KR100818332B1 - 2 New gene and polypeptides of Insulin-like growth factor-2 isolated from Cervi pantotrichum cornu - Google Patents

Abstract

The present invention relates to a novel insulin-like growth factor 2 polypeptide and its gene, and relates to a similar growth factor 2 (IGF 2) protein, an insulin isolated for deer antler. The protein promotes the growth of osteoblasts, and has shown utility as a therapeutic agent for osteoporosis. The present invention also relates to a gene encoding the IGF 2 protein of the deer, a recombinant vector containing the gene, and a host cell transformed with the recombinant vector.

IGF 2, antler, osteoporosis

Description

New gene and polypeptides of Insulin-like growth factor-2 isolated from Cervi pantotrichum cornu}

1 shows the full length sequence of IGF-2 cDNA. Initiation codon ATG and termination codon TAA of the coding region encoding IGF-2 are underlined.

Figure 2 shows the amino acid sequence translated IGF-2 cDNA, the underlined portion is a mature IGF-2 polypeptide.

Figure 3 compares the homology of the antler IGF-2 gene and human IGF 2 gene.

4 compares the homology between antler IGF-2 protein and human IGF 2 protein.

Figure 5 shows an electrophoretic picture of the overexpressed IGF-2 gene.

6 shows the results confirmed by Western blotting using an antibody against IGF-2.

FIG. 7 is a graph showing the correlation between fetal bovine serum concentration and osteoblast survival rate when 24 hours incubated with Fetal Bovine Serum (FBS) -added osteoblasts (HOS cells). The x-axis shows the concentration of fetal bovine serum and the y-axis shows the percentage survival of cells. The survival rate of cells was determined by MTS assay (SA O'Toole MSc, BL Sheppard PhD, JB Bonnar MD, EPJ McGuinness MD, NC Gleeson MD, M Yoneda MD, The MTS assay as an indicator of chemosensitivity / resistance in malignant gynaecological tumours, Cancer Detection and Prevention (2003) Vol 27, Issue 1). The line on the bar graph represents the SEM comparison using the Student Tee test. * P <0.05, ** P <0.01

Figure 8 shows the effect of deer antler IGF-2 on osteoblast cell viability at 3% FBS concentration. C: no treatment, T1: antler IGF-2 1 ng / ml treatment, T10: antler IGF-2 10 ng / ml treatment, T100: antler IGF-2 100 ng / ml treatment; * Is P <0.05, ** is P <0.01.

The present invention is an antler IGF-2 protein, an IGF-2 gene encoding the protein, a recombinant vector containing the gene, a host cell transformed with the recombinant vector, a pharmaceutical for treating osteoporosis comprising the IGF-2 protein To a red composition.

IGF (Insulin-like growth factor) is an insulin-like growth hormone, IGF-1 and IGF-2. IGF-1 is a single-chain polypeptide hormone that is structurally similar to proinsulin synthesized in liver and fibroblasts, indicating the action of growth hormone (GH). IGF-2 is a protein that is structurally similar to IGF-1. Growth hormone (GH) is secreted by the pituitary gland, which in turn produces IGF in the liver.

IGF is responsible for the formation of bone, muscle, nerve, and vascular tissues in the human body, ii) regeneration of damaged cells, ⅲ delayed aging of the pituitary gland, and blood sugar stabilization and normalization of insulin resistance. IGF has properties similar to insulin, which improves glucose and protein metabolism, helping to treat diabetes. In a double-blind clinical study of diabetes in 1997, gene binding of IGF-1 reduced insulin need in insulin-dependent diabetics (IDDM) by 45% (Internet http: //www.caregen. com / biz / igf.asp; accessed February 6, 2006).

Korean Patent Application No. 2004-046430 discloses a method for predicting the growth traits of pigs by the IGF concentration of pig blood using the properties of IGF, and Korean Patent Application No. 2004-0009660 discloses a method for the human scalp. A method for promoting hair growth and a hair growth agent for promoting hair growth have been disclosed, and in US Patent Application 2002-109971, a method for increasing the yield of recombinant protein through cell culture using IGF has been developed.

In Korean Patent Application No. 2005-7014530, growth factors such as IGF-1, IGF-2, VEGF (vascular endothelial cell growth factor) or PDGF (platelet derived growth factor) Nectin or fibronectin complexes have been reported to be effective in treating wounds and regenerating skin, and thus are useful for treating burns.

US Patent Application No. 2000-723449 discloses a therapeutic composition used for wound recovery of epithelial tissue by mixing PDGF, KGF (Keratinocyte Growth Factor) and IGF, and in Korea Patent 2005-7041530 It has been reported to inhibit cancer cell metastasis, particularly cancer cell metastasis associated with breast cancer by promoting cancer cell migration.

U.S. Patent Application 2001-972809 reports that osteoporosis is useful in the treatment of osteoclasts by inhibiting osteoclasts and activating osteoblasts that make bones. In general, osteoporosis appears to be caused by bone weakness and osteoclasts, so it is treated by inhibiting the action of osteoclasts to remove bones involved and activating the action of osteoblasts to make bones.

The present inventors have found a new IGF-2 gene through the gene analysis of antler. After mass production and purification of IGF-2 protein by genetic engineering method using the gene, administration to osteoblasts producing bone resulted in fatal reduction of fetal bovine serum (FBS). Even in this difficult situation, it was found that the survival rate of osteoblasts was significantly increased and the present invention was completed.

An object of the present invention is to provide a novel IGF-2 polypeptide and a gene encoding the polypeptide.

Another object of the present invention is to provide a recombinant vector comprising the gene and a host cell transformed with the recombinant vector.

It is another object of the present invention to provide an osteoblast function activator or composition for treating osteoporosis comprising the IGF-2 protein.

A first aspect of the invention relates to a novel insulin like growth factor (IGF) type 2 polypeptide. More specifically, the polypeptide relates to a polypeptide, characterized in that SEQ ID NO: 2.

By "functional equivalent" of an insulin-like growth factor polypeptide, by definition, it is meant a protein that exhibits substantially homogenous physiological activity with the IGF-2 protein of native deer.

“Functional equivalents” of the invention include amino acid sequence variants in which some or all of the naturally occurring protein amino acids are substituted, or some of the amino acids are deleted or added. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located in portions not directly involved in the physiological activity of insulin-like growth factors.

A “functional derivative” refers to a fragment of the IGF-2 polypeptide, a protein comprising the fragment, or a protein that has been modified to increase or decrease the physicochemical properties of the protein. The term "fragment" refers to an amino acid sequence corresponding to a portion of a protein and having a common origin element, structure and mechanism of action within the scope of the present invention. Also included within the scope of the invention are modifications to alter the stability, shelf life, solubility, etc. of the IGF-2 polypeptide or modifications made to alter the relationship with a substance that interacts with, for example, IGF-2. Methods of making functional derivatives of such proteins are known.

A second aspect of the invention provides a gene encoding an IGF-2 polypeptide, a gene characterized in that the gene comprises SEQ ID NO: 3 and a gene characterized in that the gene is derived from Cervus elaphus .

As used herein, “equivalent nucleic acid sequences” include sequences provided herein and sequences by allyl modifications, sequences by species-to-species variations, or codon degenerate sequences. The term "nucleic acid sequence group" includes native mRNAs that have been identified for selective expression, cDNA sequences complementary thereto, and nucleic acid sequences equivalent thereto.

“Equivalent nucleic acid sequences” include sequences provided herein and sequences by allyl modifications, sequences by species-to-species variations, or codon degenerate sequences. By "codon degenerate sequence" is meant a nucleic acid sequence that encodes a polypeptide that differs from the naturally occurring sequence but is identical to the naturally occurring IGF-2 polypeptide disclosed herein.

"Sequence by allylic modification" or "sequence by interspecies modification" differs from the naturally occurring nucleic acid sequence but possesses substantially the same functional properties as the polypeptides disclosed herein. Refers to a nucleic acid sequence encoding a polypeptide.

The third aspect of the present invention provides a recombinant vector containing the gene, a microorganism transformed with the recombinant vector, and a method of mass-producing IGF-2 protein by culturing the microorganism.

Particularly preferred method for producing IGF-2 protein is to use genetic recombination technology. DNA or RNA sequences encoding IGF-2 are expressed in appropriate host cells to form cell lysates or in vitro translation of IGF-2 mRNA followed by purification of IGF-2 proteins by protein isolation methods known in the art. can do. Usually, in order to remove cell debris and the like, the cell lysate or the result of in vitro translation is centrifuged, followed by precipitation, dialysis, various column chromatography, and the like. Ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, preparative SDS-PAGE, affinity columns and the like are examples of column chromatography. Affinity columns can be made using, for example, anti-IGF 2 antibodies.

Functional derivatives of IGF-2 proteins, including fragments derived from IGF-2 polypeptides of the present invention, can be prepared using one of the known organic chemical methods for peptide synthesis. Organic chemical methods of peptide synthesis include coupling the necessary amino acids by a condensation reaction on a homogeneous phase or on a so-called solid phase support. The most common methods for condensation reactions include the carbodiimide method, the azide method, the mixed anhydride method and the method using the active esters, which are described in The Peptides Analysis, Synthesis, Biology Vol. Gross, E. and Meienhofer, J.), 1979-1981 (Academic Press Inc.).

Particularly suitable solid phases include p-alkoxybenzyl alcohol resins (4-hydroxy-methyl-phenoxy-methyl-copolystyrene-1% described in Wang, J. Am. Chem. Soc., 95, 132 (1974)). Divinylbenzene resins). After synthesis the peptide can be separated from the solid phase under gentle conditions. After synthesis of the desired amino acid sequence, the detachment of the peptide from the resin is carried out using trifluoroacetic acid containing a scavenger such as triisopropyl silane, anisole or ethanedithiol, thioanisole. Reactive groups that cannot be involved in the condensation reaction are effectively protected by groups that can be removed very easily again by hydrolysis or reduction with acids, bases. Possible protecting groups are described in more detail in The Peptides, Analysis, Synthesis, Biology, Vol. 1-9 (edit by Gross, Udenfriend and Meienhofer) 1979-1987 (Academic Press Inc.).

A fourth aspect of the present invention is to provide a pharmaceutical composition for activating osteoblasts and treating osteoporosis comprising the IGF-2 polypeptide, active fragment, and / or functional derivative. The pharmaceutical composition may be prepared and administered in various formulations and methods.

For example, oral administration can be mixed with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, pressed into tablets or administered directly in the diet. For oral administration, the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.

The tablets, troches, pills, capsules and the like may be used in combination with a binder such as gum tragacanth, acacia, corn starch or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch, potato starch, alginic acid and the like; Lubricants such as magnesium stearate; And sweetening agents such as sucrose, lactose or saccharin; Or flavoring agents such as peppermint, winter green oil or cherry flavor. If the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above substances. Various other materials may be added that change the physical form of the coating or dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. Elixir syrups may contain sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes and flavoring agents such as cherry or orange flavors with the active compound. All materials used to prepare any dosage unit form must be pharmaceutically pure and substantially nontoxic in the amounts used. In addition, the active compounds may be prepared in sustained release formulations.

For oral administration, the compositions of the present invention can be used in the form of non-ingestive mouthwashes and toothpastes in admixture with excipients. Mouthwashes can be prepared by adding the active ingredient in the required amount to a suitable solvent, such as sodium borate solution. Alternatively, the active ingredient may be added to an antiseptic cleaner containing sodium borate, glycerin and potassium bicarbonate, or dispersed in toothpaste in the form of gels, pastes, powders and slurries, or water, binders, abrasives, flavoring agents, foaming agents and It may be added in a therapeutically effective amount to a paste toothpaste that may include a humectant.

Various formulations, such as for other injectable and parenteral administration, can be prepared according to the techniques described or commonly used in books well known in the art (eg, "Remington's Pharmaceutical Sciences" 15th Edition).

Dosages can vary depending on the condition of the treated patient, and the person in charge can determine the appropriate dose for each patient. In addition, for human administration, the preparations must meet sterile, pyrogenic, general safety and purity criteria in accordance with the Agency.

The invention is illustrated below by the following examples. However, the present invention is not limited to the following examples.

Example 1 Isolation of Antler mRNA

(1) Selection and Collection of Samples

The horns of the deer buses, which are considered to have the best pharmacological effect among the deer producing deer antler, were cut in the second season (early to mid-May) when the growth activity was active. The cut tissues were rapidly frozen using dry ice and ethanol, and then the growing tissues were separated and stored at -80 ° C.

(2) Total RNA Isolation from Growth Tissue

3 ml of trizol (Gibco BRL) and 3 g of growth tissue separated into a 50 ml conical tube were homogenized with a homogenizer. 27 ml of trizol was added thereto, mixed well, and allowed to stand at room temperature for 5 minutes. The mixture was divided into 7.5 ml each of four tubes, and 1.5 ml chloroform was added to each tube. After standing at room temperature for 3 minutes, the mixture was centrifuged at 12,000 rpm for 15 minutes. 3 ml of the supernatant was transferred to a new tube, and 3.75 ml of isopropanol was added to each tube, and the mixture was allowed to stand at 4 ° C. for 10 minutes and then centrifuged at 12,000 rpm for 10 minutes to remove the supernatant. The total RNA as a precipitate was washed with 7.5 ml of 75% ethanol, and again centrifuged at 7,500 rpm for 5 minutes to remove the supernatant. The total RNA was dried at room temperature and stored in 225 μl DEPC treated distilled water.

  (3) mRNA isolation from total RNA

  MRNA was isolated from the total RNA isolated as described above using an Oligotex mRNA midi kit (Qiagen).

 Example 2 cDNA Synthesis from Deer Antler mRNA and Preparation of cDNA Library

cDNA synthesis from mRNA was performed using a Gibco BRL cDNA synthesis kit, and some modification of the experimental method.

The first strand cDNA synthesis, 2 μl of Not I primer-adapter (0.5 μg / μl) was added to 9 μl of mRNA (2 μg), mixed well, and reacted at 70 ° C. for 10 minutes to denature RNA secondary structure. After cooling on ice for 2 minutes, add 4 µl of 5X first strand buffer, 2 µl of 0.1M DTT and 1 µl of 10 mM dNTP, mix well, and react for 2 minutes at 37 ° C. Then, 2 µl of Superscriptase II. After the addition was allowed to react at 37 ° C. for 60 minutes, it was allowed to stand on ice for 2 minutes.

The second strand cDNA synthesis was carried out in the reaction solution 91 μl DEPC treated distilled water, 30 μl second strand buffer, 3 μl 10 mM dNTP mixture, 1 μl E. coli DNA ligase (10 U / μl), E. coli (E. coli) 1 μl of DNA polymerase (10 U / μl) and 1 μl of E. coli RNA degrading enzyme H (2 U / μl) were added thereto, followed by reaction at 16 ° C. for 2 hours, followed by addition of 2 μl of T4 DNA polymerase (10 U). The reaction was further performed for 5 minutes. After the reaction, 10 μl of 0.5 M EDTA was added, and cDNA was extracted using phenol-chloroform and dissolved in DEPC-treated distilled water.

The EcoR 1 -BstX 1 adapters (Invitrogen) in the synthesized cDNA were ligated and, prepare cDNA with Not 1 and EcoR 1 sequence at each end of cDNA strand was cut with Not 1. Collected by removing the cDNA of a desired size through a gel filtration column, and the vector having the Not1 and EcoR1 sequence in the multi-cloning site (pDONR222, pSPORT 1, pBluescript, pPICZ, pYES3 / CT, etc.) were ligated. E. coli (ElectroMAX DH5α, Gibco BRL) was transformed using an electropenetration (electroporation) method to amplify plasmid vectors containing cDNA.

Example 3 Determination of Antler DNA Sequence and IGF-2 Selection

(1) Manufacture and method

After EST clones were isolated from cDNA library of deer antler, DNA was extracted from each clone, and DNA sequence analysis was performed in one direction using ABI's BigDye Terminator Cycle sequence Kit. Was performed. Next, after sequencing the whole clone with the initiation codon, the termination codon was found, and the sequence homology with other species was compared through NCBI, and the amino acid species homology when translated. Finally, IGF- is well known for the treatment of osteoporosis. 2 genes were found.

(2) results

1) Deer Antler IGF-2 cDNA Sequence

It consists of 681 bases in total and has an initiation codon ATG and a termination codon TAA as underlined in FIG. 1 . They have a prepro-form of 561 bases that can be fully expressed. The mature form consists of 186 amino acid sequences.

(2) Antler DNA sequencing and IGF-2 screening

1) Full length length analysis of antler DNA

It consists of 681 bases and is represented by SEQ ID NO: 1. SEQ ID NO: 1 has an initiation codon ATG and the termination codon TAA, as shown in FIG. They have 561 full length prepro-forms that can be fully expressed. The underlined ATG and ending with TAA consist of 561 nucleic acids in a mature form when comparing human IGF 2 to DNA sequences, which translates to 186 amino acids. This is represented by SEQ ID NO: 2. The electrophoresis results for this are shown in FIG. 5 .

2) Translation of Antler IGF 2 cDNA

As a result of translating the antler IGF 2 cDNA on the Internet into a translation program, it can be seen that IGF 2 starts with methionine and ends with serine, as shown in FIG. 2 .

3) Comparison of homology with other species of Deer Antler IGF 2 cDNA

It was 87% similar to the human IGF gene ( FIG. 3 ). High homology was observed between 83% and 100% (partial sequence comparison), depending on the specific portion of the nucleic acid between different species.

4) Homology Comparison with Other Species at Deer Antler IGF 2 Protein Levels

It was 77% similar to human IGF 2 protein ( FIG. 4 ) and showed homology between 44% and 100% (partial sequence comparison), depending on the specific part of the different interspecies proteins. In comparison of human and deer antler IGF 2, the 31..84 region is different and affects the effect of IGF 2, which is expected to be a better treatment for osteoporosis than human IGF 2.

 Example 4 Preparation of E. Coli Strains Containing Antler IGF-2 cDNA

E. coli strains were transformed with a plasmid vector containing antler IGF-2 cDNA using an electropenetration method. Escherichia coli cells grown from 1.3 to 1.5 at an OD ₆₀₀ were made into competent cells to allow electropermeation, and 80 µl of these cells were transformed with 5 µg of vector containing antler cDNA (1.5 kV, 25 µF, 200 Ω). , 0.2 cm cuvette, Gene-Pulser, BioRad).

 Example 5 Mass Production and Purification of IGF-2 Protein Using Transformed Escherichia Coli

  (1) Manufacture and method

The desired portion was eluted from the vector prepared according to Example 4 to express the protein of IGF-2. For this purpose, put the prepared primer (SEQ ID NO: 4) and reverse primer (SEQ ID NO: 5) 2 μl / 10 pmol, dNTP mixture, Taq-DNA polymerase, MgCl ₂ and the reaction buffer, and PCR Gel eluted.

After ligating T4 DNA ligase (3U / μl) and PCR product into a T-vector (50 ng / μl) system designed to attach the poly -A tail of the PCR product, the ligated DH5α was made by chemical method. Were transformed into a thermal shock (42 ° C.). The insert was prepared by the enzyme digestion with EcoR I and Xho I. PET28a (+) was used as a vector to express a protein, and the vector was prepared by enzyme digestion with Eco RI and Xho I. The prepared expression vector and the insert were ligated. The conditions were performed for 16 hours at 16 ° C. The resulting ligation was transformed into BL21 (DE3), and each transformant was grown to see if it was completely composed, and then the plasmid DNA obtained through the Miniprep kit (QIAGEN) was digested with EcoR I and Xho I. The size of the vector (approximately 5.3 kb) and the insert (0.7 kb) was confirmed. After confirming the above configuration for protein expression, the identified colonies were put in 3ml of LB (50 μg / ml kanamycin) in a 15ml tube, and grown overnight. The cells were placed in a 250 ml flask with 100 ml of LB medium and grown in a 37 ° C stirred incubator until the OD 600 value was 0.4-0.5. At this time, IPTG (1 mM), a protein expression derivative, is added and grown overnight at room temperature. The grown cells were harvested and resuspended in lysis buffer (50mM NaCl, 5mM MgCl ₂ , 50mM Tris-HCl / pH 7.9), and 0.1 mm PMSF and 10 mM BME (Basal Medium Eagle) were added if necessary. . The prepared solution was added to the lysozyme (1g / 10 ml) stock solution, incubated at 37 ° C. for about 30 minutes, and then sonicated. Soluble fractions were obtained and stored at 20 ° C. Ni ²⁺ was attached to the resin using an Affinity column system, followed by dialysis of the protein obtained above. First, the sticky resin was loaded on the column (2 ml), and when it passed out, the sterilized distilled water was loaded about 6 ml to check the flow rate. Next, 10 ml of charging buffer (50 mM NiSO ₄ ) was poured, followed by 6 ml of binding buffer (20 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl / pH 7.9). , When the binding buffer was passed out, 1ml of the protein sample obtained above was flowed. Since the expressed protein will express histidine, the protein to be obtained will be bound to the resin due to the binding buffer. In this step, 10 ml of binding buffer was loaded twice and flowed out, followed by washing with 10 ml of washing buffer (100 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl / pH 7.9) to wash out nonspecific binding protein. It was loaded once. Elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl / pH 7.9) was added to elute the desired protein. To use the resin again, add strip buffer solution (100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl / pH 7.9) to wash the resin thoroughly, wash with sterile distilled water, and then add 20% EtOH 10 Resin was stored in about ml (experiment related to protein purification in a cool room, 4 ° C). Dialysis was performed for desalination of the purified sample. First, prepare a buffer solution by adding 20% glycerol to the digestion buffer, add new PMSF and BME, and then put the sample in the dialysis tube so that the tube is properly submerged on a stirring plate and returned overnight in a cold room. . The protein thus prepared is quantified by the Bradford method and stored at 20 ° C until use. The expressed protein was confirmed by SDS-PAGE and western blotting.

(2) results

As shown in FIG. 6 , the recombinant antler IGF-2 obtained by separating and purifying whole protein extracted from E. coli overexpressed with antler IGF-2 on SDS-PAGE. Results confirmed by Western blotting using an antibody against IGF-2. The purified recombinant antler IGF-2 showed a molecular weight of about 20 kDa.

Example 6 Confirmation of Osteoblast Proliferation Effect of Deer Antler IGF-2

(1) Manufacture and method

1) Culture of Osteoblasts

Osteoblasts (HCL cell, TE-85 human, female, osteosarcoma; bone) were supplied from Korea Cell Line Bank (KCLB # 21543), 10% fetal bovine serum (SBS), 1% penicillin Using RPMI 1640 medium (Invitrogen Life Technologies, USA) containing stricomycin (penicillin-streptomycin, Invitrogen Life Technologies, USA), 2 g / L NaHCO ₃ (JRH Biosciences, USA), 10 mM Hepes (JRH Biosciences, USA) The cells were cultured in 37 ° C., 5% CO ₂ incubator (Nuaire, USA).

2) Measurement of osteoblast proliferation according to fetal calf serum

In order to determine the concentration of fetal bovine serum that causes inhibition of proliferation of osteoblasts, cell proliferation was measured for each concentration of fetal bovine serum. 2 × 10 ⁴ cells / ml osteoblasts were dispensed into 96-well plates and incubated in a 37 ° C., 5% CO ₂ incubator for 24 hours, followed by phosphate buffer solution (PBS, Phosphate Buffered Saline, Invitrogen Life Technologies, USA). After washing twice, fetal calf serum was diluted in medium so that the final medium concentration was 0, 0.3, 3, 5, 10%, and 100 μl each was incubated in a 37 ° C., 5% CO ₂ incubator for 24 hours. Using the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega, UK), add 20 μl of MTS solution per 100 μl into each well and in a 37 ° C., 5% CO ₂ incubator. After the reaction for 1 hour it was measured at 490nm using a microplate reader (Molecular Device, USA). Results are expressed as% cell viability based on absorbance at standard fetal bovine serum concentrations of 10%.

(2) results

To determine the concentration of fetal calf serum that inhibits the proliferation of osteoblasts, fetal calf serum was administered for each concentration and the results of measuring the proliferative capacity of osteoblasts are shown in FIG .

In general, the osteoblast proliferation ability of each low concentration of fetal bovine serum was significantly lowered based on the medium containing 10% fetal bovine serum used to culture osteoblasts. When the fetal bovine serum concentration was lowered to 3% compared to 10% fetal bovine serum, osteoblast proliferation decreased by more than 25%. In particular, 0.3% and 0% showed a significant drop of P <0.01, indicating that low concentrations of fetal bovine serum act as a stress factor to lower the proliferative capacity of osteoblasts.

Example 7 Measurement of Proliferation Capacity of Osteoblasts, Osteoblasts According to Antler IGF-2 Treatment

1) Culture of Osteoblasts

Osteoblasts (HCL cell, TE-85 human, female, osteosarcoma; bone) were supplied from Korea Cell Line Bank (KCLB # 21543), 10% fetal bovine serum (SBS), 1% penicillin Using RPMI 1640 medium (Invitrogen Life Technologies, USA) containing stricomycin (penicillin-streptomycin, Invitrogen Life Technologies, USA), 2 g / L NaHCO ₃ (JRH Biosciences, USA), 10 mM Hepes (JRH Biosciences, USA) The cells were cultured in 37 ° C., 5% CO ₂ incubator (Nuaire, USA).

2) Measurement of osteoblast proliferation according to FBS (Fetal bovine serum) concentration

In order to confirm the FBS concentration causing the proliferation inhibition of Hos cells, cell proliferation was measured for each FBS concentration. 2 × 10 ⁴ cells / ml of Hos cells were dispensed into 96 well plates and incubated in a 37 ° C., 5% CO ₂ incubator for 24 hours and washed once with phosphate buffer solution (PBS; Invitrogen Life Technologies, USA). Thereafter, 100 μl of FBS was diluted in the medium so that the final medium concentration was 3%, and cultured in a 37 ° C., 5% CO ₂ incubator for 24 hours. Using the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega, UK), add 20 µl of MTS solution per 100 µl to each well and add a 37 ° C, 5% CO ₂ incubator. After 1 hour of reaction at 490nm using a microplate reader (Molecular Device, USA) was measured. The results are expressed in% cell viability based on absorbance at 10%, the standard FBS concentration.

3) Measurement of osteoblast proliferation according to the concentration of antler IGF-2

Dispense 2 × 10 ⁴ cells / ml of Hos cells into a 96 well plate, incubate in a 5% CO ₂ incubator at 37 ° C. for 24 hours, wash once with PBS, 3% FBS and each deer Antler IGF-0 , 1, 10, 100 ng / ml aliquot and incubated for 48 hours in a 37 ° C., 5% CO ₂ incubator, followed by CellTiter 96 AQueous One Solution cell proliferation assay kit (Promega, UK) and 20 μl of MTS solution per 100 μl in each well and reacted at 37 ° C., 5% CO ₂ incubator for 1 hour and measured at 490 nm using a microplate reader. The results are expressed as cell viability (%) based on the absorbance of the sample not administered antler IGF-2 at 3% FBS concentration.

4) Results

As shown in FIG. 8 , the cell survival rate was increased by 43% and 29%, respectively, when compared with the group treated with 1 ng / ml and 10 ng / ml of IGF-2 and not treated with IGF-2. At 100 ng / ml, the survival rate was increased by 12% but there was no statistical significance. This is probably due to down regulation of the IGF-2 receptor by excessive concentrations of IGF-2.

In general, osteoporosis appears to be caused by bone weakness and osteoclast, and thus can be treated by inhibiting the action of osteoclasts to remove bones involved and activating the action of osteoblasts to make bone. As a result of mass production of novel IGF 2 protein isolated from deer according to the present invention using a gene recombination method, the concentration of FBS is fatally reduced and osteoblasts are difficult to survive. Increased survival rate. This shows that the IGF-2 protein isolated from the deer antler according to the present invention is an effective substance as a therapeutic agent for osteoporosis.

Attach sequence list electronic file

Claims (10)

Insulin-like growth factor (IGF) type 2 polypeptides having the amino acid sequence of SEQ ID NO: 2. delete A gene encoding the insulin-like growth factor (IGF) type 2 polypeptide of claim 1. 4. The insulin like growth factor (IGF) type 2 gene according to claim 3, wherein said gene consists of SEQ ID NO: 3. 4. The gene according to claim 3, which is derived from Cervus elaphus . A recombinant vector containing the gene of claim 3. A host cell transformed with the recombinant vector of claim 6. 8. The transformed host cell of claim 7, wherein said host cell is Escherichia coli. A method for producing a polypeptide according to claim 1, wherein the microorganism of claim 7 is cultured. A composition for the activation of osteoblast activity or the treatment of osteoporosis comprising the insulin-like growth factor (IGF) type 2 protein according to claim 1.

Images