WO1999063083A1

WO1999063083A1 - Novel gene and protein encoded thereby

Info

Publication number: WO1999063083A1
Application number: PCT/JP1999/002813
Authority: WO
Inventors: Akiko Ikeda; Megumi Yamashita; Katsuki Tsuritani; Makoto Yoshimoto; Seiji Arase
Original assignee: Taisho Pharmaceutical Co., Ltd.
Priority date: 1998-05-29
Filing date: 1999-05-28
Publication date: 1999-12-09
Also published as: AU3955099A; JPH11332571A

Abstract

A novel protein DERP2 originating in human hair papilla cells and having an effect of regulating the growth of hair; and a gene derp2 encoding the same. The gene derp2 encoding the novel protein DERP2 having an effect of regulating the growth of hair can be obtained by cloning from a cDNA library originating in human hair papilla cells. Because of having an effect of regulating the growth of hair, this protein is usable in developing hair growth promoting agents, etc.

Description

Specification

Novel genes and proteins encoded by them

The present invention relates to a novel protein having a function of regulating hair growth, such as DERP (dermalpapilladel erotein) 2, and a gene derp2 encoding the protein. Background art

Human hair follicles contain various epithelial and dermal cells such as keratinocytes, dermal papilla cells, fibroblasts and sebaceous cells, and the hair cycle (hair growth cycle) Is regulated through cell-cell interactions.

Among these cells, hair follicle keratinocytes produce the hair fibers, but hair papilla cells are thought to play a central role in regulating the proliferation and differentiation of these cells. Have been. In other words, it is thought that the papilla cells function as a controller of the hair cycle. In light of the above background, the analysis of the mechanism of hair cycle regulation centering on the dermal papilla is currently being actively conducted, but the molecular mechanism of hair growth has not yet been elucidated.

It is known that androgenetic alopecia develops due to excessive action of androgens on hair follicles. Androgens are the most important factors regulating hair growth, but their mechanism of action has not yet been elucidated.

Hair papilla cells express high levels of the andorogen receptor and the testosterone-metabolizing enzyme 5a-reductase, and the expression of androgen receptors in hair papilla cells is higher than that of the hair growth area. Due to high levels in the cervix, the major target cell for androgens in hair follicles is thought to be the dermal papilla cells. In other words, androgen is thought to act on the hair papilla and regulate hair growth by altering the production of factors such as hair papilla cell-derived factors.

From the above findings, it is predicted that factors derived from the dermal papilla, whose production is changed by androgen, play an important role in hair growth, but it is still unknown what this factor is. Not disclosed. As described above, factors involved in the growth of hair derived from the dermal papilla, particularly proteinaceous factors, are extremely useful for searching for a bioactive substance having a hair promoting action. An object of the present invention is to provide a novel protein and a gene thereof capable of regulating such hair growth in the process of elucidating the molecular mechanism related to hair growth. Disclosure of the invention

The present inventors aimed to identify factors involved in hair growth, and as a result of intensive studies to determine the desired protein from genes highly expressed in human hair papilla cells, the novel protein DERP 2 The present inventors succeeded in isolating the gene derp 2 encoding the gene and completed the present invention.

That is, the present invention relates to (a) a protein comprising the amino acid sequence of SEQ ID NO: 1, or (b) an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1. The present invention relates to a protein comprising a sequence and having a function of regulating hair growth. Furthermore, the present invention relates to (c) a gene comprising the DNA of SEQ ID NO: 2 or (d) a DNA that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and regulates hair growth. The present invention relates to a gene comprising DNA encoding a protein having

DERP2 of the present invention is a protein having a molecular weight of 37 kilodaltons (kd) consisting of 345 amino acid residues as shown in SEQ ID NO: 1. As a feature of the amino acid sequence, a region rich in hydrophobic amino acids is observed at a plurality of sites, and it is presumed that the region is a type of membrane protein.

Further, derp2 of the present invention is a gene consisting of 130 base pairs (bp) as shown in SEQ ID NO: 2.

The gene derp2 can be isolated as a cDNA fragment containing the gene from a cDNA library derived from human dermal papilla cells. The cDNA library used by the present inventors was prepared by extracting mRNA extracted from human papillary cells isolated according to the method of Messenger et al. (Br. J. Dermatol. 114, 425, 1986) according to a general method. Although prepared on the basis of cDNA, cDNA can also be prepared in the same manner based on human cerebral cortex mRNA commercially available from Clonetech.

Okubo and colleagues have identified a method for identifying genes that are highly expressed in human papillary cells. A method of analyzing the expression frequency of a gene by the method (Okubo et al, Nature Genet., 2, pl 73, 1992) can be used. Specifically, the following procedure is used.

Human mRNA was derived from human dermal papilla cells, and the cDNA was synthesized using type I as the primer, oligo dT bound to one end of plasmid, which was opened with an appropriate restriction enzyme. Cleavage with restriction enzymes MboI and BamHI. Since this vector was prepared using dam methylase-positive Escherichia coli as a host, the A residue of “GATC”, which is the recognition sequence of Mb0I, is methylated. Thus, Mbol only cleaves the newly synthesized cDNA portion. Since the vector has only one BamHI cleavage site near the end opposite to the end to which oligo dT is bound, the enzyme cuts the vector at one position and If the BamHI recognition sequence is present in the cDNA portion synthesized in step 2, the site is also cleaved. BamH I and Mbo I are composed of the sequence `` GATC '' and generate the same cohesive end.Cleavage with both enzymes and then closing of plasmid by the action of DNA ligase. be able to. Using the plasmid thus prepared, Escherichia coli was transformed to construct a 3′-end cDNA library. Therefore, the library includes a region from the poly A site at the 3 ′ end of each mRNA to the site where the base sequence GATC first appears in the 5 ′ side thereof. An appropriate number of recombinants are randomly selected from the 3′-end cDNA library, and the entire nucleotide sequence of the cDNA fragment in each recombinant is determined. An organ-specific gene and a high-expressing gene can be identified based on how many of the cDNA fragments having the specific sequence determined in this way are confirmed from randomly selected recombinants.

In the above-mentioned method for identifying a highly expressed gene, the total number of recombinants selected at random is suitably several hundreds to about 1,000, but if necessary, more recombinants may be processed.

The present inventors carried out the above method, determined all the nucleotide sequences of the cDNA fragments in the 789 recombinants, and determined from them the frequency of appearance as cDNA having the same sequence. The cDNA fragment of 37989 was selected as a candidate for a DNA fragment of a gene highly expressed in human papillary cells.

As described above, the cDNA fragment contains only a part of the 3 ′ end region of mRNA. Therefore, the present inventors based on the nucleotide sequence information of the region (hereinafter, 3 ′ fragment). Then, the total chain length cDNA was obtained.

A human cerebral cortex cDNA library commercially available from Clonetech is a rust type, and an oligonucleotide of an appropriate length having the sequence in the 3 ′ fragment and a comparable length having a sequence in the vector are used. Each oligonucleotide was synthesized, and PCR was performed using these as primers. As a result, a DNA fragment of about 1.5 kb could be amplified. At this time, it can also be carried out by using mRNA extracted from human cultured hair papilla cells according to a conventional method as type III and using a 5 'RACE kit of Clonetech or Gibco. In addition, screening of the human cerebral cortex or dermal papilla cell cDNA library by colony hybridization or plaque hybridization using the above 3 ′ fragment as a probe in accordance with a standard method. Can also be performed.

The cDNA fragment amplified by the above method was incorporated into vector pGEM-T commercially available from Promega, and the entire nucleotide sequence was determined. At this time, the sequence was confirmed by independently obtaining two clones of the recombinant DNA and determining the nucleotide sequence of each cDNA fragment. One protein translation region (Open Reading Flame, ORF) was found in this sequence, the gene was designated as derp2, and the protein encoded by the gene was designated as DERP2.

The gene derp 2 can be made into a recombinant gene by a general gene recombination technique using an appropriate host vector system. Suitable vectors include plasmids derived from Escherichia coli (eg, pBR322, pUC118, etc.), and plasmids derived from Bacillus subtilis (eg, pUB110, pC194). Others), yeast-derived plasmids (eg, pSH19 and others), and animal viruses such as bacteriophages, retroviruses and vaccinia viruses can be used. Upon recombination, a translation initiation codon and a translation termination codon can be added using an appropriate synthetic DNA adapter. In order to further express the gene, an appropriate expression promoter is connected upstream of the gene. The promoter to be used may be appropriately selected depending on the host. For example, when the host is Escherichia coli, the T7 promoter, 1 ac promoter, trp promoter, and λPL promoter are used, and when the host is Bacillus, the S ス 0 promoter is used. If the host is yeast, the 5 promoters, the GAP promoters, the ADH promoter, etc. In the case of vesicles, SV40-derived promoters and retrovirus promoters can be used, respectively.

It is also possible to express the gene as a fusion protein with another protein (eg, Dalphin thione S transferase, protein A, etc.). The fused DERRP2 expressed in this manner can be excised using an appropriate protease (eg, thrombin or the like).

Hosts that can be used for expression of DERP 2 include various strains of cherichia il, a bacterium belonging to the genus Escherichia, various strains of Baci 1 lus subti 1 is, a bacterium belonging to the genus Bacillus, and various strains of Saccharomyces cerevi siae as yeast. As animal cells, COS-7 cells, CHO cells and the like can be used. As a method for transforming a host cell using the above-described recombinant vector, a conventional method or a transformation method generally used for each host cell can be applied.

In the present invention, in addition to the nucleotide sequence shown in SEQ ID NO: 2, a DNA that hybridizes with the sequence and encodes a protein having a function of regulating hair growth is also included in the scope of the present invention. It is.

That is, in the full-length sequence of the gene derp 2, various artificial treatments such as site-directed mutagenesis, random mutation by treatment with a mutagen, and mutation, deletion, and ligation of a DNA fragment by restriction enzyme digestion are partially performed. Even if the DNA sequence is altered, these DNA mutants will hybridize with the gene derp 2 under stringent conditions and will encode DNA encoding a protein having a function of regulating hair growth. If present, they are within the scope of the present invention, regardless of the differences from the DNA sequence shown in SEQ ID NO: 2.

The degree of the above-mentioned DNA mutation is within an allowable range as long as it has 90% or more homology with the DNA sequence of the gene derp2. In addition, the degree of hybridization with the gene derp 2 was determined at 32 ° C when the probe was labeled under normal conditions (eg, DIG DNA Labeling kit, Bellinger's Mannheim Cat No. 1175033). Hybridize in DIG Easy Hyb solution (Behringer's Mannheim Cat No. 1603558) and remove membrane in 0.5 XSSC solution (containing 0.1% [w / V] SDS) at 50 ° C. Southern hybridization under washing conditions (1 XSSC is 0.15M NaC 0.015M sodium citrate) In this case, it is only necessary to hybridize to the gene derp2.

In addition, a protein encoded by a mutant gene having high homology to the gene derp2 as described above and having a function of regulating hair growth is also within the scope of the present invention.

In other words, even if a mutant in which one or more amino acids of the amino acid sequence of the novel protein DERP 2 has been deleted, substituted or added, the mutant has a function of regulating hair growth. The mutant is within the scope of the present invention. <The side chains of amino acids that are constituents of the protein are different in hydrophobicity, charge, size, etc., but are substantially different in the entire protein. Some highly conservative relationships in the sense that they do not affect the three-dimensional structure (also called the three-dimensional structure) are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Va1), leucine (Leu) and isoleucine (I1 e), daltamic acid (G1u) and glutamine (Gin), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine ( Ser) or A1a, lysine (Lys) and arginine (Arg), and the like.

Therefore, even if the mutant protein is a mutant protein resulting from substitution, insertion, deletion, etc. in the amino acid sequence of the novel protein DERP 2 shown in SEQ ID NO: 1, the mutation is a mutation that is highly conserved in the three-dimensional structure of the DERP 2 protein. If the mutant protein has a function of regulating hair growth like DERP2, these can be said to be within the scope of the present invention. The degree of mutation is within the acceptable range if the homology with the amino acid sequence shown in SEQ ID NO: 1 is 90% or more. Industrial applicability

Since DERP 2 has a function of regulating hair growth, it is presumed that abnormal expression of the gene derP2 or abnormal expression of DERP 2 activity affects hair growth. Therefore, a substance that regulates the expression of the gene or a substance that regulates the function of DERP 2 can be expected as a hair-growing agent or a hair-restoring agent, and the gene derp 2 and the protein DERP 2 have such physiological activities. Use for substance search Can be. For example, the effect of a test substance on gene expression can be investigated by coexisting a test substance in the transcriptional expression system of gene derp 2 and detecting the expression level of gene derp 2 by an appropriate method such as PCR. it can. It is also possible to search for a bioactive protein that directly acts on DERP 2 and controls the function of DERP 2 to regulate hair growth. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail with reference to examples. Unless otherwise specified below, various experimental methods shown in the examples, for example, extraction of recombinant cDNA, determination of the base sequence of cDNA, etc., are all methods known per se to those skilled in the art (Molecular Cloning, 2nd. ed., Cold Spring Harbor Lab. Press, 1989, and other methods described in technical manuals that introduce standard methods for those skilled in the art.

Example Cloning of gene derp2

1) Isolation and culture of dermal papilla cells

Human hair papilla cells were isolated from the hair follicles of the hair growth part scalp of a healthy male (30 years old) according to the method of Messenger et al. (Br. J. Dermatol. 114, 425, 1986) and cultured. Remove the dermal papilla from the lower part of the hair follicle, place it in a Petri dish containing MEM medium supplemented with 12% fetal bovine serum (FBS), and add 5% C02Z 95% air, 37 ° C C〇2 ink. The cells were cultured overnight for 7 days. Outgrowth cells from the dermal papilla were collected using a 0.05% trypsin-0.53 mM EDTA solution. The separated hair papilla cells were subcultured in the same medium, and the cells at the fourth and fifth passages were used for the experiment.

2) Determination of partial sequence of gene

Using the mRNA derived from human dermal papilla cells as type III, a 3′-terminal cDΔΝ library was prepared according to the method of Okubo et al. (Okuboei al. Nature Genet., 1992, 2, ρΠ3). 789 recombinants were randomly selected from the library, and the nucleotide sequence of the cDNA portion was determined. For sequencing, DNA sequencing (PRISM377, manufactured by ABI) and a reaction kit manufactured by ABI were used.

As a result of analyzing the expression frequency of each DNA fragment in the 789 recombinants, the expression frequency of the gene having the sequence (sequence-1) shown in FIG. 1 was 3Z789. 3) Cloning of cDNA fragment containing sequence 11

Cloning of the cDNA fragment containing sequence-1 was performed by the following method. First, an oligonucleotide (sequence 12 in FIG. 1) which is a reverse complementary strand to a part of sequence-1 was synthesized using a DNA synthesizer (380B manufactured by ABI). Next, an oligonucleotide (sequence 13 in FIG. 1) having a sequence in the vicinity of the cDNA insertion site of lambda phage cloning vector-1 (ADR2) was similarly synthesized. Human Brain cerebral cortex 5'-STRETCH cDNA library (manufactured by Clontech Laboratories) using λ DR 2 as a closing vector is designated as type II, and oligonucleotides of sequence 12 and sequence 13 are used as primers. The following PCR procedures were performed using the evening PCR LA PCR Kit Ver.2 and PCR Thermocycla MP (both from Takara Shuzo).

cDNA 1 ibrary (≥108 pfu / ml) 51

10X PCR buffer (including 25mM Mg ++) 5

2.5mM dNTP 1 IX

10 M array 1 2

10 / xM array 1 3 in

Water 34.b

LA Taq_Holimera-Se "0.5 i

50

PCR cycle: After holding at 94 ° C for 2 minutes, react at 98 ° C for 20 seconds, cool to 68 ° C at a rate of 1 ° C for 2 seconds, and heat at 68 ° C for 3 minutes. The holding was further performed 30 times at 72 ° C. for 10 minutes.

By the above method, a DNA fragment (about 1.5 kb) having sequence 11 was specifically amplified (FIG. 2).

4) Subcloning into a nucleotide sequence determination vector

The DNA fragment amplified in 3) was fractionated by agarose gel electrophoresis (gel concentration 1%). The gel was stained with ethidium bromide and irradiated with ultraviolet light to cut out the gel containing the target band. Extraction and purification of the DNA fragment from the agarose gel was performed using GENECLEAN II Kit (Bio101).

The extracted and purified DNA fragment was used as a base sequence determination vector pGEM-T (Promega (Fig. 3). The ligation solution was used for evening color DNA Ligation Kit Ver.2 (Takara Shuzo) and reacted at 16 C for 1.5 hours with the following composition. Extracted and purified DNA fragment l / xl (50ng)

pGEM-T 1 1 (17ng)

Water 3 1

Li gat ion solution 5 1

Total amount 10 i l

Using the solution after the above reaction, Escherichia coli K12 strain DH5 was transformed. Transformants were treated with ampicillin (Amp) 50 M g / r 1.5-Bromo-4-Chloro-3-indolyl β-D-galactose 40 g / m 1> Isopropyl—β-D-Thio It was plated on an LB agar medium containing -Ga1ac topyranosidel 100 μM and cultured at 37 ° C. The colonies that appeared in the above plate were inoculated into 10 ml of an LB liquid medium containing 50 // g / m1 Amp, cultured at 37 ° C overnight, and the cells were collected by centrifugation. After that, the recombinant DNA was purified using QIAprep Spin Plasmid Miniprep Kit (Qiagen).

5) Determination of nucleotide sequence of DNA fragment

For DNA sequencing, DNA sequencer (PRISM377, manufactured by ABI) was used, and the dye-minerator method was used. Oligonucleotides were synthesized based on the determined base sequences, and the entire base sequences of both strands were determined by the primer walking method (Fig. 4). SEQ ID NO: 3 shows the entire nucleotide sequence of cDNA of the clone. Since the nucleotide sequence contained the upstream region of sequence 12 of sequence-2 and sequence-1, it was confirmed that the target gene clerp2 was cloned.

The cDNA comprises an ORF encoding a protein consisting of 345 residues (DERP2) (SEQ ID NO: 3). Since a stop codon appeared in the same reading frame in the upstream region of the methionine residue which is the start codon of the protein, the amino acid sequence of the protein encoded by the cDNA fragment was that shown in SEQ ID NO: 3. It was confirmed that it was the only one.

Test Example 1 Antibody staining of dermal papilla cells

1) Preparation of Bo D ER P 2 peptide antibody

The anti-peptide antibody is based on the Cell Engineering Separate Volume Anti-Peptide Antibody Experimental Protocol (Onami Shinobu, (Kunio Tsujimura, Shujunsha). A peptide containing a part of the amino acid sequence of DERP2 (sequence 14 in Fig. 1) was synthesized using a peptide synthesizer (manufactured by ABI), and this was cross-linked to the carrier protein by molysin-mediated method with maleimide. And used as antigen. 0.5 mg of this antigen was injected subcutaneously into the back of a egret (Kb1: JW, 10-year-old, female). At 14, 28 and 42 days after the first immunization, further immunization was carried out using the same amount of antigen, and whole blood was collected 52 days after the first immunization. From the antiserum, a crude IgG fraction was prepared by the ammonium sulfate salting-out method, and further subjected to affinity purification using a protein A sepharose column, followed by an antigen-peptide-immobilized column, and antigen-specific. Antibodies were obtained. This antibody specifically bound to DERP2 translated and synthesized using an in vitro transcription / translation system (Promega). In addition, it specifically bound to a 37 kd protein present in a protein solution prepared by dissolving dermal papilla cells in an lm MSDS solution.

2) Bovine staining of dermal papilla cells using anti-DERP2 peptide antibody

Hair papilla cells separated and cultured in Example 1 were seeded in an 8-well chamber slide (Nunc) at 1.5 104 ce 11 s / we 11 and a MEM medium supplemented with 12% FBS. In it, it was cultured. The medium was removed and the cells were fixed with 4% paraformaldehyde-0.25% Tween 20 for 15 minutes at room temperature. This was treated with 5 μg Zm1 of the anti-DERP2 peptide at −4 ° C., reacted with a biotinylated anti-rabbit IgG antibody, and developed with an AEC staining kit (manufactured by Sigma). Was. The results are shown in FIG. The organelles around the nucleus of the dermal papilla cells (area of ER-Golgi) were strongly stained.

Test Example 2 Expression of derp2 in dermal papilla cells

A normal male (30 years old) hair papilla cell derived from the hair-growing part and a male patient with male pattern baldness (34 years old) isolated in the same manner as described in Example Total RNA was extracted by the method. After treating each total RNA l / g with 1 unit (U) of DNaseI (Gibco BRL), use 01 igo (dT) 12--18 Primer (GIBCO BRL) and Superscriptll (GIBCO BRU). CDNA was synthesized according to the protocol attached to Superscript II.The cDNA was converted into type I, and a derp2-specific primer synthesized using a DNA synthesizer (ABI 380B) (Fig. 1). The following PCR was carried out using the sequences 1 and 6) in a total amount of 40 μl. cDNA 40ng

Ex taq buffer (Takara) X 1

dNTPs (Takara) 0.16mM

[a-32P] -dCTP (NEN) 590kBq

Ex Taq (Takara) 2 U

Sequence 1 5 0.2 μΜ

Sequence 1 6 0.2M M

In the PCR cycle, after heating at 94 ° C for 2 minutes, 18 cycles of 30 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute were repeated.

The reaction mixture (6 il) was subjected to electrophoresis using a 12% polyacrylamide gel, and the gel was dried. The radioactivity incorporated into the amplified derp 2 fragment was analyzed using BAS-2000II (Fujifilm). It was measured. The amount of derp2 mRNA was expressed as a relative value to Libosomal protein S26 used as an internal standard after the measured radioactivity was corrected for the dCTP content in the amplified product. Fig. 6 shows the results.

Hair papilla cells derived from bald areas of patients with androgenetic alopecia had higher expression of derp2mRNA than hair papilla cells derived from the hair growth region of healthy individuals.

Test Example 3 Expression change of gene derp2 by testosterone treatment

In the same manner as in Example 1, a male patient with alopecia (38 years old) was isolated and cultured for hair papilla cells derived from the hair growth part. The cells at the fifth passage were seeded to a 2 × 10 5 c e 11 s 6 cm scale and cultured until they reached confluence. Thereafter, the medium was replaced with a testosterone-supplemented medium (0, 10, 50, 250 nM), and the cells were further cultured for 24 to 72 hours. After removing the medium, the cells were washed with PBS (-) to extract total RNA. The following ΔCR was carried out using 40 ng of cDNA prepared in the same manner as in 5) of Example 1 and using primers of Sequence-5 and Sequence-16 in a total amount of 201.

cDNA 40ng

Ex taq buffer (Takara) \ x

dNTPs (Takara) 0.5raM

Ex Taq (Takara) 1U

Sequence 1 50.5 ^ iM Sequence 1 6 0.5 iM

In the PCR cycle, after heating at 95 ° C for 2 minutes, 23 cycles of 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute were repeated.

The reaction solution was subjected to electrophoresis using a 2% agarose gel and stained with ethidium mouth. The results are shown in FIG. It was confirmed that the culture to which testosterone was added at 50 nM or more increased the expression level of the gene derp2 in the dermal papilla cells. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows nucleic acids or peptides used in the examples.

SEQ ID NO: 1 is the 3'-terminal cD obtained from human papillary cells by the method of Okubo et al.

This represents the nucleotide sequence of the DNA fragment identified at a frequency of 3/789 from the NA library.

Sequence-2 shows the sequence of the reverse complement of a part of Sequence-11.

SEQ ID NO: 13 shows the sequence of an oligonucleotide having a sequence near the cDNA insertion portion of the lambda phage cloning vector.

Sequence-4 is the DERP used to prepare the anti-DERP2 peptide antibody.

2 shows the sequence of the antigen peptide containing the partial sequence of 2.

Sequence-5 is a subsequence for extending derp2 by PCR.

Sequence-6 is a partial sequence for amplifying .derp2 by PCR.

FIG. 2 shows the PCR for a cDNA library containing sequence-1. FIG. 3 shows a scheme in which the gene derp2 is recombined into the cloning vector pGEMT.

FIG. 4 shows the outline of the primer-walking method.

FIG. 5 shows a diagram in which hair papilla cells separated and cultured are immunostained using an anti-DERP2 peptide antibody.

FIG. 6 shows a diagram comparing the expression levels of the gene derp2 in hair papilla cells derived from a normal male hair growth site and hair papilla cells derived from a bald patient with androgenetic alopecia.

FIG. 7 shows the expression of the gene derp2 in the presence of various concentrations of testosterone. Arrangement table

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¹ to GTQ

― CTQ

OP

GTCJ

Val Gly Gly Leu Ser Thr Val Ala Met Cys Ala Pro Ser Glu Lys

230 235 240

111 ctg aac atg ggt gca ccc c tg gga gtg ggc ctg ggt etc gtc 835 Phe Leu Asn Met Gly Ala Pro Leu Gly Val Gly Leu Gly Leu Val

245 250 255 ttt gtg tec tea ί tg gga tc t atg 111 c 11 cca cc t acc acc gtg 880 Phe Val Ser Ser Leu Gly Ser Met Phe Leu Pro Pro Thr Thr Val

260 265 270 gel ggt gcc act c 11 tac tea gtg gca aig lac ggt gga t ta gt t 925 Ala Gly Ala Thr Leu Tyr Ser Val Ala Met Tyr Gly Gly Leu Val

275 280 285 c 11 t tc age atg t tc c 11 ctg tat gat acc cag aaa gta ate aag 970 Leu Phe Ser Met Phe Leu Leu Tyr Asp Thr Gin Lys Val lie Lys

290 295 300 cgt gca gaa gta tea cca atg tat gga gt t caa aaa tat gat ccc 1015 Arg Ala Glu Val Ser Pro Met Tyr Gly Val Gin Lys Tyr Asp Pro

305 310 315 at t aac tcg atg ctg agt ate tac atg gat aca t ta aat ata ttt 1060 l ie Asn Ser Met Leu Ser lie Tyr Met Asp Thr Leu Asn lie Phe

320 325 330 atg cga git gca act atg ctg gca ac ί gga ggc aac aga aag aaa 1105 Met Arg Val Ala Thr Met Leu Ala Thr Gly Gly Asn Arg Lys Lys

335 340 345 tga agtgac t cage t tc tgg ct lctctgct acatcaaata tc t tgt t taa tggggcagat 1165 atgcat taaa tagt t tgtac aagcagc 111 cgt tgaagt t tagaagataa gaaacatgtc 1225 atcatat t ta aatgt tccgg laatgtgcc gca tgg ccca

Claims

The scope of the claims

1. The following protein (a) or (b):

(a) SEQ ID NO: a protein consisting of the amino acid sequence of 1;

() SEQ ID NO: A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of 1, and which has a function of regulating hair growth.

2. DNA of (a) or (b) below

(a) SEQ ID NO: DNA comprising the nucleotide sequence set forth in 2

(b) a DNA that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and encodes a protein having a function of regulating hair growth.