i) have mitogenic activities, which in turn stimulate cellular proliferation; ii) have angiogenic activities and thus stimulate the growth of new blood vessels; iii) have chemotactic activities, attracting inflammatory cells and fibroblasts to the wound; iv) influence the synthesis of cytokines and growth factors by neighboring cells; and v) effect production and degradation of the extracellular matrix.

PDGF is an important mitogenic growth factor which is detected in serum, but quiescent in plasma (Antoniades et al., Proc. Nat' 1 Acad. Sci. USA, vol. 72 (1975), 2635-2639; and Ross and Vogel, Cell, vol. 14 (1978), 203-210). It was discovered upon the observation that serum is superior to plasma in stimulating the in vitro proliferation of fibroblasts (Balk et al., Proc. Nat'l Acad. Sci. USA, vol. 70 (1973), 675-679).

PDGF is a major mitogen for most mesenchymally derived cells as well as connective tissue cells (Pierce and Mustoe, Annual Review of Medicine, vol. 46 (1995), 467- 481) and acts as a potent chemoattractant for neutrophils, monocytes and fibroblast cells (Lepisto et al., Eur. Surg. Res., vol. 26 (1994), 267-272). Circulating monocytes and fibroblasts, which migrate into a wound due to the chemotactic activity of PDGF, mature into tissue macrophages and are themselves able to secrete PDGF. Besides chemotaxis, it has been shown that PDGF-BB is known to induce the expression of tissue factor, the initiator of the clotting cascade, in human peripheral blood monocytes (Ernofsson M., and Siegbahn, A., Thromb. Res., vol. 83 (1996), 307-320). In addition, PDGF mediates the induction of extracellular matrix synthesis, including the production of hyaluronic acid and fibronectin (Robson, M. C. Wound Rep. Reg., vol. 5 (1997), 12-17). Further, collagenase, playing a critical role in wound matrix remodeling, is produced in response to PDGF (Steed, D. L. Surg. Clin. North Am., vol. 77 (1997), 575-586) . PDGF is also involved in pathological conditions, such as tumorogenesis, arteriosclerosis, rheumatoid arthritis, pulmonary fibrosis, myelofibrosis or abnormal wound repair (Bornfeldt et al., Ann. NY Acad. Sci., vol. 766 (1995), 416-430; Heldin, C. H., FEBS Lett., vol. 410 (1997), 17-21) and acts as a mitogen for bone cells, which stimulate the proliferation of osteoblastic cells (Homer et al., Bone, vol. 19 (1996), 353-362.

PDGF is used as a supplement to cell culture media. The treatment of cancer through the regulation of PDGF signaling is currently under intensive study. In fact, PDGF expression suppressors (refer to, e.g., Japanese Laid-Open Application No. 10-59850), inhibitors of binding between PDGF and PDGF receptor α (refer to, e.g., Japanese Patent Laid-Open Publication No. Hei. 8—500010), tyrosine kinase inhibitor of PDGF receptor α (refer to, e.g., Japanese Patent National Publication of International Patent Application No. 2002-514228), polynucleotides, substances for suppressing expression, agents for suppressing expression, and cancer therapeutic agents for use in methods for suppressing expression that enable the selective suppression of PDGF signals specific to cancer cells (refer to, e. g., Korean Patent Application No. 2005- 7008784), and a protein complex comprising PDGF for the inhibition of cancer cell metastasis (refer to, e.g., Korean Patent Application No. 2005-7014530) have been proposed as therapeutic agents for such malignant diseases. Also, PDGF is used along with other growth factors, such as IGF-I, IGF-II or VEGF, or receptors thereof, for stimulating or inducing cell migration and/or proliferation which may have use in wound healing, tissue engineering, cosmetic and therapeutic treatments such as skin replacement and skin replenishment and the treatment of burns where epithelial cell migration is required (refer to, e.g., Korean Patent Application No. 2005-7014530). PDGF has been known as a fibroblast mitogen, but was recently found to be the most abundantly expressed in embryonic and adult brains, with both PDGF-A and PDGF-B secreted from nerve cells.

Mammalian brains can perform their complex functions only after the systemic neural network has undergone development through the division, differentiation, survival and apoptosis of neuronal stem cells and the formation of synapses. Nerve cells undergo apoptosis unless they are supported by target-derived survival factors, such as nerve growth factors, in the course of differentiation and synapse formation. The apoptosis of nerve cells, attributable to stress and cytotoxic agents, is a major cause of neurodegenerative diseases .

Serving as a neurotrophic factor inducing striatal neurogenesis, PDGF can be used as a therapeutic for various neurodegenerative diseases, ischemic neurological disorders, and neurological damage (refer to, e.g., Korean Patent Application No. 2002-044770) .

Deer velvet antlers, annually regenerating bone tissues of deer, are used as a medicinal material for invigoration and immunity enhancement in herbal medicine. Many research reports on the medicinal efficacies of deer antlers have been published in Korea. In "Shennong bencao jing", the oldest Chinese medicine book in the world, deer antlers are described as being useful in the treatment of fatigue, deficient positive-energy (qi) , womb coldness, and a syndrome in which the spleen fails to manage blood, thereby invigorating and restoring the body. According to many recent reports, extracts from deer antlers are proven to have various healthful effects including growth stimulation, haematogenesis, cardio-stimulation, hepatoprotection, regeneration of liver tissues, activation of hepatic enzymes, enhancement of hormone functions, osteoporosis treatment, wound healing, etc.

Leading to the present invention, intensive and thorough research into the medicinal effects of deer velvet antlers, conducted by the present inventors, resulted in the finding that the PDGF-B obtained from deer velvet antlers affords rapid wound healing when it is applied to wound fibroblast cells, and is superior to human PDGF-B in terms of the phosphorylation of ERK 1/2.. Disclosure of the Invention

It is therefore an object of the present invention to provide a novel PDGF gene from deer velvet antlers and a polypeptide thereof. In order to accomplish the above object, there are provided a novel PDGF B gene and a polypeptide thereof.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic view illustrating the strategy of searching for a novel platelet-derived growth factor polypeptide. FIG. 2 is a photograph of a deer platelet-derived growth factor B gene separated by electrophoresis.

FIG. 3 is a photograph showing the electrophoresis results of deer platelet-derived growth factor B polypeptides, along with a marker. Lane M: a marker from Bio-Rad, Lane 1: a pET 28 (a) (+) vector sample before induction, Lane 2: a pET 28 (a) (+) vector sample after induction, Lanes 3-4 : PDGF B sample before induction, Lanes 5-6: a PDGF B sample after induction, Lane 7: purified polypeptide (M) .

FIG. 4 is a full-length PDGF-B cDNA sequence of deer antler with an initiation codon ATG and a termination codon TAA underlined in the coding region, consisting of 729 bases in a completely expressible prepro-form and 327 bases in a mature form which is translated into a polypeptide consisting of 111 amino acid residues.

FIG. 5 is a deer PDGF-B amino acid sequence and PDGF- B nucleotide sequence. FIG. 6 is a view showing the comparison of homology of the PDGF-B gene between deer and humans .

FIG. 7 is a view showing the comparison of homology of the PDGF-B polypeptide between deer and humans .

FIG. 8 is a photograph showing human fibroblast cells treated with 1, 10 and 100 ng/ml of deer PDGF B (P) and 1, 10 and 100 ng/ml of human PDGF B (Sigma) (S) for wound healing.

FIG. 9 shows the comparison of healing effects on wound human fibroblast cells between deer PDGF B (P) and human PDGF B (S) when the cells are treated with 0, 1, 10 and 100 ng/ml of each of the PDGF B proteins.

FIG. 10 shows the healing effects of deer PDGF B on wound human fibroblast cells 24 hours (A) and 72 hours (B) after the treatment thereof. FIG. 11 shows the PDGF B-induced ERK 1/2 phosphorylation. (A) non-treated and undamaged normal cells, (B) 24 after treatment with PDGF B, (C) 30 min after treatment with PDGF B, (D) 6 hours after treatment with PDGF B; M: standard sample, PO: treated with 0 ng/ml of deer PDGF, Pl: treated with 1 ng/ml of deer PDGF, PlO: treated with 10 ng/ml of deer PDGF, PlOO: treated with 100 ng/ml of deer PDGF, SO: treated with 0 ng/ml of human PDGF, Sl: treated with 1 ng/ml of human PDGF, SlO: treated with 10 ng/ml of human PDGF, SlOO: treated with 100 ng/ml of human PDGF.

Best Mode for Carrying Out the Invention

In accordance with an aspect thereof, the present invention pertains to a novel platelet-derived growth factor-B (PDGF-B) polypeptide set forth in SEQ ID NO. 2 and a functional equivalent or functional derivative thereof. The novel PDGF-B polypeptide according to the present invention is sourced from deer antlers.

As used herein, the term "functional equivalent" of the novel PDGF-B polypeptide is intended to refer to a polypeptide exhibiting physiological activity substantially identical to that of the natural novel PDGF-B polypeptide from deer.

In detail, the "functional equivalent" of the novel PDGF-B polypeptide useful in the present invention refers to a polypeptide, occurring naturally or generated artificially, which is different in amino acid sequence from the wild-type due to the deletion, insertion, or conservative or non-conservative substitution of amino acids or combinations thereof. Preferable is conservative substitution. Examples of conservative substitutions of naturally occurring amino acids include aliphatic amino acids (GIy, Ala, Pro) , hydrophobic amino acids (lie, Leu, VaI) , aromatic amino acids (Phe, Tyr, Trp) , acidic amino acids (Asp, GIu) , basic amino acids (His, Lys, Arg, GIn, Asn) and sulfur-containing amino acids (Cys, Met) . As for the deletion of amino acids, it preferably occurs at a position that is not directly responsible for the physiological activity of the insulin-like growth factor.

The term "functional derivative", as used herein, is intended to refer to a fragment of the PDGF polypeptide, a protein containing the fragment, or a modified form of the PDGF-B polypeptide protein, so as to improve or disable the native physiochemical properties thereof. By the term "fragment" of the PDGF-B polypeptide, it is meant that an amino acid sequence corresponding to a part of the PDGF polypeptide has a common origin element, structure, and operational mechanism within the scope of the present invention. The functional derivative is modified so as to improve or decrease the stability, preservability, solubility and other properties of the protein or, for example, to alter the relationship between the PDGF and a material interacting therewith, which also falls within the scope of the present invention. Methods for preparing such functional derivatives are well known in the art.

Preferably, the production of the PDGF protein may resort to a gene recombination technique. A PDGF-B-encoding DNA or RNA sequence is expressed in a suitable host cell, which is then lysed to isolate the PDGF-B polypeptide. Alternatively, PDGF-B mRNA is translated in vitro, followed by the isolation of the PDGF-B protein. For the isolation of protein, typically, cell lysates are first centrifuged to remove cell debris, and then precipitation, dialysis and various kinds of column chromatography may be applied to the cell lysates or in-vitro translation reactions. Examples of typical column chromatography include ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, prep SDS-PAGE, and affinity chromatography. As for affinity chromatography, it may utilize an anti-PDGF-B antibody.

Functional derivatives of the PDGF polypeptide, including fragments derived from the PDGF polypeptide of the present invention, can be prepared using one of the organochemical techniques for peptide synthesis which are well known in the art. In a typical organochemical technique for peptide synthesis, amino acids required therefor are coupled through condensation on a homogeneous phase or a solid-phase support. For the condensation, methods using carbodiimide, azide, combined anhydrates and active ester may be used, and are disclosed in The Peptides Analysis, Synthesis, Biology Vol. 1-3 (edited by Gross, E. and Meienhofer, J.)_* 1979-1981 (Academic Press Inc.). A suitable solid phase is p-alkoxybenzyl alcohol resin (4-hydroxy-methyl-phenoxy-methyl-copolystyrene-l% divinylbenzene resin) , which is disclosed in (Wang, J. Am. Chem. Soc, 95, 132 (1974)). After synthesis, the resulting peptide may be released from the solid phase under mild conditions. In this regard, trifluoroacetic acid, in combination with a scavenger such as triisopropyl, anisole ethanedithiol, or thioanisole, may be used for the separation of the polypeptide of interest from the solid phase. Groups which are to be prevented from participating in condensation are protected through a hydrolysis and reduction reaction by removable protecting groups such as acids or bases. For details of the protecting groups, reference may be made to The Peptides, Analysis, Synthesis, Biology, Vol. 1-9 (edited by Gross, Udenfriend and Meienhofer) 1979-1987 (Academic Press Inc.).

In accordance with a second aspect thereof, the present invention pertains to a nucleic acid sequence encoding selected from a group of nucleic acid sequences, each comprising the genetic information of the novel PDGF-B polypeptide according to the present invention, set forth in SEQ ID NO. : 1. The term "a group of nucleic acid sequences", as used herein, is intended to refer to a group consisting of a gene encoding the novel PDGF-B polypeptide, a gene comprising the nucleotide sequence of SEQ ID NO. : 1, a native mRNA thereof, identified as being selectively expressed, a cDNA sequence complementary thereto, and an equivalent nucleotide sequence thereof.

The term "equivalent nucleotide sequence", as used herein, is intended to include the nucleotide sequence provided according to the present invention, an allelic variation thereof, an interspecies variation thereof, or a codon degenerate sequence thereof. The term "codon degenerate sequence" of the nucleotide sequence refers to a nucleotide sequence which is different from the naturally occurring nucleotide sequence but codes for a polypeptide identical to the naturally occurring PDGF polypeptide according to the present invention.

The terms "allelic variation" and "interspecies variation" of the nucleotide sequence, as used herein, are intended to refer to a nucleotide sequence which is different in sequence from naturally occurring nucleotide sequences, but encodes a polypeptide having functional properties substantially identical to those of the wild- type polypeptides. In accordance with a third aspect thereof, the present invention is directed to a pharmaceutical composition useful in wound healing and in the treatment and prevention of neurodegenerative diseases, ischemic neurological diseases and diseases causing neurological damage, through cell proliferation, development and differentiation, comprising the novel PDGF-B polypeptide, an active fragment thereof and/or a functional derivative thereof as an active ingredient. The pharmaceutical composition may be formulated into various preparations and administered in various manners. For oral administration, for example, the pharmaceutical composition may be mixed with an inactive diluent or an edible carrier, loaded within a hard or soft gelatin capsule, pressurized into tablets, or directly eaten with meals. Among the available oral dosage forms of the pharmaceutical composition according to the present invention are tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.

For the formation of tablets, troches, pills, and capsules, the active ingredient of the pharmaceutical composition according to the present invention may be an admixture with a binder, such as gum, tragacanth, acacia, corn starch or gelatin; an expedient, such as calcium diphosphate; a disintegrant, such as corn starch, potato starch, or alginic acid; a lubricant, such as magnesium stearate; a sweetener, such as sucrose, lactose or saccharin; or a flavor such as peppermint, wintergreen oil or cherry flavor. An administration unit in the form of capsules may include a liquid vehicle in addition to the above additives. Further, a coating agent or other various agents adapted to change the physical conformations of administration units may be added. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. Elixir syrup may comprise sucrose as a sweetener, methylparaben and propylparaben as preservatives, pigment, and a cherry flavor or orange flavor, in addition to the active ingredient. All of the materials which are used in the preparation of any single dosage form should be not only pharmaceutically pure, but also substantially nontoxic in the amount used. In addition, the pharmaceutical composition of the present invention may be formulated into sustained release forms .

Also, the pharmaceutical composition of the present invention may be in the form of a non-ingestible mouthwash or toothpaste. For mouthwash, the active ingredient may be dissolved in, for example, a sodium borate solution (Dobell's solution). In an alternative, the active ingredient is mixed with a preservative detergent comprising sodium borate, glycerin and potassium bicarbonate. Also, the active ingredient may be dispersed in dentifrice in the form of a gel, paste, powder or slurry, or may be added in a therapeutically effective amount to a toothpaste composition which may include water, a binder, a grinder, a flavor, a foaming agent and a wetting agent.

Various dosage forms for injection or parenteral administration may be prepared using techniques well known in the art (e.g., "Remington's Pharmaceutical Sciences" 15th Edition) or typically available techniques.

The suitable administration dose of the pharmaceutical composition of the present invention depends on the state of the patients to be treated, and can be readily determined by physicians. Of course, ingredients used in the pharmaceutical composition for use in humans must meet the requirements of the FDA in various aspects including sterility, pyrogenicity, general safety and purity.

When it was applied to fibroblast cells of a wound, the novel PDGF-B protein isolated from deer velvet antlers was observed to almost perfectly heal the wound within 48 hours. Also, the treatment of normal fibroblast cells with the novel PDGF-B according to the present invention increased the of phosphorylated ERK 1/2, as compared to treatment with human PDGF-B, indicating that the protein of the present invention can be used as a more effective wound or ulcer healing agent.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[EXAMPLE 1]

Isolation of mRNA from Deer Antler

(1) Material and Method

Velvet antlers were taken from Cervus elaphus. Reagents for cell culture media, RNA isolation and cDNA were purchased from Gibco BRL, for mRNA isolation from Quigen, and for expression in E. coli from Invitrogen. The other reagents used in the present invention were all at molecular biological levels. An overall scheme for gene isolation is outlined in FIG. 1.

(2) Test Material and Sampling

Velvet antlers of Cervus elaphus, known to have antlers superior in pharmaceutical effectiveness to those of other deer, were cut in the first and the second part of May, during which deer most actively grow their antlers. The cut antlers were quickly frozen with dry ice and ethanol, and growing tissues were separated from the antlers and stored at -8O⁰C before use.

(3) Isolation of Total RNA from Growing Tissue To a 50 ml conical tube were added 3 ml of Triazole (Gibco BRL) and 3 g of the separated growing tissue, followed by homogenation in a homogenizer. After mixing the homogenate with an additional 27 ml of Triazole, the mixture was allowed to stand for 5 min at room temperature and aliquoted into 4 tubes in an amount 7.5 ml per tube. 1.5 ml of chloroform was added to each tube. The tubes were allowed to stand at room temperature for 3 min before centrifugation at 12,000 rpm for 15 min. The supernatant was aliquoted into new tubes in an amount of 3 ml per tube. 3.75 ml of isopropanol was added to each tube, after which they were allowed to stand at 4⁰C for 10 min and then centrifuged at 12,000 rpm for 10 min. The pellet thus obtained was washed with 7.5 ml of 75% ethanol and centrifuged at 7,500 rpm for 5 min. The RNA pellet thus obtained was dried at room temperature and dissolved in 225 μl of DEPC-treated water for storage.

(4) Isolation of mRNA from Total RNA Using Oligotex mRNA midi kit (Qiagen) , mRNA was isolated from the total RNA obtained.

(5) Synthesis of cDNA from the mRNA of Deer Antler and Cloning For the synthesis of cDNA from the mRNA, a cDNA synthesis kit (Gibco BRL) was used in a modified manner. The synthesis of the first strand of cDNA started with denaturing the secondary structure of the mRNA through the reaction of 9 μl of the mRNA (2 μg) with 2 μl of Not I primer-adaptor (0.5 μg/μl) at 70°C for 10 min. The reaction mixture was cooled on ice for 2 min, mixed with 4 μl of a 5X first strand buffer, 2 μl of 0.1 M DTT and 1 μ of 10 mM dNTP at 37⁰C for 2 min and was allowed to react at 37⁰C for 60 min in the presence of 2 μl of Superscriptase II, followed by leaving on ice for 2 min. For the synthesis of second strand cDNA, the reaction mixture was subjected to reaction at 16°C for 2 hours, along with 91 μl of DEPC-treated water, 30 μl of a second strand buffer, 3 μl of a 10 mM dNTP mixture, 1 μl of E. coli DNA ligase (10 U/μl) , 1 μl of E. coli DNA polymerase (10 U/μl), and 1 μl of E. coil RNase H (2 U/μl), followed by additional reaction for 5 min in the presence of 2 μl of T4 DNA polymerase (10 U) . After the addition of 10 μl of 0.5 M EDTA thereto, the resulting reaction mixture was treated with phenol-chloroform to extract cDNA, which was then dissolved in DEPC-treated water for use.

The synthesized cDNA was provided at opposite ends with a Wotl sequence and an EcoRl sequence, respectively, by ligating an EcoRl-BstXl adaptor (Invitrogen) thereto and cutting with Notl. Desired sizes of the cDNA were separated and recovered through gel filtration columns and inserted into the multi-cloning site of a vector (pDONR222, pSPORT 1, pBluescript, pPICZ, pYES3/CT, etc.) which was previously treated with restriction enzymes Notl and EcoRl . The recombinant plasmid vector thus obtained was transformed into E. coli (ElectroMAX DH5α, Gibco BRL) by electroporation, followed by amplification through cell culture .

(6) Determination of Base Sequence of DNA from Deer Antler and Selection of PDGF-B Gene EST clones were obtained from the cDNA library of deer antlers, and were used to extract DNA therefrom. The DNA was base sequenced in a single direction with the aid of a BigDye Terminator Cycle Sequence kit, commercially available from ABI . Clones with the initiation codon were analyzed for the possession of termination codons. Clones with both initiation and termination codons were detected. Interspecies base and amino acid homology searches were easily conducted on the clones using the program BLAST from NCBI, resulting in the discovery of the PDGF-B gene, which is well known for its wound healing activity. A vector carrying the gene of SEQ ID NO. : 1 was selected.

(7) Preparation of E. coli Strain Containing Deer Antler PDGF-B cDNA A plasmid vector carrying deer antler PDGF-B cDNA was transformed into E. coli using electroporation. In this regard, E. coli cells grown to an OD₆oo of 1.3 ~ 1.5 were made able to receive foreign DNA through electroporation. 80 μl of the competent cells were transformed with 5 μg of the vector carrying the deer antler cDNA (1.5kV, 25μF, 200Ω, 0.2cm cuvette, Gene-Pulser, BioRad) .

[EXAMPLE 2] Expression of Deer Velvet Antler FDGF-B Gene

(1) Overexpression of Deer Velvet Antler PDGF-B Protein and Purification thereof

A desired portion suitable for expressing PDGF-B was eluted from the vector constructed in Example 1. In this regard, PCR was performed with a reaction mixture containing a forward primer (SEQ ID NO. : 3) and a reverse primer (SEQ ID NO.: 4), each used at 2 μl/10 pmol, a dNTP mix, a Taq-DNA polymerase, MgCl₂, and a reaction buffer, followed by the gel-elution of a desired band (see FIG. 2) .

The PCR product was ligated to a T-vector system (50 ng/μl), designed to tag a target with a poly-A tail, in the presence of T4 DNA ligase (3 U/μl) . The resulting vector was transformed into DH5α through heat shocking (42⁰C) . After amplification, the vector was digested with EcoRI and Xho I to prepare an insert to be expressed. pET28a(+), as an expression vector, was also treated with the same restriction enzymes. The ligation of the insert to the digested vector was performed for 16 hours at 16°C. The recombinant expression vector was introduced into BL21 (DE3) . In order to confirm the transformation, the transformant was cultured, and the plasmid DNA was prepared using a MiniPrep kit (QIAGEN) and digested with EcoRI and Xho I. Two bands (about 5.3 kb for the vector, 0.7 kb for the insert) were visualized in a gel.

A colony of the transformant was cultured overnight in 3 ml of LB (kanamycin 50 μg/ml) in a 15 mL tube. This cell culture was incubated at 37⁰C to an O.D.₆oo of 0.4 ~ 0.5 in 100 ml of an LB medium in a 250 ml flask. In this way, the incubation was performed overnight in the presence of IPTG (1 πiM) to induce the expression of the protein of interest. After being harvested, the cells were re- suspended in a lysis buffer (50 mM NaCl, 5 mM MgCl₂, 50 mM Tris-HCl/pH 7.9), optionally in the presence of 0.1 mm PMSF and 10 mM BME (Basal Medium Eagle) . To the suspension was added a lysozyme (1 g/10 ml) solution, and incubation was conducted at 37⁰C for 30 min before ultrasonication. The cell lysate was stored at 20⁰C. Resins were attached with Ni²⁺ using an affinity column system, and were loaded with the proteins. First, resins (2 ml) were loaded in the column. Thereafter, 6 ml of sterile distilled water was poured onto the resins to estimate the flow rate. Then, 10 ml of a charging buffer (50 mM NiSO₄) and 6 ml of a binding buffer (20 mM imidazole, 0.5 M NaCl, 20 mM Tris- HCl/pH 7.9) were charged in the column in the order. After completion of the passage of the binding buffer, 1 ml of the protein sample was loaded on the resins. The expressed protein was attached to the resins thanks to the binding of the histidine residues to the nickel ions of the binding buffer. In this regard, after 10 ml of the binding buffer was loaded twice, 10 ml of a washing buffer (100 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl/ pH 7.9) was poured three times so as to wash off non-specific binding proteins. The bound protein was eluted with an elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl/pH 7.9). For reclamation, the resins were completely cleaned with a strip buffer (100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl/pH 7.9), washed with sterile distilled water, and stored in 10 ml of 20% EtOH (protein purification was performed at 4°C in a cold room) . The protein in each step of the electrophoresis is shown in FIG. 3. The protein sample thus obtained was desalted through dialysis. First, the lysis buffer was added with 20% glycerol and supplemented with PMSF and BME to give a dialysis buffer. A dialysis tube containing the sample was immersed and dialyzed overnight against the dialysis buffer on a plate with a stirrer in a cold room. The protein thus obtained was quantitatively analyzed using a Bradford method, and stored at 20°C before use. The expressed protein was also confirmed through SDS- PAGE and Western blotting (refer to FIG. 3) . (2) Base sequencing of Deer Antler DNA and Selection of PDGF-B

1) Base sequencing of full-length DNA of deer antler The full-length DNA of deer antler was determined to consist of the 1,110 bases set forth in SEQ ID N0.:l. It has ATG as the initiation codon and TAA as the termination codon, as shown in FIG. 8. Its prepro-form has 729 bases, all of which can be expressed. When compared to the DNA base sequence of human PDGF-B, the 327 bases extending from the underlined mid ATG to the termination codon ACC were assumed to have been translated into a polypeptide consisting of 111 amino acid residues. This is given in SEQ ID NO. : 5.

2) Translation of deer antler PDGF-B cDNA

When the deer antler 2 cDNA was translated virtually using a translation program, the resulting polypeptide started with methionine and terminated in serine, as shown in FIG. 9 (underlined portion) . Mature PDGF-2 starts with serine and terminates with threonine, as expressed in bold in FIG. 9.

3) Homology comparison of PDGF-B cDNA between deer and other species

Deer antler PDGF-B cDNA was analyzed to have 88% homology with the human PDGF-B gene (FIG. 10) . Interspecies gene homology was found to range from 84 to 98% when partial sequences thereof were compared.

4) Homology comparison of PDGF-B peptide sequence between deer and other species

The deer antler PDGF-B peptide was determined to share 68% homology with human PDGF-B (FIG. 11) . Interspecies peptide homology was found to range from 32 to 98 % when partial sequences thereof were compared. Different from human PDGF-B in the 95..182 region (in detail, amino acid sequences in 146..159 beta-strand region, 160..161 hydrogen bond region, 162..175 beta-strand region are different) , the deer antler PDGF-B is expected to have a greater positive influence on the phosphorylation of PDGF-beta receptor and the activation of its downstream signaling molecule ERK 1/2, and thus on wound healing, than the human PDGF-B.

[EXAMPLE 3]

Wound Healing Effect of Deer Antler PDGF-B

(1) Material and Method

For the assay of the deer antler PDGF-B polypeptide activity, first, human fibroblast cells were purchased and cultured. After the cells were grown in a suitable medium for 2 days to 70% confluency, the medium was removed and cells were washed twice with PBS. The cells were damaged with a scraper in four predetermined regions of the culture dish. Two rounds of washing with PBS were conducted before the medium was supplied to the cells. The cells were observed before and after damage.

24 hours after the damage, the medium was replaced with fresh medium supplemented with 10 ng/ml of a purified PDGF-B(M) polypeptide. The cells were monitored for morphological change during 24~72 hours of incubation. For comparison of growth factor activity (in terms of dose effect and time course) between human and deer PDGF-B, the cells were treated separately with 0, 1, 10 and 100 ng/ml of the polypeptide obtained from recombinant human PDGF-B gene (SIGMA) and the PDGF-B polypeptide according to the present invention. Using Western blotting with a phosphor- ERK 1/2 (phospho-extracellular signal-regulated kinase 1/2) antibody, the phosphorylation of ERK 1/2, mediated by PDGF- B receptor binding, was assayed.

(2) Results

During 24-72 hours of incubation, none of the damaged cells were observed to undergo natural wound healing. In the presence of a PDGF-B polypeptide, the cells spread and started to grow around the wounds starting 24 hours after the incubation. On the third day of PDGF-B treatment, it was observed that the void spaces were filled with the cells. They were grown to 80% confluency of the void spaces on the fifth day of the treatment.

From 24 hours and within 72 hours after application to the damaged human fibroblast cells, the PDGF-B of deer velvet antlers exhibited a wound healing effect (FIG. 6) .

In order to determine whether the deer antler PDGF-B or the human PDGF-B has a more effective wound healing effect on undamaged normal cells, the activation ERK 1/2 was assayed using Western blotting with an anti-phospho-ERK 1/2 (phosphor-extracellular signal-regulated kinase 1/2) . There was no significant difference between human and deer PDGF-B polypeptides upon treatment with 1 and 10 ng thereof for 30 min and 6 hours. However, the activation was increased significantly upon treatment with 1 and 10 ng for 24 hours (respectively by 44% and 72%) and the deer PDGF-B further increased phosphorylation compared with the human PDGF-B (by 44% and 72%, respectively) (FIG. 7 and Table 1).

TABLE 1

Comparison of Effects of Deer PDGF B and Human PDGF B on Phosphorylation of Erk 1/2

As seen in Table 1, treatment with 100 ng/ml of PDGF- B was found to decrease phosphorylation. It is inferred that an excessive agonist concentration caused the down- regulation of PDGF-B receptors. Therefore, 10 ng/ml was determined to be suitable for treatment for promoting wound healing.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

[Sequence List: Text]

<110> Purimed Co.

<120> New gene and polypeptides of platelet derived growth factor B

<160> 5

<170> Kopatentln 1.71

<210> 1 <211> 1110 <212> DNA <213> Cervus elaphus

<220> <221> CDS <222> (78) .. (803)

<400> 1 cggaattccc gggctcgacc cacgcgtccg ctcggtccac gcgtccgggg ccccgcgggg 60

ccgggcccgg agtcggc atg aat cgc tgc tgg gcg etc ttc ctg tet etc 110

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu

1 5 10 tgc tgc tac ctg cgt ctg gtc age gcc gag gga gac cec att cec gag 158 Cys Cys Tyr Leu Arg Leu VaI Ser Ala GIu GIy Asp Pro lie Pro GIu 15 20 25 gag etc tac gag atg ctg agt gac cac tec ate cgt tec ttc gat gac 206 GIu Leu Tyr GIu Met Leu Ser Asp His Ser lie Arg Ser Phe Asp Asp

30 35 40 etc cag cgc ctg ctg cac gga gac tec gta gat gaa gac ggg get gaa 254 Leu GIn Arg Leu Leu His GIy Asp Ser VaI Asp GIu Asp GIy Ala GIu

45 50 55 ttg gac ctg aat ttg ace egg tec cat tct gga ggc gag ttc gag age 302 Leu Asp Leu Asn Leu Thr Arg Ser His Ser GIy GIy GIu Phe GIu Ser 60 65 70 75 tta tec cgt ggg aga agg age eta ggt tec ccg acg gtt gca gca gag 350 Leu Ser Arg GIy Arg Arg Ser Leu GIy Ser Pro Thr VaI Ala Ala GIu 80 85 90 eta gcc atg atg gcg gag tgc aag acg cgc acg gag gtg ttt gag ate 398 Leu Ala Met Met Ala GIu Cys Lys Thr Arg Thr GIu VaI Phe GIu He 95 100 105

teg egg cgc etc ata gac cgc acg aac gcc aac ttc ctg gtg tgg ccg 446 Ser Arg Arg Leu He Asp Arg Thr Asn Ala Asn Phe Leu VaI Trp Pro HO 115 120

ccc tgc gtg gag gtg cag cgc tgc tec ggc tgc tgc aac aac cgc aac 494 Pro Cys VaI GIu VaI GIn Arg Cys Ser GIy Cys Cys Asn Asn Arg Asn 125 130 135

gtg cag tgc egg ccc aca cag gtg cag gac egg aag gtg cag gtg aaa 542 VaI GIn Cys Arg Pro Thr GIn VaI GIn Asp Arg Lys VaI GIn VaI Lys 140 145 150 155 aag att gag ate gtg egg aag agg aaa ate ttt aag aag gcc aca gtg 590 Lys He GIu He VaI Arg Lys Arg Lys He Phe Lys Lys Ala Thr VaI

160 165 170 ace ttg gtg gac cac ctg gag tgc agg tgt gag acg gtg gtg gcg cga 638 Thr Leu VaI Asp His Leu GIu Cys Arg Cys GIu Thr VaI VaI Ala Arg

175 180 185 gcg gtg ace gga ace ccg ggg agt tec cag gag cag cga gca get agg 686 Ala VaI Thr GIy Thr Pro GIy Ser Ser GIn GIu GIn Arg Ala Ala Arg 190 195 200

acg ccc caa act egg gtg ace att egg acg gtg cga gtc cgc egg ccc 734 Thr Pro GIn Thr Arg VaI Thr He Arg Thr VaI Arg VaI Arg Arg Pro 205 210 215 ccc aag ggg aag cac egg aag ttc aag cac aca cat gaa aag acg gcg 782 Pro Lys GIy Lys His Arg Lys Phe Lys His Thr His GIu Lys Thr Ala 220 225 230 235 egg aag gag ace etc gga gee taggggc atctgcagga gagtgtgggc 830 Arg Lys GIu Thr Leu GIy Ala 240

agggttattt aatatggtat ttgctgtatt gcccccatgg ggtccttgga gtaaagggaa 890

aagatcccca gggcccccta ggggtggggt ctgacctccc acctcccttt ccaccttcta 950

ctgcactttc cctcttccct gctgtctccc aaatctgctt ccttcagttt gtaaagtcgg 1010

tgattatatt tttgggggct ttccttttat tttttaaatg taaaatttat ttatattccg 1070

tatttaaagt tgtaaaaaaa aaataaccac aaaacaaaac 1110

<210> 2

<211> 242

<212> PRT

<213> Cervus elaphus

<400> 2

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 Leu VaI Ser Ala GIu GIy Asp Pro lie Pro GIu GIu Leu Tyr GIu Met

20 25 30

Leu Ser Asp His Ser lie Arg Ser Phe Asp Asp Leu GIn Arg Leu Leu

35 40 45 His GIy Asp Ser VaI Asp GIu Asp GIy Ala GIu Leu Asp Leu Asn Leu

50 55 60

Thr Arg Ser His Ser GIy GIy GIu Phe GIu Ser Leu Ser Arg GIy Arg 65 70 75 80

Arg Ser Leu GIy Ser Pro Thr VaI Ala Ala GIu Leu Ala Met Met Ala 85 90 95

GIu Cys Lys Thr Arg Thr GIu VaI Phe GIu He Ser Arg Arg Leu He

100 105 HO

Asp Arg Thr Asn Ala Asn Phe Leu VaI Trp Pro Pro Cys VaI GIu VaI

115 120 125 GIn Arg Cys Ser GIy Cys Cys Asn Asn Arg Asn VaI GIn Cys Arg Pro

130 135 140

Thr GIn VaI GIn Asp Arg Lys VaI GIn VaI Lys Lys He GIu He VaI 145 150 155 160

Arg Lys Arg Lys He Phe Lys Lys Ala Thr VaI Thr Leu VaI Asp His 165 170 175

Leu GIu Cys Arg Cys GIu Thr VaI VaI Ala Arg Ala VaI Thr GIy Thr

180 185 190

Pro GIy Ser Ser GIn GIu GIn Arg Ala Ala Arg Thr Pro GIn Thr Arg

195 200 205 VaI Thr He Arg Thr VaI Arg VaI Arg Arg Pro Pro Lys GIy Lys His

210 215 220

Arg Lys Phe Lys His Thr His GIu Lys Thr Ala Arg Lys GIu Thr Leu 225 230 235 240

GIy Ala

<210> 3

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

*267<223> Deer Antler Velvet forward primer

<400> 3 ttaagaattc agcctaggtt ccccgacggt tgcagcagag 40

<210> 4

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Deer Antler Velvet reverse primer <400> 4 ttaactcgag ctaggtcacc gctcgcgcca ccaccgtctc aca 43

<210> 5

<211> 111

<212> PRT

<213> Cervus elaphus

<400> 5

Ser Leu GIy Ser Pro Thr VaI Ala Ala GIu Leu Ala Met Met Ala GIu 1 5 10 15

Cys Lys Thr Arg Thr GIu VaI Phe GIu lie Ser Arg Arg Leu lie Asp

20 25 30 Arg Thr Asn Ala Asn Phe Leu VaI Trp Pro Pro Cys VaI GIu VaI GIn

35 40 45

Arg Cys Ser GIy Cys Cys Asn Asn Arg Asn VaI GIn Cys Arg Pro Thr

50 55 60

GIn VaI GIn Asp Arg Lys VaI GIn VaI Lys Lys lie GIu lie VaI Arg 65 70 75 80

Lys Arg Lys lie Phe Lys Lys Ala Thr VaI Thr Leu VaI Asp His Leu

85 90 95

GIu Cys Arg Cys GIu Thr VaI VaI Ala Arg Ala VaI Thr GIy Thr

100 105 110

Claims

1. A novel platelet-derived growth factor-B protein having an amino acid sequence set forth in SEQ ID NO. : 2.

2. A gene coding for the platelet-derived growth factor-B protein of claim 1.

3. The gene as set forth in claim 2, wherein the gene consists of a nucleotide sequence extending from base 78 to base 803 of SEQ ID NO.: 1.

4. A composition for wound healing and for treatment and prevention of neurodegenerative diseases, ischemic neurological diseases and diseases causing neurological damage, through cell proliferation, development and differentiation, comprising the platelet-derived growth factor-B protein of claim 1 as an active ingredient.