JP2005046135A - Dog follistatin and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently produce high purity dog follistatin. <P>SOLUTION: Dog follistatin specifically inhibiting basic secretion of follicle-stimulating hormone by acting to the dog pituitary gland without inhibiting basic secretion of luteinizing hormone and having binding ability with activin and ability as its antagonist.A DNA sequence coding for dog follistatin, a recombinant vector containing the DNA sequence, a transformant prepared by transforming a host with the recombinant vector are claimed. The method for producing dog follistatin comprises culturing or breeding the transformant and harvest dog follistatin. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The object of the present invention is to mass-produce canofolistatin whose primary protein structure is derived from canine genetic information by genetic manipulation technology, and to use it as a veterinary drug (hepatic or renal disease). , DNA sequences, recombinant vectors, transformants, canofollistatin and methods for producing the same.

  Follistatin is a glycoprotein having a molecular weight of about 35,000-32,000. Both follistatins with 315 and 288 amino acids have the ability to bind to activin and have the effect of suppressing follicle stimulating hormone (FSH) production in pituitary cultured cells.

  Tumor-derived Madin-Darby Canine kidney (MDCK) cells are cultured in type I collagen to form cysts, but hepatocyte growth factor (HGF) is added to the lumen Formation is seen (Non-Patent Document 1). However, activin inhibited this effect in a concentration-dependent manner. Follistatin, an activin antagonist, induced tube formation even in the absence of HGF.

  Many kinds of follistatin genes have been isolated so far by genetic engineering techniques (Non-patent Document 2), for example, having 91% homology between humans (Homo sapiens) and mice (Mus musculus). There are no reports of canine follistatin cDNA cloning.

  It is known that canine diseases include acute hepatitis-like liver diseases, and in many cases, liver dysfunction is observed. Steroid preparations and health foods are used for canine liver disease, but no fundamental treatment has been established.

In addition, as a purification method for follistatin, a protein is prepared directly from a purification method using a column prepared by crosslinking activin A capable of binding to follistatin with a suitable carrier (Non-patent Document 3) or electrophoresis gel. Although there are methods (Non-patent Document 4), these methods are not preferable as a means for developing pharmaceuticals and the like, and it is necessary to establish a more versatile method that can be purified in large quantities.
Maeshima et al .: Cytokine Growth Factor Rev. 2001 12 (4) 289-98 Shimazaki et al .: Proc Natl Acad SciUSA. 1988. 85 (12) 4218-22 De Winter JP et al .: Molecular, Cell Endocrinol 1996.116 (1) .105-114 Yokoyama Y et al .: Journal Clinical End Clinology Metabolization (J Clin Endocrinol Metab) 1995.80 (3) .915-921

  It is an object of the present invention to provide a therapeutic drug for canine liver disease having an action different from the conventional one by producing canine follistatin.

That is, it binds to activin, which is expected to be highly expressed in severe liver disease, and inhibits the production of extracellular matrix and tissue fibrosis induced by activin, thereby regenerating liver regeneration and tissue damage. It is intended to enable the delay of the process.
In addition, when a useful protein is used as a pharmaceutical from a culture solution or the like, it is an important issue to separate and fractionate the target component by purification.

  Therefore, if it is possible to establish a method for easily separating and fractionating canofolistatin containing impurities in the culture solution with high purity, it is expected that the development of a pharmaceutical or the like will be opened.

  In order to solve the above-mentioned problems, the present inventor has succeeded in cloning a gene encoding catforistatin from canine cDNA for the purpose of cloning canine follistatin cDNA and mass production using the same. In addition, the inventors succeeded in producing a cell that produces canifolistatin using a plasmid in which these are linked to an expression vector, and thus established a method for easily producing canifolistatin in large quantities, and further, two types of columns Using a chromatographic carrier, high-purity production is possible, and thus the present invention has been completed.

  That is, the present invention acts on the pituitary gland of dogs to specifically suppress basal secretion of follicle stimulating hormone, does not suppress basal secretion of luteinizing hormone, has an ability to bind to activin, and has the ability as an antagonist. A canine follistatin, a DNA sequence encoding the canofollistatin, a recombinant vector containing the DNA sequence, a transformant obtained by transforming a host cell with the recombinant vector, and culturing or transforming the transformant A method for producing inufolistatin, comprising breeding and collecting highly pure inufolistatin by column chromatography purification.

  If a method for easily separating and fractionating canofolistatin with high purity can be established, it is expected that the development of pharmaceuticals and the like will be opened.

The canofolistatin of the present invention includes both natural types and gene recombinant types, and has the following functions (1) to (3).
(1) It acts on the pituitary gland of dogs to specifically suppress basal secretion of follicle stimulating hormone.
(2) Does not suppress basal secretion of luteinizing hormone.
(3) It has an ability to bind to activin and has an ability as an antagonist.

  Here, whether or not it has an ability to bind to activin can be confirmed by a binding assay of activin labeled with activin. Activin is expected to be overexpressed in canine liver disease.

  The amino acid sequence of inufolistatin can be determined by a conventional method, and the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are examples thereof. The sequence of SEQ ID NO: 2 is the sequence of SEQ ID NO: 1 with the C-terminal 27 amino acid residues deleted. As long as the functions of (1) to (3) above are substantially retained, even if one or several amino acids in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 are deleted, they are replaced with other amino acids Or one or several amino acids may be added.

  Among the DNA sequences encoding canifolistatin of the present invention, preferred are those encoding the above SEQ ID NO: 1 or SEQ ID NO: 2, particularly the DNA sequence represented by SEQ ID NO: 3, 4 or 5, or A DNA sequence that hybridizes.

  A recombinant vector incorporating a DNA encoding the canofolistatin protein of the present invention can be produced, for example, as follows. That is, after extracting poly (A) RNA from canine cells, cDNA is synthesized, and polymerase chain reaction (hereinafter abbreviated as PCR) is performed using primers based on gene sequences encoding mouse and human follistatin. By doing so, it is possible to clone a gene exhibiting canifolistatin activity. In addition, a canine follistatin can be cloned by preparing a phage library from the synthesized cDNA recombinant and performing plaque hybridization with two gene fragments obtained by PCR.

  Examples of methods for obtaining RNA from canine organs and cells, MDCK derived from canine kidney cells, and the like include conventional methods such as polysome separation, sucrose density gradient centrifugation, and electrophoresis. As a method for extracting RNA from canine cells, guanidine thiocyanate-cesium chloride method in which CsCl density gradient centrifugation is performed after guanidine thiocyanate treatment (Chirwin et al .: Biochemistry 1979. 18. 5294-5304) , A method of performing phenol extraction after treatment with a surfactant in the presence of a ribonuclease inhibitor using a vanadium complex (Berger et al .: Biochemistry 1979. 18. 5143-49), guanidine thiocyanate-hot Select an appropriate method from among the phenol method, guanidine / thiocyanate-guanidine hydrochloride method, guanidine / thiocyanate-phenol / chloroform method, and guanidine / thiocyanate treatment followed by treatment with lithium chloride to precipitate RNA. Can be done.

  MRNA is isolated from the obtained RNA by lithium chloride / urea method, guanidine isothiocyanate method, oligo dT cellulose column method, etc., and cDNA is synthesized from the obtained mRNA using reverse transcriptase or DNA polymerase with primers.

  The canofolistatin gene can be cloned by PCR using the cDNA as a template and primers based on mouse or human nucleotide sequences. The synthesized cDNA is ligated to a λ phage vector, and then packaged by mixing with a coat protein of λ phage in vitro, and the generated phage particles are infected with E. coli serving as a host. Escherichia coli infected with λ phage is lysed, and each clone is recovered as a plaque. The plaque can be transferred to an appropriate filter, and the canofolistatin gene can be cloned by hybridization using a pre-labeled oligomer or a gene obtained by PCR as a probe.

  Prokaryotes or eukaryotes can be used as the host. E. coli can be used as an example of a prokaryotic organism. For example, animal cells or insect cells can be used as eukaryotes.

  As an expression vector, a plasmid, phagemid, cosmid, phage, retrovirus and the like can be used. The promoter and drug selectable marker in the expression vector are selected depending on the host cell. For example, lac promoter in bacteria, baculovirus promoter in insect cells, SV40 promoter in animal cells, etc. The transformant can be cultured according to a conventional method.

  The produced inufolistatin has a molecular weight of about 35,000 to 32,000 by SDS-PAGE under reducing conditions.

  In the present invention, it is important to use a cation exchange carrier, more preferably further to use a heparin carrier, in purifying inifolistatin. By using both column carriers, it is possible to obtain highly pure inforistatin.

  Examples of the cation exchange carrier used for separation and fractionation of inifolistatin include anions by chemically stable ether bonds via hydrophilic spacers on agarose, cellulose, sepharose, synthetic polymer gels, etc. Those having a functional group introduced may be mentioned. Preferably, SP Sepharose (Amersham-Pharmacia) and the like are used. Examples of the heparin carrier include those obtained by introducing a heparin group into agarose, cellulose, sepharose, synthetic polymer gel or the like through a chemically stable ether bond via a hydrophilic spacer. Preferably, heparin Sepharose (manufactured by Amersham-Pharmacia) or the like is used.

  In the purification step in the present invention, a method in which a solution containing inufolistatin is brought into contact with a cation exchange carrier and a heparin carrier, and the adsorbed material on the carrier is eluted with an eluent is preferably employed.

  Elution from the cation exchange carrier can be separated and fractionated by gradually increasing the salt concentration, canceling ionic interaction with the cation exchange group that is the ligand, and by the difference in ionic strength of the adsorbed protein. . In addition, elution from the heparin carrier can gradually increase the salt concentration to cancel the specific interaction with the heparin group that is a ligand, and can be separated and fractionated. In general, when proteins are produced in various production systems, not limited to follistatin, impurities such as a culture solution composition in a culture solution and a protein in serum are mixed. It is not easy to separate only inifolistatin from these components. The cation exchange carrier and heparin carrier used in the present invention can separate and fractionate inifolistatin from impurities in the culture solution, depending on the concentration of the eluent.

  In the purification operation using a cation exchange carrier and a heparin carrier, the composition of the eluent, the amount of liquid, etc. are not particularly limited, and the optimum separation conditions are the amount of contaminating protein present, the amount of each component of useful protein, It is determined appropriately according to the dimensions of the column.

  The present invention will be described more specifically with reference to the following examples.

Example 1
Cloning of canine follistatin gene (1) Preparation of canine cDNA library Canine kidney-derived cells MDCK were cultured in 150 cm ² flasks for 3 to 4 days to prepare cell precipitates of 5 × 10 ⁷ cells. MRNA was prepared with a messenger RNA isolation kit (Strtagene). That is, a denaturing solution containing guanidine thiocyanate was added to the MDCK precipitate, and the DNA mixed with the 21G needle was sheared. Next, the eluate (elution buffer) was added and centrifuged to obtain the supernatant. The obtained supernatant was applied to an oligo dT cellulose column, and the column was washed. Then, poly (A) RNA was eluted with an eluate preheated to 68 ° C. Next, using the obtained poly (A) RNA, a cDNA library was synthesized by a cDNA synthesis kit (TOYOBO). That is, a double-stranded cDNA was synthesized according to the kit manual.

(2) Cloning of canofollistatin gene N-terminal and C-terminal nucleotide sequences of human follistatin (Ueno et al .: Proc Natl Acad SciUSA. 1984. 84. 8282-86) Based on the above, two kinds of primers of SEQ ID NO: 6 and SEQ ID NO: 7 were synthesized with a DNA synthesizer. Add 1 μL of the MDCK cDNA from (1) above to a 0.5 mL microtube, add 20 pmol of each primer, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgSO ₄ , 25 mM KCl, 50 μM each dNTP, and KOD. 50 μL. 35 cycles of DNA denaturation conditions 94 ° C, 30 seconds, primer annealing conditions 60 ° C, 30 seconds, primer extension conditions 68 ° C, 1 minute using Bio-RAD DNA thermal cycler I let you. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 900 bp was prepared according to a conventional method (Molecular Cloning Cold Spring Harbor Laboratory. New York. 1982).

  This DNA fragment was ligated to pUC108 (Amersham Pharmacia) using DNA Ligation kit Ver.2. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, it was confirmed that the PCR fragment was inserted into this plasmid by PCR under the same conditions as described above, and restriction enzyme treatment with EcoRI and HindIII, and ABI PRISM 377XL (Applied Biosystems) was used. Cycle Sequencing (Molecular Cloning Cold Spring Harbor Laboratory. New York. 1982), the nucleotide sequence of the DNA considered to be inforistatin was determined. This sequence is shown in SEQ ID NO: 3.

(3) Cloning of the 5 ′ end of the canine follistatin gene by 5′RACE method Based on the partial base sequence of canofollistatin obtained in (2), three types of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 The primers were synthesized with a DNA synthesizer. 2 μL of the MDCK mRNA of (1) above was placed in a 0.5 mL microtube, and DNA containing the 5 ′ end of canofolistatin was prepared using a 5 ′ RACE kit (manufactured by GIBCO). That is, DEPC water for inactivating MDCK mRNA and RNase and 25 pmol of primer (SEQ ID NO: 8) were added and treated at 70 ° C. for 10 minutes. Next, 10 × PCR Buffer, 25 mM MgCl ₂ , 10 mM dNTP, and 0.1 M DTT were added on ice for 1 minute, respectively, and heated at 42 ° C. for 1 minute. Next, SuperScript II RT (SuperScript IIRT) was added, and the treatment was further continued at 42 ° C. for 30 minutes, followed by treatment at 70 ° C. for 15 minutes to perform reverse transcription to cDNA. Finally, RNaseMix was added and treated at 37 ° C. for 20 minutes to degrade RNA. Using the sample thus obtained, the cDNA reverse transcribed by Spin Cartridge was purified. That is, a binding solution (6M NaI) was added to a sample, and after setting in a Spin cartridge, centrifugation was performed at 13,000 G for 20 seconds. Washing was repeated with 400 μL wash buffer, and further washing with 70% ethanol was performed. Then, sterilized water heated to 65 ° C. was added in advance, and centrifuged at 13,000 G for 20 seconds. The eluted sample (10 μL), DEPC-treated water (6.5 μL), tailing buffer (5 μL) and 2 mM dCTP (2.5 μL) were mixed, treated at 94 ° C. for 3 minutes, and allowed to stand on ice for 1 minute. Next, add 1 μL of terminal deoxynucleotidyl transferase (terminal deoxynucleotidyl transferase) of 5'RACE kit (manufactured by GIBCO), treat at 37 ° C for 10 minutes, further treat at 65 ° C for 10 minutes, and the adapter primer and base sequence described below. Was added to the 5 ′ end of the cDNA. Using the obtained sample, 20 pmol of primer (SEQ ID NO: 9), 20 pmol of adapter primer, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgSO ₄ , 25 mM KCl, 50 μM each dNTP and KOD were added, and the total amount was 50 μL. It was. DNA denaturation conditions 94 ° C., 30 seconds, primer annealing conditions 62-50 ° C., 30 seconds, primer extension conditions 68 ° C., 1 minute each using a Bio-RAD DNA thermal cycler, 35 A cycle reaction was performed. Using the obtained PCR product, 20 pmol of primer (SEQ ID NO: 10), 20 pmol of adapter primer, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgSO ₄ , 25 mM KCl, 50 μM each dNTP, KOD were added, and the total volume was 50 μL. It was. DNA denaturation conditions 94 ° C., 30 seconds, primer annealing conditions 62-50 ° C., 30 seconds, primer extension conditions 68 ° C., 1 minute each using a Bio-RAD DNA thermal cycler, 35 A cycle reaction was performed. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 400 bp was prepared according to a conventional method.

  This DNA fragment was ligated to pUC108 (Amersham Pharmacia) using DNA Ligation kit Ver.2. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, it was confirmed by PCR under the same conditions as described above that the PCR fragment had been inserted into this plasmid, and the 5 ′ end was determined by the BigDye Terminator Cycle Sequencing method using ABI PRISM 377XL (Applied Biosystems). The nucleotide sequence presumed to be a part of the DNA of canofolistatin was determined.

(4) Cloning of the 3 ′ end of the canine follistatin gene by 3′RACE method Based on the partial base sequence of canofollistatin obtained in (2), two primers of SEQ ID NO: 11 and SEQ ID NO: 12 were used as DNA. Synthesized with a synthesizer. 2 μL of the MDCK mRNA of (1) above was placed in a 0.5 mL microtube, and DNA containing the 3 ′ end of canofolistatin was prepared using a 3′RACE kit (GIBCO). That is, DEPC water for inactivating MDCK mRNA and RNase and 25 pmol of primer (SEQ ID NO: 11) were added and treated at 70 ° C. for 10 minutes. Next, it was placed on ice for 1 minute, 25 mM MgCl ₂ , 10 mM dNTP, and 0.1 M DTT were added and heated at 42 ° C. for 1 minute. Next, Superscript IIRT (SuperScript IIRT) was added, followed by further treatment at 42 ° C. for 30 minutes, followed by treatment at 70 ° C. for 15 minutes to perform reverse transcription to cDNA. Finally, RNaseMix was added and treated at 37 ° C. for 20 minutes to degrade RNA. Using the sample thus obtained, the cDNA reverse transcribed by a spin cartridge was purified. That is, a binding solution (6M NaI) was added to a sample, and after setting in a spin cartridge, it was centrifuged at 13,000 G for 20 seconds. Washing was repeated with 400 μL wash buffer, and further washing with 70% ethanol was performed. Then, sterilized water previously heated to 65 ° C. was added, followed by centrifugation at 13,000 G for 20 seconds. The eluted sample 50 μL, DEPC-treated water 6.5 μL, tailing buffer 5 μL, and 2 mM dCTP 2.5 μL were mixed, treated at 94 ° C. for 3 minutes, and allowed to stand on ice for 1 minute. Next, add 1µL of terminal deoxynucleotidyl transferase (terminal deoxynucleotidyl transferase) from 3'RACE kit (GIBCO), treat at 37 ° C for 10 minutes, and further at 65 ° C for 10 minutes. Was added to the 3 ′ end of the cDNA. Using the obtained sample, 20 pmol of primer (SEQ ID NO: 12), 20 pmol of adapter primer, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgSO ₄ , 25 mM KCl, 50 μM each dNTP and KOD were added, and the total amount was 50 μL. It was. DNA denaturation conditions 94 ° C., 30 seconds, primer annealing conditions 62-50 ° C., 30 seconds, primer extension conditions 68 ° C., 1 minute each using a Bio-RAD DNA thermal cycler, 35 A cycle reaction was performed. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 400 bp was prepared according to a conventional method.

  This DNA fragment was ligated to pUC108 (Amersham Pharmacia) using DNA Ligation kit Ver.2. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, it was confirmed by PCR under the same conditions as described above that the PCR fragment had been inserted into this plasmid, and the 3 ′ end was determined by the BigDye Terminator Cycle Sequencing method using ABI PRISM 377XL (Applied Biosystems). The nucleotide sequence presumed to be a part of the DNA of canofolistatin was determined.

(5) cDNA sequence of canifolistatin The cDNA sequence of canifolistatin was clarified by combining the determined nucleotide sequences according to the above (2), (3) and (4) (SEQ ID NO: 5 ).

  As a result of homology search of the obtained canofolistatin gene, horse (Equus caballus): 95%, sheep (O aries): 94%, pig (Porcine): 92%, human (Homo sapiens): 93% , Rat (Rattus norvegicus): 92%, mouse (Mus musculus): 92%.

Example 2
Production of canifolistatin (1) Preparation of canine follistatin recombinant virus Expression vector pVL1392 was cleaved with restriction enzymes BglII and EcoRI, and the end was dephosphorylated with bovine-derived alkaline phosphatase. On the other hand, for the canine follistatin DNA fragment, a primer added with two cleavage sites (BglII and EcoRI) of SEQ ID NO: 8 and SEQ ID NO: 9 was prepared, and the canofolistatin DNA prepared in Example 1 (2) was used as a template. 35 cycles using DNA-biocycler manufactured by Bio-RAD under conditions of denaturation of DNA at 94 ° C for 30 seconds, primer annealing at 60 ° C for 30 seconds, primer extension at 68 ° C for 1 minute Reacted. After ethanol precipitation, it was cleaved with a restriction enzyme, and a DNA fragment of about 1 kbp was prepared by 1% agarose gel electrophoresis.

  This DNA fragment was phosphorylated with T4 polynucleotide kinase and then ligated to pVL1392 using DNA Ligation kit Ver.2. Using this, E. coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, the insect cell Sf9 was co-transfected with this plasmid and AcNPV according to a conventional method to obtain a recombinant virus expressing canofollistatin.

  Furthermore, the base sequence of inufolistatin DNA in the prepared expression plasmid was confirmed.

(2) Expression of canofolistatin in insect cell Sf9 The recombinant virus obtained in (1) above was infected with Sf9 cultured at 27 ° C. After culturing at 27 ° C. for 5 days, the culture supernatant was collected and then centrifuged at 2,000 G for 10 minutes to collect the supernatant.

(3) Measurement of activity of inufolistatin The activity of inufolistatin produced in the above (2) was measured as follows. Suppression of hemoglobin release action by activin A was confirmed using K562 cells (Molecular Cloning Cold Spring Harbor Laboratory. New York 1982).

In a 96-well plate, K562 cells (2 × 10 ⁵ cells / mL) were added with RPMI-1640 medium containing 10% FBS in the absence or presence of activin A (20 ng / mL), in the absence or presence of canofollistatin Culturing was performed for 4 days. Next, the cells were washed twice with PBS, suspended in 50 uL of sterilized water, and 40 μL of the supernatant fraction was obtained by centrifugation. To the obtained supernatant, 200 μL of a citrate buffer solution containing 0.5 mg / mL O-phenylenediamine and 0.03% hydrogen peroxide was added and treated in the dark for 15 minutes. Thereafter, 50 μL of 2.5 M sulfuric acid was added, and the absorbance at 490 nm was measured. As a result, hemoglobin production was significantly suppressed in the presence of canofolistatin (FIG. 1).

Example 3
2. Purification method of inufolistatin (1) Purification of inufolistatin by cation exchange carrier A cation exchange carrier in which the culture supernatant obtained in Example 2 (2) is pre-equilibrated with 20 mM phosphate buffer. It was introduced into SP Sepharose and washed again with 20 mM phosphate buffer. Next, the activity was measured by immunoblotting using the eluate obtained by elution by the stepwise method using 20 mM phosphate buffer containing 0 to 1 M sodium chloride and the method according to Example 2 (3). However, inofollistatin was detected in the fraction containing 0.5M sodium chloride.

(2) Purification of inifollistatin with heparin carrier The eluate obtained above was introduced into a heparin carrier previously equilibrated with 20 mM phosphate buffer containing 0.2 M sodium chloride, and again 20 mM containing 0.2 M sodium chloride. Washing was performed using a phosphate buffer. Next, using a 20 mM phosphate buffer containing 0.2 M to 1 M sodium chloride, elution was carried out by the stepwise method, and the activity was measured by immunoblotting and the method according to Example 2 (3). As a result, inofollistatin was detected in the fraction containing 0.8 M sodium chloride.

  It is an object of the present invention to provide a therapeutic drug for canine liver disease having an action different from the conventional one by producing canine follistatin.

  If a method for easily separating and fractionating canofolistatin with high purity can be established, it is expected that the development of pharmaceuticals and the like will be opened.

FIG. 1 is a diagram showing suppression of hemoglobin releasing action by activin A using inufolistatin.

Claims (10)

Canine follistatin that acts on the pituitary gland of the dog to specifically suppress the basic secretion of follicle-stimulating hormone, does not suppress the basic secretion of luteinizing hormone, has the ability to bind to activin, and has the ability as an antagonist . The inforistatin according to claim 1, comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. A DNA sequence encoding the canofollistatin according to claim 1 or 2. The DNA sequence represented by SEQ ID NO: 3, 4 or 5, or the DNA sequence according to claim 3, which hybridizes with the DNA sequence. A recombinant vector comprising the DNA sequence according to claim 3 or 4. A transformant obtained by transforming a host cell with the recombinant vector according to claim 5. A method for producing canofollistatin, comprising culturing or raising the transformant according to claim 6 and collecting canofollistatin. 7. Inufolistatin, wherein the iniferinstatin contained in the culture medium in which the transformant according to claim 6 is cultured is brought into contact with a cation exchange carrier, and the adsorbate adsorbed on the carrier is eluted with an eluent. High-purity manufacturing method. A high purity production method of inufolistatin, which comprises contacting the solution containing the eluted inufolistatin according to claim 8 with a heparin carrier, and eluting the adsorbate on the carrier with an eluent. The method for producing highly pure inufolistatin according to claim 8 or 9, wherein the eluent is a sodium chloride solution.

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