JPH07267993A - New protein shbp-10 - Google Patents

Abstract

PURPOSE:To obtain a new protein serum heparin binding factor protein-10 (SHBP-10), available from blood of a fetus and a neonate of mammals including humans, having a specific molecular weight and a specified amino acid sequence, capable of manifesting growth promoting activities for fibroblasts and useful as a therapeutic agent, etc., for wounds. CONSTITUTION:This new protein SHBP-10 is obtained from blood of a fetus and a neonate of mammals including humans and has about 8-12 kDa molecular weight measured by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and an amino-terminal sequence having an amino acid sequence expressed by the formula. Furthermore, the protein is capable of manifesting growth promoting activities for fibroblasts and useful as a therapeutic agent, etc., for wounds. This protein is prepared by adding sodium chloride to a serum of a bovine neonate, passing the resultant mixture through a column immobilizing a heparin, eluting the adsorbed substance. then treating and purifying the obtained eluate according to the reversed phase liquid chromatography.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel protein, and more particularly to a protein having heparin-binding ability and fibroblast proliferation activity. The protein of the present invention is SHBP-10 (Serum Heparin Binding Protein-
It is named 10) and can be obtained from the blood of fetuses and newborns of mammals including humans.

[0002]

It is known that various growth factors are involved in the process of wound healing (Dijke et al., Bi
otechnology, vol.7, p793-798, 1989). In particular, TGF
-Β (transforming growth factor-β) and PDGF (p
latelet-derived growth factor) is known to proliferate fibroblasts, attract cells to the lesion site, and promote wound repair. However, it has not been reported until now that the substance of the present invention proliferates fibroblasts and is effective for treating wounds.

[0003]

Therefore, an object of the present invention is to provide a protein which can be used as a new therapeutic agent effective for treating wounds.

[0004]

Means for Solving the Problems It is considered that the blood of fetuses and newborns of mammals including humans contains various growth factors that stimulate the growth of cellular tissues of rapidly growing fetuses and newborns. To be We attempted to purify and isolate a new protein factor with fibroblast proliferation activity using the bovine neonatal serum, which is easily available quantitatively, as a starting material. For the measurement of fibroblast proliferation activity, for example, Balb / 3T3 cell line can be used.

Since various growth factors were known to easily bind to heparin, first, heparin affinity column chromatography was used to separate the fractions bound to the column, and then, further fine phase separation was performed by reverse phase liquid chromatography. It can be divided into fractions. Fibroblast proliferation activity was measured for each of these fractions. Thus, the N-terminal amino acid sequence and amino acid composition of one of the obtained active fractions was determined, and it was examined whether or not it was a known protein with reference to the protein and gene sequence database. I knew there was.

This protein was named SHBP-10. SHBP-10 provided by the present invention is a novel protein isolated from animal blood, which has the following physical properties and N-terminal amino acid sequence. (1) Molecular weight: about 8-12 KDa (SD under reducing conditions
(By S-PAGE method) (2) N-terminal amino acid sequence: amino acid sequence shown in SEQ ID NO: 1 in the sequence listing (3) Properties: heparin-binding and fibroblast proliferation action. SHBP-10 does not lose the fibroblast proliferation effect,
It can be expected that a protein further suitable for a preparation can be obtained by slightly changing amino acid residues, adding chemical modification, or performing fragmentation.

SHBP-10 is highly water-soluble, and the most suitable method for treating wounds is to mix it with a suitable water-soluble base and apply it directly to the affected area locally. It can be administered as an intravenous injection or a subcutaneous administration agent, and as an aerosol formulation of fine particles,
It can be administered nasally or pulmonary. The dose is 1 to 100 μg / administration site / person / day for local administration, and 0.1 to 10 mg / kg / day for systemic administration. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0008]

【Example】

Example 1 Fibroblasts from bovine neonatal serum (Bal
b / 3T3 cells) Purification of growth promoting factor 1) Crude purification by heparin affinity chromatography method 10.5 liters of bovine newborn serum (purchased from GIBCO Laboratories) was added with sodium chloride (20.5 g). This newborn serum was used in advance in Tris buffer A (20 mM Tris-HC
l, pH 7.5, 0.5M NaCl) equilibrated with heparin
Develop on a Toyopearl column (diameter 5 cm x length 5.5 cm, Tosoh Corporation) at a flow rate of 3 ml / min. Then, the column is thoroughly washed with Tris buffer A. After washing, the peptide or protein adsorbed on the heparin-Toyopearl column was washed with Tris buffer B (20 mM, Tris-HCl, pH 7.5, 1.0 MN).
elute with aCl). For the eluate, use an absorptiometer and 2
Monitor the absorbance at 80 nm,
About 300 ml was collected.

2) Purification by the reverse phase liquid chromatography method The eluate obtained in the operation 1) was previously equilibrated with water containing 0.1% trifluoroacetic acid (TFA), and the Cosmosir 5C18-300 column was used. (Diameter 4.6 mm x length 250 mm, Nakarai Tesque, Inc.), 0.1% T
It was washed thoroughly with water containing FA. After that, the adsorbed peptide or protein is adjusted to an acetonitrile concentration of 0 to 8
Elute with a 0% linear gradient. The eluate is
Monitored by absorbance at 214 nm and collected peak by peak. The elution pattern is shown in FIG.

Example 2 Measurement of Fibroblast Growth Promoting Activity 96 Fibroblast cell line Balb / 3T3 cells (purchased from ATCC, Catalog No. CCL163-Balb / 3T3-clone A31) were used.
Seed 5 x 10 ³ cells per well in a well culture plate and add 10% fetal bovine serum to Dulbecco's modified Eagle ME.
Add 100 μl of M medium (hereinafter DME), and add 2 in the incubator.
Incubated at 37 ° C. for 4 hours. Then, after removing the culture medium and washing the cells, a low serum medium (DM containing 0.2% fetal calf serum was used.
E) 100 μl was added, and the cells were further cultured for 3 days. There,
10 μl of each fraction obtained in Example 1 was added and cultured for 15 hours. Then, ³ H-thymidine was added to 74 KBq / ml, and the mixture was cultured for 6 hours. After the culture, the medium was removed, the cells were collected, and the amount of ³ H-thymidine incorporated into the cells was measured. As a result, a cell growth promoting activity was observed in the fraction of peak 1 (arrow) in FIG.

Example 3 Measurement of physicochemical properties of peak 1 peptide 1) N-terminal amino acid sequence analysis For the peptides of the fractions whose activity was confirmed in Example 2, N
Terminal amino acid sequence is amino acid sequencer, model 4
When analyzed by 77A (Aplite Biosystems), the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing was confirmed. This sequence is
When it was examined whether or not there was a match, it was confirmed to be a novel protein.

2) Amino acid composition analysis The amino acid composition of the peptide in the fraction of peak 1 was examined by an amino acid analyzer. Table 1 shows the results of the amino acid composition analysis.

[0013]

[Table 1] Amino acid analysis results (* 1) Asp 10.4 (10) Glu 11.6 (12) Ser 4.8 (5) Gly 5.0 (5) His 3.8 (4) Arg 4. 0 (4) Thr 5.9 (6 ) Ala 4.3 (4) Pro 3.5 (4) Tyr 1.7 (2) Val 6.2 (6) Met 0 (0) 1/2 Cys 0 ( 0) Ile 5.5 (6) Leu 9.6 (10) Phe 4.4 (4) Lys 8.9 (9) Trp N.D. (* 2) (* 1) Calculated as a molecular weight of 10,000 Daltons (* 2) undecided

3) Analysis by Electrophoresis The molecular weight of the peptide of peak 1 is determined by SDS-P under reducing conditions.
When confirmed by AGE, it showed a molecular weight of approximately 8-12 KDa (FIG. 2). FIG. 2 shows the electrophoresis pattern of the peptide of peak 1, and in FIG. 2, lane 1 shows peak 1
Shows the band of the peptide, and lane 2 shows the molecular weight marker.

From the results shown in the above 1), 2) and 3), it was confirmed that the peptide of peak 1 having a fibroblast proliferation activity was a novel protein.

Example 4 Measurement of SHBP-10 Fibroblast Proliferation Activity Further, in order to examine the dose-dependent cell growth promoting activity of the present protein obtained in Example 2, SHBP-10 was added to each well in an amount of 0.03. ³ H-thymidine incorporation was measured by adding according to the method of Example 2 so that the concentration was -10 μg / ml. Table 2 shows the results of the measurement of the fibroblast proliferation activity of SHBP-10. The data show the mean and standard deviation of each group (4 cases in 1 district).

[0017]

Table 2 Additive compound dose (μg / ml) ³ H-thymidine incorporation (cpm) vs. control group-100 SHBP-10 0.025 99.4 ± 26.6 0.25 136.0 ± 25.0 2 .5 4005.0 ± 217.0

From these results, it was confirmed that SHBP-10 has a cell growth promoting activity in a dose-dependent manner.

[0019]

EFFECT OF THE INVENTION Protein SH provided by the present invention
BP-10 has a fibroblast proliferation promoting activity and is therefore useful as a wound healing agent.

[0020]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin: Organism name: Bos taulus Tissue type: Serum Sequence characteristics: None Sequence: Thr Lys Leu Glu Asp His Leu Glu Gly Ile Ile Asn Ile Phe His Gln Tyr 1 5 10 15 Ser Val Arg Val Gly His Phe Asp Thr Leu Asn Lys Arg 20 25 30

[Brief description of drawings]

FIG. 1 shows a pattern in which a fraction eluted by binding to heparin affinity column chromatography was further developed by reverse phase liquid chromatography. The arrow indicates the active fraction containing this protein.

FIG. 2 is a diagram showing an electrophoresis pattern of a peptide of peak 1.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area C07K 14/47 8318-4H

Claims (4)

[Claims] 1. A protein SHBP-10 having a molecular weight of about 8-12 KDa as analyzed by SDS-PAGE under reducing conditions and having an amino terminal sequence having the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. The protein SHBP-10 of claim 1, which is derived from mammalian blood. 3. The protein SHBP-10 of claim 1 or 2 which is heparin-binding. 4. A wound healing agent containing the protein SHBP-10 according to any one of claims 1 to 3.