WO2000027885A1 - Novel chimeric polypeptide - Google Patents

Abstract

A novel chimeric polypeptide comprising klotho protein and immunoglobulin bonded thereto which remains stable in the blood and is usable in treating or preventing diseases wherein klotho gene participates; a DNA encoding this chimeric polypeptide; a process for producing the chimeric polypeptide; remedies/preventives with the use of the chimeric polypeptide or the DNA encoding the same; a method for detecting and quantitating a molecule interacting with the klotho protein; and a method for cloning a gene encoding a protein interacting with the klotho protein.

Description

 Description New Chimera Polypeptide Technical Field

 The present invention relates to a chimeric polypeptide in which a closoprotein and an immunoglobulin are bound, a DNA encoding the chimeric polypeptide, a method for producing the chimeric polypeptide, a DNA encoding the chimeric polypeptide or the chimeric polypeptide. Therapeutic and prophylactic drugs using Background art

 The Klotho gene is located at the site of transgenic mouse insertion in a transgenic mouse that exhibits remarkable and various premature aging symptoms, such as shortened life span, calcification of various organs, arteriosclerosis, and atrophy of reproductive organs. It is a gene identified as a causative gene for aging symptoms [Nature, 390, 45 (1997)]. The human closogene has also been cloned using the mouse closogene, and its structure has been elucidated.

Analysis of Hitokuroso c DNA, two mRNA force by differences in splicing from Hitokuroso ^genes? Transcribed, two proteins from the mRNA is translated (hereinafter, including these translated protein, the closo gene The encoded protein is called a closoprotein).

Of the above two types of mRNA, one of the closoproteins is a type 1 membrane protein (hereinafter referred to as a “type 1 membrane protein” having a structure having an N-terminal signal sequence region, an extracellular domain region, and a C-terminal transmembrane domain region). The cell-domain I is composed of two domains (KL1, KL2) that are homologous to bacterial or plant β-glucosidase. On the other hand, the other protein is a secretory protein having a signal sequence region at the N-terminal and a KL1 domain region (hereinafter referred to as a secretory protein). GBiochem. Biophys. Res. Commun., 242, 626 (1998)].

 Between the KL1 and KL2 domains of the membrane-bound closoprotein, Lys Lys Arg Lys, an amino acid sequence commonly found in sites that are cleaved by peptide hormone precursors, etc., is recognized. It has been suggested that the KL1 domain may be secreted following cleavage of the ligated closoprotein after translation.

 Although the expression of the Kokuso gene is high in the kidney, the severe aging symptoms caused by the Kokuso gene mutation affect systemic organs including the lungs, bones, stomach wall, and skin. It is presumed that some secretory factor s exists in the mechanism and exerts its effects [Nature, 390, 45 (1997)]. However, the mechanism by which the liposomal gene mutation causes various aging symptoms—the molecular function of liposomal protein has not yet been elucidated.

The aging of Croso mice has been achieved by cross-breeding experiments between Chloro // mouse and transgenic mice expressing the Closo gene, and experiments in which Closo proteins are expressed in Closo mice using an adenovirus vector incorporating membrane-bound Closo cDNA. Because of the prevention of the onset of symptoms, it is thought that the treatment of diseases caused by dysfunction of the stomatoprotein can be achieved by expressing the closo gene in the patient's body by gene therapy [Nature, 390, 45 (1997), W098 / 29544). However, by administering directly from outside the Klotho蛋white matter, no report about whether the treatment or prevention ^force? Possible of such diseases.

Various closoproteins such as membrane-bound closoprotein, secreted closoprotein, and closoprotein having only the KL1 region are administered from the outside, and the therapeutic or preventive effects of the above-mentioned diseases are analyzed, whereby the activity of the closoprotein is improved. It is thought that important areas can be analyzed. As a method of administering such a biological activity and a desired protein to a living body, a method of administering the protein directly into the blood by a technique such as intravenous injection is common. Shin power shin power? According to the method, the administered bioactive ⁵ ′ expected protein is cleared by the kidney glomerulus. Due to clearance and degradation by proteases in the blood, the half-life in the blood is shortened, and the desired effect may not be expected.

 As a technique for extending the half-life in blood, a method for producing chimeric polypeptides in which the target protein is linked to the constant region of immunoglobulin by using the long half-life of immunoglobulin in blood [W094 / 04689, Nature, 337, 525 (1990), J. Immunol., 149, 4096 (1992), Mol. Immunol., 31-1345 (1994)]. In these techniques, a constant region of immunoglobulin is bound to the C-terminal side of the extracellular domain of the membrane protein, thereby converting the membrane protein into a stable soluble protein.

 The constant region of the immunoglobulin to be bound includes a light chain (CL) and a heavy chain (consisting of a CH1-hinge-CH2-CH3 domain). The domain below the hinge excluding the CH1 domain of the heavy chain for the following reasons: It is commonly used.

 (1) The blood stability of immunoglobulins is comparable between the fragment below the hinge (Fc fragment) and the original immunoglobulin, and the light chain and CH1 are not directly involved in the stability [J. Exp. Med. , 121, 323 (1965)].

 ② Efficiency of secretion from cells is higher than when CHI is present [Nature, 344. 667 (1990)] o

 (3) The hinge portion has a flexible structure, and it is highly likely that the binding of the foreign protein at the hinge portion can maintain the activity of the foreign protein and immunoglobulin without impairing the original three-dimensional structure [Trends in Biotechnology, 14 52 (1996)].

 There has been no report on the production of a chimeric polypeptide by binding a closoprotein to an immunoglobulin and the effect of the chimeric polypeptide.

Disclosure of the invention

To examine whether the administration of closoprotein from the outside can treat or prevent the onset of diseases associated with the closogene, There is a need for proteins. There is also a need for an administrable closoprotein capable of treating or preventing the onset of a disease associated with the closogene.

 A chimeric polypeptide of a closoprotein and an immunoglobulin is expressed in animal cells by a gene recombination technique, and the purified chimeric polypeptide is administered to a closo mouse or a model animal of various diseases, whereby the disease model animal The present inventors have completed the present invention by confirming that the symptom is improved.

 That is, the present invention provides the following (1) to (24).

 (1) A chimeric polypeptide in which immunoglobulin is bound to a closoprotein.

(2) Klotho protein ^force?, Signal sequence and the extracellular domain of Klotho protein, Klotho protein having a region selected signal sequence and KL 1 region of click nitroso protein, the extracellular domain of Klotho protein, and the KL 1 of Klotho protein The chimeric polypeptide according to (1) above.

 (3) The chimeric polypeptide according to the above (1) or (2), which is a closoprotein derived from human or mouse.

(4) The chimeric polypeptide according to (1), wherein the chimeric polypeptide is an immunoglobulin having only immunoglobulin strength ^s and a constant region of the immunoglobulin.

 (5) The chimeric polypeptide according to (4), wherein the immunoglobulin constant region is an Fc region of human IgG (hinge-CH2-CH3).

 (6) The chimeric polypeptide according to (1), (4) or (5), wherein the immunoglobulin is a human or mouse-derived immunoglobulin.

 (7) The human IgG1 Fc region or human I8〇4 at the C-terminal side of the human closoprotein having the chimeric polypeptide power, signal sequence and KL1 region? (The chimeric polypeptide according to the above (1), which is a chimeric polypeptide obtained by combining regions.

(8) The chimeric polypeptide according to any one of (1) to (7) above, wherein the chimeric polypeptide is a chimeric polypeptide in which an immunoglobulin is bound via a linker peptide to the C-terminal side of a closoprotein. Petit (9) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the polypeptide, and a closoprotein A polypeptide having the activity of:

 The deletion, substitution or addition of the above amino acids can be performed by the methods described in Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci "USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids. Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci., USA, 82, 488 (1985), etc., using the site-directed mutagenesis method described in SEQ ID NO: 2 or 4. This can be done by introducing site-specific mutations into the DNA encoding the polypeptide, or one or several amino acids, as many as can be deleted, substituted, or added by site-directed mutagenesis. Means amino acid.

 (10) A DNA encoding the polypeptide according to any one of the above (1) to (9).

(11) a DNA comprising a nucleotide sequence selected from the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, and 6, or a DNA that hybridizes with the DNA under stringent conditions; and DNA encoding a polypeptide having an activity.

The above-mentioned `` DNAJ that hybridizes under stringent conditions '' refers to a colony hybridisation method, a plaque, '' It means DNA obtained by using the hybridization method or Southern blot hybridization method, and more specifically, using a filter on which DNA derived from colonies or plaques is immobilized. After hybridization at 65 ° C in the presence of Omol / 1 NaC1, 0.1- to 2-fold concentration of SSC (saline-sodium citrate) solution (composition of 1-time concentration SSC solution) DNA can be identified by washing the filter at 65 ° C using 15 mmol Zl sodium chloride and 15 mmol Zl sodium citrate). Can be given.

 Hybridization is based on Molecular Cloning, A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular-Cloning 2nd Edition), -Current Protocols in Molecular Biology, Supplement 1 ~ 38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current-protocols 'in' molecular biology sub-units 1-38, DNA and loningl: Core iechniques, A Practical Approach, Second Edition, Oxford University Press (1995) and the like.

 Specific examples of the hybridizable DNA include a DNA having at least 60% homology with the nucleotide sequence selected from the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, and 6, and preferably a homology of 80% or more. And more preferably a DNA having a homology of 95% or more.

 (12) A recombinant DNA obtained by incorporating the DNA of (10) or (11) above into a vector.

(1 3) is recombinant DNA force pKA TEXKLL ₇ 1 or pKANTEXKLL § 4, the (1 2) recombinant DNA according.

 (14) A transformant having the recombinant DNA according to the above (12) or (13).

 (15) The transformant according to (14), wherein the transformant is a transformant selected from a microorganism, an animal cell, a plant cell, an insect cell, a non-human transgenic animal, and a transgenic plant. .

 (16) The transformant according to the above (15), wherein the animal cell is a CHO cell or a YB2Z0 cell.

(17) A transformant containing a recombinant DNA obtained by incorporating a DNA encoding the polypeptide according to any one of the above (1) to (9) into a vector is used as a culture solution. And producing the polypeptide in the culture, collecting the polypeptide from the culture, and producing the polypeptide.

 (18) breeding a non-human transgenic animal having a recombinant DNA obtained by incorporating a DNA encoding the polypeptide according to any of the above (1) to (9) into a vector; A method for producing the polypeptide, comprising producing and accumulating a peptide in the animal, and collecting the polypeptide from the animal.

 (19) A therapeutic agent for a disease selected from renal disease, cachexia and osteoarthritis, comprising the polypeptide according to any one of the above (1) to (9).

 (20) A preventive agent for a disease selected from renal disease, cachexia and osteoarthritis, comprising the polypeptide according to any one of the above (1) to (9).

 (21) A gene therapy vector comprising the DNA of (10) or (11) above. (22) A method for detecting a molecule that interacts with a closoprotein, using the polypeptide of (1) to (9) above.

 (23) A method for cloning a gene encoding a protein that interacts with a closoprotein, using the polypeptide of (1) to (9) above.

 (24) A method for quantifying a molecule that interacts with a closoprotein, using the polypeptide of (1) to (9) above.

 (25) A method for producing an antibody recognizing a closoprotein, comprising immunizing a non-human animal using the polypeptide of (1) to (9) above as an antigen.

 (26) An antibody that recognizes the polypeptide of (1) to (9). Hereinafter, the present invention will be described in detail.

 [1] Construction of a gene encoding a chimeric polypeptide in which a closoprotein and an immunoglobulin are linked

DNA coding for a chimeric polypeptide (hereinafter referred to as Closo Ig) obtained by binding a closoprotein and immunoglobulin (hereinafter referred to as Closo ZIg DNA) ) Consists of the DNA encoding the closoprotein and the DNA encoding the immunoglobulin.

 As the Closoprotein in Closo Ig, any protein may be used as long as it is a protein having the activity of Closoprotein, but a Closoprotein containing a KL1 domain and having no transmembrane domain is preferable. . Such closoproteins include, for example, fragments of only the extracellular domain excluding the transmembrane domain of secretory closoproteins and membrane-bound closoproteins.

 Specific examples include secretory closoproteins derived from mouse or human, and fragments of only the extracellular domain excluding the transmembrane domain of the membrane-bound closoprotein.c Plasmid containing DNA encoding these proteins Specific examples of pNKH106 (FERM BP-5767) containing DNA encoding human secretory closoprotein, pNKH102 (FERM BP-5766) containing DNA encoding human membrane-bound closoprotein, mouse PNKM112 (FERM BP-6184) containing DNA encoding secretory closoprotein, and ρΝΚΜΙΟΙ (FERM BP-5765) containing DNA encoding mouse membrane-bound closoprotein (W098 / 29544).

The immunoglobulin I g G, I gM, I gA, neither I gE can be used, it ^mosquito? Preferably using the constant region of the immunoglobulin, the domains of the hinge one CH2 - CH3 H chain of I gG It is more preferable to use a region including the region (hereinafter, referred to as Fc region).

 Plasmids containing DNA encoding human IgG include phBSC4 (W097 / 10354), pKANTEX93 (W097 / 10354), pBRHIGl [Nucl. Acid. Res., 10, 4071 (1982). PATHG3 [Nucl. Acid. Res.,, 1779 (1986)].

When closo-ZI g is administered to humans as a prophylactic or therapeutic agent for diseases associated with closo-gene, human-derived closoprotein and immunoglobulin in closo / Ig can be used as anti-closo-ZIg antibodies. Is considered difficult to make, desirable.

 As DNA encoding the Closo Ig, specifically, a fragment obtained by cutting pNKH106, which encodes a human secretory closoprotein, with Ec0RI and PvuII, and SacII of pKANTEX93, DNA encoding human secreted closonohuman IgG1 Fc chimeraboli peptide obtained by isolating the BamHI fragment and linking both via synthetic DNA can be mentioned. The synthetic DNA includes a closoprotein which has been deleted by restriction enzyme digestion, and a DNA which encodes an amino acid sequence which complements the amino acid sequence of IgG1Fc and binds both. It is a thing. The DNA fragment encoding the closoprotein or immunoglobulin should be obtained by amplifying a DNA fragment by PCR using primers corresponding to the base sequence of the portion to be amplified by converting the plasmid containing each gene into type II. Can also be prepared.

Chimeric polypeptides in which liposomal proteins and immunoglobulins are bound can be prepared by directly binding the N-terminus of the immunoglobulin to the C-terminus of the closoprotein portion. Changes in the three-dimensional structure of both proteins due to force binding. In order to reduce the size, the closoprotein and the immunoglobulin can be bound via an appropriate length peptide (hereinafter, such an appropriate length peptide is called a linker peptide). Linker peptides are chimeric polypeptides of the IL-5 receptor chain and the IgG constant region [J. Biol. Chem., 268, 6581 (1993)] and the Η chain variable region of single-chain antibodies. Natl. Acad. Sci. USA, 85, 5879 (1988), Nature, 339, 394 (1989)] and the length of 4 to 20 residues were used. in order group, the force used Quy glycine Ya serine to get a specific secondary structure as an amino ^acid? desirable.

Also, taking the closo ZI g ^force? Right conformation, as can undergo modifications glycosylation, etc., described later DNA encoding a polypeptide signal peptide is added to the N-terminal to the [2] insert expressed base Kuta foremost, it ^mosquitoes? preferred to secrete expressed Kurosono I g in animal cells. The signal peptide used here is closogene A signal peptide consisting of 33 amino acids which the child originally has may be used, or a signal peptide of another secreted protein such as human IgG and human growth hormone may be used.

The Closo / Ig DNA prepared as described above can be incorporated into a plasmid vector capable of autonomous replication in a host organism for amplification and maintenance. This plasmid vector has a restriction enzyme site that can easily insert a foreign gene, and can express a gene such as a drug resistance gene to select a host organism into which the vector has been introduced ^. Used.

 As the host organism, E. coli Escherichia, which is a gram-negative bacterium, is preferably used because it is easy to introduce and isolate the vector, and the host organism is easy to culture and grow quickly.

 Plasmid vectors used in E. coli include pBluescriptll SK (-) (Stratagene) and pZErO-1 (Invitrogen)

And pUC19 [Gene, 33, 103 (1985)] and pBR322 [Gene, 2, 95 (1977)].

[2] Closo Z Ig production

 In order to express the Closo ZIg DNA of the present invention prepared in [1] in a host cell and to produce Closo / Ig of the present invention, Molecular Cloning 2nd Edition, Current. Protocols. The method described in Molecular Biology Supplements 1 to 38 and the like can be used.

 That is, by constructing a recombinant vector in which the DNA of the present invention is inserted downstream of a promoter of an appropriate expression vector, and introducing the vector into a host cell, a trait that expresses the polypeptide of the present invention. The polypeptide of the present invention can be produced by obtaining the transformant and culturing the transformant.

An expression vector is capable of autonomous replication in a host cell or Those that are capable of integration and contain a promoter that can transcribe mRNA from Closo / Ig DNA in the host cell are used. As the host cell, any prokaryotic cell, yeast, animal cell, insect cell, plant cell, or the like can be used as long as it can express the gene of interest. In addition, animal and plant individuals can be used.

 Both clonal exoproteins and immunoglobulin are originally secreted and produced in vertebrate cells, so when expressed secreted and expressed in animal cell hosts or animal individuals, closo ZIg retains the correct three-dimensional structure. In addition, modification of sugar chains and the like is performed, and it is considered that they have an action similar to that of closoprotein.

 When an animal cell is used as a host, an expression vector that contains a promoter for transcription in the host animal cell, Closo ZIg DNA, and a signal sequence for termination of transcription and polyadenylation of the transcript is used. Can be In addition, in order to facilitate the production and maintenance of the vector, it is desirable to use a vector capable of expressing a drug resistance gene that is capable of autonomous replication and gene transfer.

 As the promoter, any promoter can be used as long as it can perform transcription in animal cells. The early promoter of SV40, the promoter of the immediate early (IE) gene of human cytomegalovirus (CMV), and the like. Enhancer, Rous sarcoma virus (RSV), human immunodeficiency virus (HIV), Moloney mouse leukemia virus (MMLV), etc. Long-term repeats, and other promoters of animal-derived genes, such as the metamouth thionein gene, actin gene, and elongation factor-1.

A permanent closo / Ig-expressing cell in which chroso-ZI g DNA is integrated into the host chromosomal DNA is a host cell containing a closo / g expression vector containing a sequence capable of expressing a resistance gene to a drug such as G418 or hygromycin. In the presence of drugs And can be selected by culturing. In addition, in order to increase the amount of Closo / Ig produced in the host cell, a mouse / Ig constitutive expression vector containing a sequence capable of expressing the dihydrofolate reductase (dhfr) gene was prepared. Amplifying the copy number of closo / \ gDNA together with the dhfr gene by culturing while introducing the dhfr inhibitor, methotrexate, into a host cell and increasing the concentration of the dhfr inhibitor methotrexate. Can also. As a host cell for performing gene amplification using the dhfr gene, a cell in which the dhfr gene does not function, for example, CHO / dhfr- (ATCC: CRL-9096) is used.

 Specific vectors used for preparing the above-mentioned Closo ZIg expression vector include, for example, pAGE107 [JP-A-3-22979, Cytotechnology, 3, 133, (1990)], pAS3-3 (special Kaihei 2-227075), pCDM8 [Nature, 329, 840, (1987)], pcDNA3.1 (+) (Invitrogen), pREP4 (Invitrogen), pBK-RSV (Stratagene), p SVK3 [Amersham Pharmacia Biotech], cDNA l / Amp (Invitrogen), pc DNA1.1 (Invitrogen), pAGE103 Q. Biochem., 101, 1307 (1987), FERM BP-1312], pAMo [J. Biol. Chem., 268, 22782 (1993)], pAMoA Q. Biol. Chem., 268, 22782 (1993) )) And so on.

 Host cells include mouse / myeloma cells such as SP2 / 0, NS0, etc., rat-myeloma cells such as YB2 / 0, etc., african green monkey kidney cells such as COS-1, COS-7, and 293 etc. Human fetal kidney cells, hamster cells such as CH0 cells, BHK spores, etc.

 The host cell should be one that does not produce immunoglobulin by itself before the introduction of the vector. Purification of Clos Zig, and isolation is preferred.

As a method for introducing the recombinant vector, any method for introducing DNA into animal cells can be used. For example, the elect-portion method [ Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA, 84.7413 (1987)], and the like.

 When insect cells are used as host cells, a baculovirus expression system [

Bacuiovirus Express ion Vectors, A Laboratory Manual , WH Freeman and Company, New York (1992), Bio / Technology, 6, 47 (1988) ] ^force? Used. That is, after inserting the croso-Ig DNA into a vector called a transfer vector, the vector and the baculovirus are simultaneously introduced into insect cells, and placed under the promoter of the polyhedrin gene, which is a strong promoter. Closono / I Eight clones of recombinant baculovirus were prepared by homologous recombination, and the recombinant baculovirus was again infected into insect cells to express closono Ig. Can be.

Baculoviruses include Autographa cal i fornica nuclear polyhedrosis virus and silkworm nuclear polyhedrosis virus. As insect cells, Spodoptera frugiperda cells Sf9 and Sf21 [Bacuiovirus Expression Vectors, A Laboratory Manual, New York (1992)], Trichoplusia ni cells High 5 (manufactured by Invitrogen Corporation), and the like can be used. . The silkworm larva can also be used as it is. The transfer vector contains a polyhedrin promoter and a baculovirus-derived sequence for causing the same recombination, as well as maintenance and propagation of the vector and genetic manipulation such as integration of foreign genes in E. coli. sequences included (autonomously replicable sequence and drug resistance genes in E. coli) force, in particular pVL1392, pVL1393, _P BlueBac4 (both Invitrogen) and the like. Closo ZIg can also be produced using an individual animal. For example, according to a known method [American Journal oi Clinical Nutrition, 63.639S (1996), American Journal of Clinical Nutrition, 63, 627S (1996), Bio / Technology, 9, 830 (1991)]. To produce closo / Ig in animals transfected with closo / Ig DNA. it can.

The promoter, the force can be used, so long as it can function in an animal ^s, for example, non-casein promoter which is a mammary gland cell-specific promoters one

-, /? Casein promoter, y3 lactoglobulin promoter, whey acid promo-promoter, etc. Suitable for use.

 When a plant cell or a plant individual is used as a host, a known method [tissue culture,

20 (1994), Tissue Culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)].

 Any promoter that can express the gene in plant cells can be used as the promoter for the expression of Closo Ig DNA. For example, the 35S promoter of cauliflower mosaic virus (CaMV) and the inactin 1 promoter In addition, by inserting intron 1 of maize alcohol dehydrogenase gene between the promoter and the Closo ZI g DNA to be expressed, the expression efficiency of the Closo ZI g DNA can be increased. Can also be given.

 Examples of the host cell include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, wheat, barley, rye, Alfalpha, flax and the like.

 As a method for introducing the recombinant vector, any method for introducing DNA into plant cells can be used. For example, agrobacterium, electroporation [Cytotechnology, 3, 133 (1990) )], Particle gun

(Gene gun), a method using, and the like.

 Cells and organs of a plant into which Closo ZIgDNA has been introduced can be cultured in large quantities using a jar fermenter. In addition, by redifferentiating the transfected plant cells, plant individuals (transgenic plants) into which the Closo ZIgDNA has been introduced can also be constructed.

When using a prokaryote such as a bacterium as a host cell, use a Closo Ig expression vector. Is used, which is capable of autonomous replication in a host prokaryote and in which closo // Ig DNA is arranged downstream of a promoter having a ribosome binding sequence. It is preferable that the distance between the ribosome binding sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases in the case of an E. coli host vector). Although not necessary, it is better to place the transcription termination sequence directly below the Closo / 1 g DNA. In addition, for the selection of the transformant, a sequence that expresses a gene that can be the most important, such as a drug resistance gene, should be included.

Any promoter can be used as long as it can be expressed in a host cell. If for example where the E. coli and inn production, t卬promoter (Ptrp), lac pro motor-(Pi¾ £), P _L promoter one, T7 promoter one coater, such as [rho _kappa promoter, promoters derived from Escherichia coli or phage, etc. I can give first class. Also, artificially designed and modified promoters such as a promoter (P ^ 2 x 2), a promoter, a T71ac promoter, and a let I promoter in which two P trps are connected in series can be used. When Bacillus subtilis is used as a host, examples thereof include promoters of SP01 and SP02 which are phages of Bacillus subtilis, pen P promoter and the like. Examples of expression vectors include pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), and pKYP10 (manufactured by Tokukai Sho 58-1) 106 000), p KYP 200 Agric. Biol. Chem., 48, 669 (1984)], p LSA 1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL 1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescript II SK (-) (STRATAGENE), pT rs30 (FERM BP—5407), pTrs3 2 (FERM BP-54 08), p GHA 2 (FERM B P-400), p GKA 2 (FERM B-6998), pTer 2 (Japanese Unexamined Patent Publication No. 3-22997) , US 4 6 6 1 9 KUS 4 9 3 9 0 94, US 5 1 6 0 7 3 5), p KK 2 2 3-3 (Amersham-Pharmacia 'Biotech), p GEX (Pharmacia) ), PET system (Novagen), pSupex, pTrxFus (Invitrogen), pMAL-c 2 (New England Biolabs).

 As host cells, microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia col i XLl-Blue, Escherichia col i XL2-Blue , Escherichia col i DH1, Escherichia col i MC1000> Escherichia col i—KY3276, Escherichia col i W1485, Escherichia col i 09, Escherichia col i concealed 01, Escherichia col i No. 49, Escherichia col i W3110, Escherichia col i NY Serrat if icaria> Serrat ia font icola, Serrat ia 1 iquefaciens, Serrat ia marcescens.

Corynebacterium glutamicum ATCC140, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophi lum ATCC13870, Microbacterium ammoni aphi lum ATCC15354, Pseudomonas sp. D-0110 and the like.

 Any method for introducing the recombinant vector can be used as long as it is a method for introducing DNA into the host cell described above. For example, electroporation (Nucleic Acids Res., 16, 6127 (1988)), USA, 69, 2110 (1972)], a protoplast method (JP-A-63-248394), Gene, 17, 107 (1982), and Molecular & General Genet. ics, 168, 111 (1979).

When yeast is used as a host cell, the expression vector includes a promoter that performs transcription in the host yeast, Crosono Ig DNA, a transcription termination sequence, and a gene that becomes a transformation marker in yeast, such as a drug resistance gene and Those containing a sequence capable of expressing an amino acid synthesis system gene such as TRP1, HIS3, LEU2, etc. are used. In addition, in order to facilitate the production and maintenance of the expression vector, it is desirable to have autonomous replication and expression of a drug resistance gene that is a gene transfer marker in coli. Any promoter can be used as long as it can perform transcription in yeast.For example, the alcohol dehydrogenase gene ADH1 of Saccha genus yces cerevisiae and the galactokinase gene GAL1 GAL10 can be used. Promoter-PH05 promoter, PGK promoter, GAP promoter, heat shock protein promoter, MF α1 promoter, CUP 1 promoter, Pichia pastor is alcohol oxidase gene A 0 X 1 promoter Etc. are used.

 Examples of the host cell include yeast strains belonging to the genera Saccharomyces, Schizosaccharomyces, Kluybium mycetes, Trichosporon, Schizinomyces, Pichia and the like. Yes, concrete white 3 contains SaccharomYces cerevisiae

Sch i zosaccharomyces ombe-Kluyveromyces lactis Trichosporon pul lulans Schwann iomyces alluvius Pichia pastor is available.

 As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method (Methods.Enzymol. 194, 182 (1990)) and a spheroplast method [ Natl. Acad. Sci. USA, 81, 4889 (1984)], lithium acetate method [Journal of Bacteriology, 153, 163 (1983)], and the like.

 A transformant derived from a microorganism, animal cell, or plant cell having a recombinant vector incorporating a DNA encoding the Closo ZIg of the present invention is cultured according to a conventional culture method. By producing and accumulating Closo ZIg and collecting the Closo / Ig from the culture, the Closo ZIg can be produced.

RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)] Eag 1e MEM Medium [Science, 122, 501 (1952)] D MEM medium [Virology, 8, 396 (1959)] 1 99 Medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] A medium or the like to which fetal serum or the like has been added can be used. However, the fetal bovine serum because it contains immunoglobulins of cattle, it force not use if you row the isolation and purification of closo / g from the culture ^medium? Desirable. If necessary, an antibiotic such as benicillin-strebtomycin may be added to the medium.

Culture, usually pH6~8, 30~40t :, 5% C0 ₂ present in conditions such as below:! Perform for ~ 7 days.

 If necessary, an antibiotic such as kanamycin or penicillin may be added to the medium during the culture.

 As a medium for culturing a transformant obtained by using insect cells as a host cell, generally used TNM-FH medium (Pharmingen), Si-900 II SFM medium (Life Technologies) ), ExCel 1400, EXCe 1405 (all manufactured by JRH Biosciences), Grace's Insect Medium [Grace, T., Nature, 195, 788 (1962)) Etc. can be used.

 Culture conditions are preferably pH 6-7, culture temperature 25-30 ° C, and culture time is usually 1-5 days.

 If necessary, an antibiotic such as gentamicin may be added to the medium during the culture.

 When the transformant is an animal or plant individual, it is bred or cultivated according to a usual method to produce and accumulate the Closo ZIg of the present invention, and collect the Closo / Ig from the animal or plant individual By doing so, the Closo / Ig can be produced.

That is, in the case of an animal individual, for example, a non-human transgenic animal having the Closo / ⁷ Ig DNA of the present invention is bred, and the recombinant DNA encoding Closo / 1 g is added to the animal. By producing and accumulating it in an animal and collecting the Closo ZIg from the animal, the Closo / Ig can be produced. Production and storage in the animal For example, milk, eggs and the like of the animal can be mentioned.

 In the case of a plant individual, for example, a transgenic plant having the Closo ZIg DNA of the present invention is cultivated, the Closo ZIg of the present invention is produced and accumulated in the plant, and the Closo / Ig is extracted from the plant. By collecting, the Closo ZIg can be produced.

 A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium can be used as long as the medium can efficiently culture the cells.

 The carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Acids, alcohols such as ethanol and propanol, and the like can be used.

 Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., ammonium salts of inorganic or organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn starch, etc. Plyka, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, and digests thereof can be used.

 As the inorganic substance, potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.

The culture is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The cultivation temperature is preferably 15 to 40 ° C, and the cultivation period is usually 16 to 96 hours. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, antibiotics such as ampicillin-tetracycline may be added to the medium during the culture period. Good.

 When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using a promoter, cultivate a microorganism transformed with an expression vector using tnp promoter overnight, for example, isopropyl-1 /?-D-thiogalactobyranoside. Sometimes, acrylic acid or the like may be added to the medium.

 In order to isolate and purify Closo ZIg accumulated in the culture of the above-mentioned transformant, the following ordinary protein isolation and purification methods may be used.

When it is secreted outside closo ZI g force ^s cells, closo / I g is it accumulated in the medium. Therefore, after the culture is completed, only the medium without cells is collected by a method such as centrifugation. Normal protein isolation and purification methods from the culture medium, ie, solvent extraction, salting out using ammonium sulfate, desalting, precipitation with organic solvents, getylaminoethyl (DEAE) — Cephalos, DIAI0N HPA -Anion exchange chromatography using resins such as 75 (Mitsubishi Chemical), Mono-Q (Amersham-Pharmacia's Biotech), and resins such as SP Sepharose (Amersham-Pharmacia-Biotech) Cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose, phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectrics A purified sample can be obtained using an electrophoresis method such as point electrophoresis alone or in combination. If the immunoglobulin constant region of Closo / Ig is derived from IgG, a purified sample can be easily obtained by affinity chromatography using protein A or protein G, which has the property of specifically binding to IgG. be able to. When Closo / Ig accumulates in the cells of the transformant, the cells of the transformant are collected from the culture after the completion of the culture by centrifugation or the like, and suspended in a buffer. Crush cells with a sonicator or French press to obtain a cell-free extract. K When Roso-ZIg is present in a dissolved state in the cells, a purified sample is obtained from the supernatant obtained by centrifuging the cell-free extract in the same manner as in the above-mentioned purification and isolation from the medium. Obtainable. When Crosono Ig is present as an insoluble form in the cells, the cell-free extract is centrifuged, and the insoluble form of Closo // Ig is collected as a precipitate fraction. After solubilizing the inclusion body of the Kurosono I g a protein denaturing agent, dilution or dialysis the solubilized solution, to a dilute solution extent that the concentration force ^s proteins or protein denaturing agent does not contain a protein denaturing agent does not denature Then, after the Closo Ig is restored to a normal three-dimensional structure, a purified sample can be obtained by the same isolation and purification method as described above. In addition, according to a known method [J. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)], in vitro transcription 'translation system Can be used to produce the Closo / Ig of the present invention.

 Furthermore, Closo Ig of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tB0c method (t-butyloxycarbonyl method). can do. In addition, Advanced ChemTech, Parkin Elma, Pharmacia Biotech, Protein Technology Instrument (Synthecel l-Vega) Chemical synthesis can also be performed using a peptide synthesizer such as PerSeptive, Shimadzu Corporation.

Structural analysis of the closo / ^/ I g of the purified invention, how commonly used in protein chemistry, for example, Protein Structure Analysis for Gene Cloning (Hirano Hisacho, Tokyo Chemical Dojin, 1 9 9 3 years) Can be performed by the method described in (1).

 [3] Use of Closo // Ig

 (1) Since aging of Croso mice can be restored by administration of Closono Ig, remedy / prevention of aging symptoms such as arteriosclerosis, alopecia, soft tissue calcification, etc. Can be used as medicine.

(2) Administration of Closo / Ig can relieve symptoms in renal disease model mice Because it can be used, it can be used as a therapeutic or prophylactic drug for kidney disease.

 (3) The administration of Closo ZIg can relieve the symptoms of cachexia disease model mice, so that it can be used as a therapeutic or preventive drug for cachexia.

(4) The administration of Closo / Ig can restore the symptoms of osteoarthritis model mice, and can be used as a therapeutic or prophylactic agent for osteoarthritis.

Pharmaceutical comprising the protein of the present invention, the therapeutic agent or prophylactic agent as the protein alone be administered can also be a certain force ^s, usually one is had the higher of allowed the protein pharmacological It is desirable to mix it with a carrier and provide it as a pharmaceutical preparation produced by any of the methods well-known in the art of pharmacy. Preferably, a sterile solution which is dissolved in an aqueous carrier such as water or an aqueous solution of salt, glycine, glucose, human albumin or the like is used. Also, pharmacologically acceptable additives such as buffering agents and tonicity agents to bring the formulation solution closer to physiological conditions, such as sodium acetate, sodium chloride, sodium lactate, potassium chloride, quencher Sodium acid or the like can also be added. It can also be lyophilized for storage and dissolved in an appropriate solvent before use.

 It is desirable to use the most effective route for treatment and prevention. Usually, the parenteral route, for example, the subcutaneous, intramuscular, intravenous, or respiratory route is used.

The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., and is usually 10 _μg / kg to 20 mg / kg per day for an adult.

 (5) Since the extracellular domain of a closoprotein is presumed to exert its function by interacting with other molecules, identifying the molecule that interacts with the protein requires the function and mechanism of action of the closoprotein. It is important to elucidate.

For molecules that bind to and interact with the extracellular domain of Croso, Can be used in the same manner as antibodies against the molecule [Cell, 73, 447 (1993), Pro Natl. Acad. Sci. USA, 92, 1866 (1995)]. That is, when the immunoglobulin constant region of Closo ZI g is labeled with fluorescence, peroxidase, radioisotope, or the like, the Closo Ig can be easily detected using the label. Similarly to the antibody that recognizes, it is possible to detect the above molecule that binds to the Closo ZIg, or to isolate the cell using a cell sorter if the molecule is present on the cell surface.

 When the molecule is a protein, the cell expressing the molecule to be bound is screened using Closo / Ig against a cell group into which the expressed cDNA library has been introduced, whereby the cloned ZIg is obtained. It is also possible to clone the cDNA of the molecule to which it binds. This method has the following advantages over the method using a closoprotein and an anti-closo antibody.

 (1) Since the closoprotein portion that binds to the molecule and the immunoglobulin portion used for detection are different domains, the detection of the molecule is not hindered even if the molecule binds to closo // Ig.

 (2) Labeled anti-immunoglobulin antibodies (FITC-labeled anti-human IgG antibodies, etc.) are commercially available and easily available.

 (6) The molecule obtained in (5) above can be quantified by the following method using labeled Closo / Ig.

① Kurosono I _g to labeled with a radioactive isotope or an enzyme, such as Paokishidaze such ¹²⁵ 1. At this time, it is desirable to label a region of closo / 1 g in which the binding between the above molecule and closo ZIg is not inhibited. The region includes the immunoglobulin portion of Closo Ig. A sufficient amount of the labeled Closo ZIg is added to a test sample and reacted with the molecules in the sample. After the reaction, an excess of the molecule in which the molecule is immobilized on beads or the like is added to the reaction solution in excess, and free closo ZIg present in the reaction solution is bound to the molecule in which the molecule is immobilized, and centrifugation is performed. Remove. After removal, it remains in the reaction solution. The amount of the molecule bound to the labeled Closo / Ig is determined by measuring the existing labeled Closo ZIg by a detection method according to the label, such as radioactivity or coloring of a substrate that develops a color with an enzyme.

(2) Immobilize Closo ZIg on beads or the like. Label the test sample with a radioisotope such as ^125I . A fixed amount of the labeled molecule is added to 1 g of immobilized Closo ^ and reacted with a limited amount of immobilized Closo / Ig. In this case, the amount of the molecule that binds to 1 g of Crosono is limited to a certain amount, and therefore the amount of the labeled molecule that binds to Ig of Crosono decreases as the amount of the molecule in the sample increases. The immunocomplex of the immobilized Closo ZIg and the labeling molecule is separated from the solution by centrifugation or the like, and the amount of the labeling molecule bound to the antibody is measured by measuring the radioactivity. In this case, the immobilized antibody against the immunoglobulin constant region used for Crosono Ig was used to separate the immunocomplex of Crosono Ig and the labeled molecule from the solution without immobilizing Closo ZIg. You can also.

 In both (1) and (2) above, a solution of the molecule with several known concentrations is prepared, the solution is used as a standard sample, a calibration curve is created from the measured values, and the measured values of the sample to be measured are measured. By comparing with, the amount of the molecule can be quantified.

(7) By immunizing Closo Ig of the present invention as an antigen, an antibody recognizing Closoprotein can be obtained. However, when Crosono Ig is used as an antigen, an antibody that recognizes the immunoglobulin portion of Closo ZIg is usually produced simultaneously in the serum of the immunized animal. Is prepared, and a monoclonal antibody recognizing the closoprotein is selected from among them. As a method of immunization, the Closo ZIg of the present invention is administered subcutaneously, intravenously or intraperitoneally to a non-human mammal such as a heron, a goat, a rat, a mouse, a hamster and the like. The closo 1 g of may be administered as it is force ⁵ ', Freund's complete adjuvant (Complete Freund' s Adjuvant), aluminum hydroxide gel, pertussis vaccine and the like, it forces ^s preferably is administered with a suitable adjuvant.

The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. After each dose Blood is collected from the fundus venous plexus on the 3rd to 7th days, and whether or not the serum reacts with the Closo ZIg used for immunization is determined according to an enzyme immunoassay (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Check by measuring the antibody titer. A non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells.

 As the antibody-producing cells, spleen cells, lymph nodes, and antibody-producing cells in peripheral blood, particularly splenocytes, are suitably used.

As myeloma cells, 8-azaguanine-resistant mouse (derived from BALBZc) myeloma cell line P3—X63Ag 8—U1 (p3-Ul) strain [Current Topics in Microbiology and Immunology, 18, 1 (1978) )), P3-NS1 / 1 / Ag41 (NS-1) strain [European J. Immunology, 6, 511 (1976)], SP2Ό-A14 (SP_2) strain (Nature , 276, 269 (1978)], P3-X63-Ag8653 (653) strain [J. Immunology, 123, 1548 (1979)], P3-X63-Ag8 (X63) strain [Nature, 256 , 495 (1975)], and the like, cell lines ^mosquitoes? that suitably used o derived from mouse

 The hybridoma can be prepared by the following method.

 The antibody-producing cells and myeloma cells are mixed, suspended in HAT medium (medium containing hypoxanthine, thymidine, and aminopterin in normal medium), and cultured for 7 to 14 days. After cultivation, a portion of the culture supernatant is removed, and those that react with the antigen but do not react with the protein containing no antigen are selected by enzyme immunoassay or the like. Next, single cloning is performed by the limiting dilution method, and those with a stable high antibody titer determined by the enzyme immunoassay are selected as monoclonal antibody-producing hybridomas.

In addition, the selected hybridomas are cultured, and the culture supernatant is used as the primary antibody, and the sample containing the closoprotein is subjected to stamping to detect the absorptive force of the same molecular weight as the kokusoprotein. Is selected as an antibody-producing hybridoma that recognizes the closoprotein. W098 / 29544 as a sample containing closoprotein The culture supernatant of CHO cells into which the closoprotein produced by the method described in (1), for example, the human secretory closoprotein expression plasmid pYS111 has been introduced, can be used.o

 ゥ Estane blotting can be performed according to the method described in Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

 Monoclonal antibodies can be obtained from a culture solution obtained by culturing hybridoma cells, or a liquid obtained by injecting hybridoma cells into the peritoneal cavity of an animal and causing the animal to undergo water tumor. Can be prepared by separating and purifying from

 Methods for separating and purifying monoclonal antibodies include centrifugation, ammonium sulfate precipitation, caprylic acid precipitation, or chromatography using DEAE-Sepharose columns, anion exchange columns, protein A or G columns, or gel filtration columns. And the like can be used alone or in combination.

 Closoproteins can be immunologically detected or quantified using the above-mentioned monoclonal antibodies recognizing closoproteins, as in the case of antibodies using liposomal proteins as antigens.

 Examples of the method for immunological detection include an ELISA method using a microtiter plate, a fluorescent antibody method, a western blot method, and an immunohistological staining method.

As a method of immunologically quantifying, San Deutch ELISA method Epitopu of monoclonal antibodies using two different monoclonal antibodies reactive with Klotho protein in the liquid phase, labeled with a radioisotope such as ¹²⁵ 1 The radioimmunoassay method using a Closoprotein and an antibody that recognizes the protein of the present invention can be used. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a view showing the structures of brassmids pKANTEXKLLα1 and pKANTEXKLLα4 for expressing Closo / IgG animal cells. ? Closo / I g G l (: is Kurosono 1 ₈ DNA encoding G 1 F c, closo I GG4 Ji DNA encoding Kurosono 12_Rei_4 F c, P _M0 is the Moroni one murine leukemia virus is a promoter LTR, dhfr is dihydrofolate reductase gene, G418I or animal cells antibiotic G 418 resistance gene, Amp ¹ "is a transformation marker in represents the antibiotic ampicillin resistance gene in the form transformants marker one in E. coli.

 Figure 2 shows the results of SDS-PAGE of purified Closo ^^ IgG 1 (: and Closo 1304 Fc. The results under non-reducing conditions are shown in lanes 1 and 2, and the results under reducing conditions. The results are shown in lanes 3 and 4. Lanes 1 and 3 show the results with Closo / IgG1Fc, and lanes 2 and 4 show the results with Closo ZlgG4Fc, respectively. Indicates the molecular weight of the protein, the arrow on the left shows the value of each molecular weight (kDa), and the arrow on the right shows the position of each band of Closo IgGFc and the comparison with the marker. The approximate molecular weight (kDa) is shown.

 FIG. 3 is a view showing the results of evaluation of purified Closono IgG1 Fc and Closo ZIgG4 Fc by the ELISA method. The vertical axis indicates the absorbance at 415 nm, and the horizontal axis indicates the concentration of each Closo / IgGFC. 〇 shows the results of Kuroso ZIgG1Fc and the fist shows KuguchisoIgG4Fc.

FIG. 4 is a graph showing the results of ゥ ヱ stamp lotting of human secreted closoprotein using the culture supernatant of the hybridoma of mouse 2 and clone 5 as the primary antibody. The band on the right side is a human secreted auxoprotein of about 70 kDa detected with a force of ⁵ kDa. The lines and numbers on the left indicate the position of the band of the protein molecular weight marker and the value of the molecular weight (kDa).

 FIG. 5 is a diagram showing the results of immunohistochemical staining of rat kidney using culture supernatants of hybridomas of mouse 2 and clone 5. A shows the result at a magnification of 12.5 times with an optical microscope, and B shows the result at a magnification of 50 times. The part that looks dark (actually brown) is the part that has reacted with the force antibody.

Fig. 6 Body weight of Closo mice administered with Crosono IgGl Fc after 4.5 weeks of age It is a figure which compared a progress with a control group. Kigakuroso ZI gG 1 F c administration group

(4) mean body weight (g), □ indicates mean body weight (g) of PBS-administered control group (6).

 FIG. 7 is a graph comparing organ weights (g) of thymus, spleen and testis of Closo mice 7 weeks after administration of Crosono IgG 1 Fc with the control group. The black bar indicates the average organ weight of the Croso IgG 1 Fc-administered group (4 animals), and the shaded bar indicates the average organ weight of the PBS-administered control group (6 animals). Hereinafter, the present invention will be specifically described with reference to examples.

BEST MODE FOR CARRYING OUT THE INVENTION

 Example 1 Preparation of Human Secreted Closoprotein Human IgG Fc Region Chimeric Polypeptide Expression Plasmid

 Plasmids pKANTEXKLL 71 (IgG1 type) and pKANTEXKLLy4, which secrete and express human secreted closoprotein / human IgGFc region chimeric polypeptide (hereinafter referred to as crossoZIgGFc) in animal cells. (IgG4 type) was prepared as follows.

 (1) Construction of plasmid pBS-hKLLinker, which encodes human secreted closoprotein and linker peptide

Almost the entire human secretory closo cDNA obtained by digesting the human secretory closo cDNA clone pNKH106 (FERM BP-5767: WO 98/29544) with the restriction enzymes EcoRI and PvuII. Approximately 1.6 kb of EcoR I-PVu II fragment (corresponding to amino acid sequence 1 to 536 containing 33 amino acids of signal peptide of human secreted closoprotein) and SEQ ID NOS: 7 and 8 Of the synthetic DNA consisting of two synthetic DNAs having different base sequences is inserted between the Ec0RI_HindIII of the cloning vector pBIuescript SK (-) (Stratagene). , PBS-hKLCF were prepared. The P vu II -Hind III linker encodes the C-terminus of human secreted closo (amino acid sequence 537-549) and the FLAG peptide (amino acid sequence Asp Tyr Lys Asp Asp Asp Asp Lys), the joint of P vu II- H ind III closo cDNA linker in one and F LAG B sp HI site ^solved? it exists. An Ec0RI-BspHI fragment containing human secreted closo cDNA obtained by digesting the pBS-hKLCF prepared above with restriction enzymes EcoRI and BspHI, and SEQ ID NO: 9 and A DNA encoding a linker peptide (amino acid sequence Gly Ser Gly Gly Ser Gly) composed of two synthetic DNAs having the nucleotide sequence shown in FIG. 10 was transferred to the EcoRI / vector pBluescriptSK (-). Plasmid pBS-KLLinker containing DNA encoding human secretory closoprotein and linker peptide was prepared by inserting it between SacI.

(2) Preparation of plasmid pBS-Mroly 1 coding for human IgGI Fc region Four primers having the nucleotide sequences shown in SEQ ID NOS: 11 to 14 and having the mutually annealing base sequences DNA PCR was performed using the primers as a primer and a double-stranded DNA encoding a part of the IgG1 Fc hinge and CH2 (up to the SacII site in the cDNA). K pn I site and S a I site solved ^terminal? exists.) were amplified.

 The KpnI-SacII fragment obtained by digesting the amplified DNA with restriction enzymes JLnI and SacII, and the remaining portion of IgGIFC (part of CH2 and CH3) were The SacII-BamHI fragment of pKANTEX93, which encodes, was inserted between the KpnIZBamHI sites of the closing vector pBluescript SK (-), and human IgGlFC (hinge-CH2-CH3 ) Containing the plasmid pBS-Mrolγ1.

 (3) Preparation of plasmid pMKE01 encoding human IgG4Fc

By performing PCR using four primers DNA having the nucleotide sequences shown in SEQ ID NOs: 15 to 18 and having the nucleotide sequences that anneal to each other as a primer and 錶 type, the IL-5 receptor chain is Up to C-terminal below SacI site Plastid, linker one peptide and human I g G 4 part of F c (to hinge hereinafter P ma CI site) S ac I site and E c 0 RI ^cytokines? Exists in double-stranded DNA (end encoding ) Was amplified.

The SacI-EcoRI fragment obtained by cleaving the amplified DNA with restriction enzymes SacI and EcoRI was converted into a plasmid pAI297 (WO 97-10354) having human IL-5 receptor α chain cDNA. ) And human IL-5 receptor Ec0RI-SacI fragment containing the _α- chain cDNA, to prepare plasmid pMK01.

 A HindIII-PmaCI fragment encoding the human IL-5 receptor α-chain, linker peptide and human IgG4 hinge of pMK01, and a human IgG4

The PmCI-BamHI fragment encoding the rest of the IgG4Fc of the Fc cDNA clone pBShCy4 was ligated to HindIII / PAGE210 (WO 98/29544), a vector for animal cell expression. Plasmid pMKEX01, which encodes the human IL-5 receptor << chain human IgG4Fc chimeric polypeptide, was prepared by inserting between BamHI.

 (4) Preparation of plasmid encoding Closo IgG Fc

(2) a Mr 0 I- B amH I fragment containing the fabricated pBS- MroI ₇ 1 human I g G 1 F c DNA encoding the region of pBS- KLLinker prepared in (1) M ro I / Plasmid pBSKLLy1, which encodes Crosono IgGlFc, was prepared by inserting it between BamHI sites.

Similarly, insert the Mr0I-BamHI fragment containing DNA encoding human IgG4 Fc of pMKEX01 prepared in (3) between the MroIZBamHI sites of pBS-KLLinker. it was thus produced a plasmid pBSKLL _y 4 that co one de the closo / I gG4 F c.

 (5) Preparation of plasmid for animal cell expression of Closo ZlgGFC

Includes the DNA encoding the Closo / IgG1Fc of pBSKLLα1 prepared in (4). The EcoRI—BamHI fragment is ligated to the Ec0RI-BamHI fragment containing a part of the plasmid pKA TEX93 for expression in animal cells, resulting in a Closo IgG1Fc animal cell. An expression plasmid pKANTEXKLLα1 was prepared. SEQ ID NO: 5 shows the nucleotide sequence encoding Closo ZI gG 1 Fc in pKANTEXKLLy1. Similarly, the Ec0RI-BamHI fragment containing the DNA encoding the closo "IgG4Fc of pBSKLL4 prepared in (4) is used to contain the animal cell expression vector portion of the plasmid pKANTEX93 ^ by coupling with 2_R I one B AMH I fragment, encoding the closo / I g G 4 F c of closo / / I GG4 the F c animal cell expression plasmid PKANTEXKLLy 4 of the work made. pKANTEXKLL ₇ 4 The nucleotide sequence is shown in SEQ ID NO: 6.

 The structures of pKANTEXKLLα1 and pKANTEXKLLγ4 are shown in FIG.

transformant Escherichia coli DH5 a / pKANTEXKLL ₇ 4 containing a transformant Escherichia coli DH5 Fei / pKANTEXKLL γ 1, and pKANTEXKLL § 4 containing pKANTEXKLL § 1, 1998 September 25, dated in industrial technology FERM BP-6526 and FERM BP-6527 have been deposited at the Institute of Biotechnology, Industrial Science and Technology, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan (zip code 305-8566). Example 2 Production of Closo ZI gG F c in Animal Cells

 (1) Introduction of Closom / IgGFC expression brasmid into animal cells

Rat myeloma cell line ΥΒ 2Z0 (ATCC No. CRL-1662) and Chinese hamster ovary epithelial cell line (¾0 / — () were used as host cells for the vector for expressing the Closo IgG Fc prepared in Example 1. (: 101½0644) (hereinafter referred to as DG 44) YB 2Z0 was cultured in 10% fetal bovine serum (FBS) and 0.1% 2-mercapto for each cell culture. RPMI 1640 medium supplemented with ethanol, DG44 is a modified Iscove's Dulbecco's medium supplemented with 10% FBS and 10% HT Sabment (Life 'Technologies). Iscove's modified Dulbecco's medium; I MDM) were cultured in 5% C 0 ₂ conditions with and grown.

To each 1 × 10 ⁷ cells, 30 g of pKANTEXKLLy1 was introduced by an electoral port method using Gene Pulser (Bio-Rad).

 pKANTEXKLLy4 was similarly introduced into two types of cells. The electroporation was performed according to the method specified by Gene Pulser, except that the voltage was 0.2 kV, 250 / F for YB20, and 0.35 kV, 250 ^ F for DG44.

 (2) Selection of cells expressing high levels of Closo Zl gG F c

 After electoral port poration, YB2 / 0 and DG44 were each suspended in 40 ml of the medium described in (1), and 200 / y1 was dispensed into a 96-well plate and cultured. On the second day of culture, YB2 / 0 sucked out 100 u1 of the supernatant of 2001, added 1001 of YB20 medium supplemented with 1 mg Zm1G418, and cultured on day 3. Then, 100 1 1 of 200 1 was sucked, and 100 I of a medium for YB 20 supplemented with 0.1 mg of SmgZm 1 G418 was added. The operation performed on the third day was repeated for another two days, followed by culturing.

 For DG44, on day 2 of culture, aspirate 1001 of 2001 supernatant, add 1 mg Zm 1G418 and 10% dialyzed FBS!], Add HT Not IMDM added 10 O 1. The operation was repeated for another 3 days to culture. After the culturing, the plate was periodically observed, and for the holes where the cell growth potential was observed, Closo Ig Fc was detected by the ELISA method described below using the supernatant 501.

Closo / IgFc secreted in the supernatant (pKANTEXKLL γ1 / YB2 / 0 16 cells, pKANTEXKLL 4 / YB2 / 0 33 cells, pKANTEXKLLa 1 / DG44 37 cells, pKANTEXKLL 74 / DG44 (26 cells), the cells were transferred to 24-well plates and cultivation was continued. [Quantification of Closo / Ig GF c by ELISA method]

 An anti-human clonal monoclonal antibody KM207 (FERM BP—61197: WO988 / 29554) produced in mice using the N-terminal peptide of human closoprotein as an antigen at a concentration of 10 g / m1 Dissolve 50 μl of PBS (phosphate buffered saline) solution in a 96-well plate each time, and let it stand still in step 4 to immobilize KM 2 076 on the bottom of the hole. did. After discarding the solution, PBS100 ^ 1 containing 1% bovine serum albumin (BSA) was dispensed and allowed to stand at room temperature for 1 hour to perform blocking to prevent non-specific binding. Discard the solution and add 0.05% 6% After washing with 85 (PBS-Tween), 500 1 of the culture supernatant as a sample was added, and allowed to stand at room temperature for 1 hour to react with KM206.

 Discard the solution, wash with PBS-Tween, and dilute horse radish peroxidase (HRP) -labeled anti-human IgG1 antibody (for quantification of 1 gG1FC of closo) or HRP label Anti-human IgG4 antibody (when quantifying Closo IgG4Fc) 501 was added and left at room temperature for 1 hour to react with bound Closo / IgGFc. Discard the solution, wash with PBS-Tween, add ABT S solution 501 that develops color with HRP, allow it to stand at room temperature for 15 minutes, and use a plate reader to reduce the intensity of color development using a plate reader. Measured as absorbance in nm.

 2 For cells cultured in a 4-well plate, add the dhfr inhibitor methotrexate (MTX) to a final concentration of 50 nM and culture. Gene amplification was carried out by sequentially increasing the order to 2000 nmol Zl and 200 nmol 1/1. For the cells that acquired the resistance of 200 nmo 1/1 MTX, the amount of closo / IgGFc in the supernatant was quantified by ELISA in the same manner as described above. The cell with the highest secretion was selected for each strain, and the strain was cloned singly by the serial dilution method.

 (3) Culture of cell clones with high expression of Closo Ig G F c

For the Closo // IgGFc high-expressing cell clone selected in (2), Nourishment. The host YB2Z0 clone was cultured in a cell culture flask in 150 ml of GIT medium (Wako Pure Chemical Industries) until it became confluent. After becoming confluent, 0.5 mg of ZmlG418 and 200 nmolZ1MTX were added, and culture was continued for 4 days. For the clone of the host DG44, culture in the same medium (150 ml) for DG44 used in (1) until confluence, followed by addition of lmgZml G4 18 and 200 nmol 1 MTX to CH0. The medium was changed to -S-SFII medium (Life Technologies Inc.) and cultured for 4 days.

 (4) Purification of Crosono IgG Fc

 The medium after the culture of (3) was collected, and passed through a ProsepA column (Bioprocessing, Bioprocessing; volume: 1 m1) at a flow rate of 100 m1 / hour in step 4, The secreted Closo gG F c was adsorbed in the medium. Elution was carried out with 0.1 mol Zl glycine-HC1 (pH 3.0), and the eluate was collected by fractionating 1 ml each. The fractionated eluate was immediately neutralized by adding 1 mo 1 Tris 50/1, and the amount of protein in the eluate was detected by absorbance at 280 nm. Collected pictures.

 (5) Analysis of purified Closo / IgG Fc by electrophoresis

The 2. 5 / g of 5〃 g and closo ^/ / I gG4 F c of closo / I gG 1 F c purified in (4), a known method [Nature, 227, 680, (1970)] one 4 according SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 20% gradient gel was performed to examine the molecular weight and the degree of purification. The results are shown in FIG. As shown in FIG. 2, purified Closo // IgG1 and Closo ZIgG4 have a molecular weight of about 200 kilodaltons (hereinafter referred to as kDa) under non-reducing conditions. Below, a band of about 100 kDa was observed. Under non-reducing conditions, a dimer was formed, and under reducing conditions, disulfide bonds in the Fc portion were cleaved and the molecular weight was confirmed to be about half the molecular weight.

(6) Evaluation of purified Closo ZI gG F c FIG. 3 shows the results of analyzing Closono IgG 1 (:, Closo 13〇4 Fc) purified in (4) according to the ELISA method described in (2) of Example 2. FIG. As shown in the figure, Closo IgG G Fc and Closo / Ig G4 Fc were detected by ELISA in a concentration-dependent manner, respectively. ^ iKM2076 has been shown to have both a closo moiety recognized by the anti-human IgG antibody and a Fc moiety Example 3. Closo ^ lg GFc as immunogen Preparation and evaluation of monoclonal antibodies recognizing closoprotein

 (1) Preparation of monoclonal antibody using Closo / ^ lgGFC as immunogen

 Three mice were immunized once a week (total of 5 times) with Closo ZIgG1Fc (100y «g) purified in (2) of Example 2. The preparation and selection of the hybridoma was carried out according to a conventional method described in Antibodies-A Laboratory Manual, Cold spring Harbor Laboratory (1988) and the like. That is, spleen cells of the immunized mouse were fused with mouse myeoma cells (P3-U1) to prepare a hybridoma. The obtained hybridoma was cultured, and the culture supernatant was used as a sample to select and clone a hybridoma producing an antibody against Closo IgG 1 Fc by the ELISA assay. The ELISA method uses a 96-well plate immobilized with 10 μg / ml of Closo IgG 1 Fc, and uses peroxidase-labeled anti-mouse immunoglobulin antibody as the primary antibody for detection. (DAK0) was used in the same manner as in Example 2, (4), ELI SA method.

Furthermore, from the antibodies produced by the selected hybridomas, an antibody that specifically recognizes the human secretory closoprotein expressed in CH0 cells was selected by Western blotting as follows. According to the method described in W09 8/2954, SDS-PAGE was performed using the culture supernatant 101 of CH0 cells expressing human secreted closoprotein, and PVDF (polyvinylidene difluoride) was obtained from the gel. C) Transferred to the membrane. After blocking for 1 hour with 1% BSA, the cells were reacted for 1 hour with the culture supernatant of each hybridoma selected above (containing 2 to 4 gZml of antibody). After washing with PBS-Tween for 1 hour, a peroxidase-labeled anti-mouse immunoglobulin antibody (DAKO) was reacted as a second antibody for 1 hour at room temperature. After washing with PBS-Tween for 1 hour, autoradiography was performed by reacting with ECL ゥ ヱ stamp lotting detection reagent (Amersham 'Pharmacia' Biotech). A human secretory closoprotein of about 70 kDa in which bandoca was detected was selected as an antibody that specifically recognizes the closoprotein. FIG. 4 shows the results of Western blotting using one of the antibodies thus selected (mouse 2 and clone 5). Ri by the above method, the antibody force against human secreted Klotho protein of native ^type? To be able to effectively get the divide.

 (2) Immunohistochemical staining of rat kidney using antibodies against Crosono IgG Fc as antigen

A 4-week-old SD rat (female, weighing about 100 g, Nippon S.L.C.) was anesthetized with 50 mg Zkg Nembutal, perfused and fixed with PLP (periodate-lysine-paraformaldehyde) fixative, and kidney Was extracted.

 After the kidneys were minced, post fixation was performed overnight in the same fixative at 4 ° C.

 The fixed tissue was washed with PBS containing 5% and 10% sucrose for 4 hours each, and then washed with PBS containing 15% sucrose at-° C and 4 ° C.

 The washed tissue was embedded in a 0. CT compound (Sakura Fine Technical Co., Ltd.) and frozen with liquid nitrogen. Six-meter-thick frozen sections were prepared on a cryostat and mounted on silane-coated slides.

 To remove endogenous peroxidase activity, the sample sections were treated with 0.3% hydrogen peroxide in methanol at room temperature for 10 minutes and washed with PBS. Non-specific adsorption of antibodies was prevented by treating with ProteinBlock Serum Free (DAKO) at room temperature for 20 minutes.

The sample section was incubated with the hybridoma culture supernatant of the antibody selected in (1) overnight at 4 After the reaction and washing with PBS, a biotinylated anti-mouse IgG1 goat antibody (Amersham Pharmacia Biotech) was reacted as a second antibody for 30 minutes at room temperature. The sample slice was washed with PBS, reacted with Streptavidin-HRP conjugate Strept ABCompe 1 ex / HRP (DAKO) for 30 minutes at room temperature, and then washed with PBS.

 After washing, a brown color was formed by reacting with a DAB (diaminobenzidic acid) reagent at room temperature for 1 minute. After nuclear staining with hematoxylin, dehydration with an ascending ethanol series and xylene, the cells were sealed, and observed under a light microscope.

 Figure 5 shows the results of immunohistochemical staining using the culture supernatant of the hybridoma of one of the antibodies selected in (1) (mouse 2 and clone 5). In the tubules.

 The same operation as above was performed using the anti-closoprotein antibody KM 2070 (WO 98/29544). However, unlike the case of mouse 2. clone 5, no reaction was observed in smooth muscle. Example 4 Suppression of Senile Symptoms of Closo Mice by Administration of Closo / ^^ IgG Fc Each animal was intraperitoneally administered at 10 μm. The administration was performed daily for 2 weeks from the start of administration, and once every 3 days thereafter.

 As a control, PBS was administered to 6 one-week-old Kuroso mice at the same administration interval.

Weaning was performed 2 weeks after the start of administration, and body weight was measured over time after 4 weeks of age. Seven weeks after administration, necropsy was performed and various aging symptoms (loss of weight gain, atrophy of the thymus, spleen and testes, loss of abdominal adipose tissue, and ectopic calcification of the thoracic ribs) were observed in black mice. Examined. In other words, thymus, spleen and testis, abdominal adipose tissue, and thoracic ribs are observed, and thymus, spleen and testis are also measured for organ weight. Was.

 FIG. 6 shows the weight progress of the Kuroso mice in each group. The weight gain was significantly higher in the group treated with Crosono IgG 1 Fc than in the control group, and the body weight increased with the age of the week, compared to the control group, which showed almost no weight gain. Was seen.

As shown in FIG. 7, in the closo ZI g G 1 F c administration group, thymus, organ weight in the spleen and testis obviously many as compared with the control group, by necropsy observations, closo / ^/ I It was found that atrophy was clearly suppressed in the gG1Fc administration group as compared to the control group.

 Furthermore, in the control group, no abdominal adipose tissue was observed, and ectopic calcification of the thoracic ribs was observed, whereas in the Crosono IgG 1 Fc administration group, abdominal adipose tissue was observed. No ectopic calcification of the thoracic ribs was seen. From the above, it was shown that various aging-like symptoms observed in the Kuroso mouse were suppressed by administration of Closo IgG 1 Fc. In addition to this, in addition to expressing the closogene in vivo by gene therapy, it is possible to prevent or treat diseases caused by dysfunction of the closoprotein by directly administering the closoprotein from outside. Was confirmed. It was also shown that the KL1 region of the closoprotein may be sufficient for the prevention and treatment of such a disease caused by the closoprotein. Example 5 Improvement of Cachexia Symptoms by Administration of Crosono IgGlFc

 (1) Preparation of cachexia model mouse

A cachexic model mouse was prepared by transplanting a tumor into a nude mouse and allowing it to survive as follows. The human melanoma strain SEKI [In vitro culture of human cancer cells, Asakusa Shoten, Tokyo (1975)] was expanded according to the method of Iseki et al. [Jpn. J. Cancer Res., 86, 562 (1995)]. Thereafter, a 7-week-old male nude mouse BALB / c-nu / nu was subcutaneously implanted into the flank using an injection needle to form and maintain a tumor. The formed tumor was excised, cut into 2 mm squares, and subcutaneously transplanted into the flank of a plurality of nude mice (7-week-old male, BALB / c-nu / nu) using a transplantation needle. One week later, 10 mice in which tumor engraftment was confirmed were selected as cachexia model mice, and subjected to a Closo / 1 gG1Fc administration test. As described in (2), these cachexia model mice were compared with model mice with normal serum glucose concentrations (140 to 170 mg / dl) as the tumor grew. It is a mouse that has a severely reduced and cachectic trait.

 (2) Closo IgG1Fc administration test

 The Crosono IgGI Fc purified in (4) of Example 2 was diluted with PBS to a concentration of 50 gZml, and this was diluted with a cachexia model mouse 1 week after cancer transplantation prepared in (1). Twenty-five Z animals were intraperitoneally administered to 5 mice at a time. The administration was performed twice a week every 3 to 4 days, and the administration period was 3 weeks.

 As a control, PBS was administered to 5 cachexia model mice at the same administration interval and period.

 After the administration period, blood was collected from each cachexia model mouse, and the glucose concentration in the serum was measured. Glucose concentration was measured using a commercially available kit (Kyowa Medex, Detamina-I L, GLU II). As a result, in the cachexia model mouse to which the control PBS was administered, the serum glucose concentration was 70.2 mg / d1, whereas in the cachexia model mouse to which It was shown that in the model mice of liquid quality recovered to 8 3. Omg / d1, and the state of cachexia was improved. Industrial applicability

According to the present invention, a secretory closoprotein stable in blood is provided, and treatment or prevention of the onset of a disease involving the closo gene becomes possible. PT / J

"Sequence List Free Text"

SEQ ID NO: 1 Description of an artificial sequence: DNA encoding a chimeric protein of human secreted auxoprotein and a constant region of human IgG1 heavy chain (hinge-CH2-CH3) SEQ ID NO: 2-artificial Sequence description: Chimeric protein between human secreted auxoprotein and the constant region of human IgG1 heavy chain (hinge-CH2-CH3)

SEQ ID NO: 3—Description of Artificial Sequence: DNA encoding chimeric protein of human secreted closoprotein and constant region of human IgG4 heavy chain (hinge-CH2-CH3) SEQ ID NO: 4 Description of artificial sequence: Chimeric protein of human secreted closoprotein and constant region of human IgG4 heavy chain (hinge-CH2-CH3)

SEQ ID NO: 5—Description of Artificial Sequence: DNA encoding chimeric protein of human secreted closoprotein and constant region of human IgG1 heavy chain (hinge-CH2-CH3) SEQ ID NO: 6—Description of artificial sequence : DNA encoding a chimeric protein of human secretory closoprotein and the constant region of the human IgG4 heavy chain (hinge-CH2-CH3) SEQ ID NO: 7—Description of artificial sequence: Human secretory closoprotein and FLAG Sense strand of linker designed to encode amino acid residues 537-549 of peptide

SEQ ID NO: 8-Description of artificial sequence: antisense strand of linker designed to encode amino acids 537-549 of human secreted closoprotein and FLAG peptide

SEQ ID NO: 9 Description of Artificial Sequence: Linker Sense Strand Encoding Designed Linker Peptide

SEQ ID NO: 10—Description of artificial sequence: antisense strand of linker encoding the designed linker peptide

SEQ ID NO: 11—Description of artificial sequence: Sense primer designed to amplify DNA encoding part of the hinge and C H2 regions of IgG1 heavy chain

SEQ ID NO: 12—Description of artificial sequence: Copies hinge and part of CH2 region of IgG1 heavy chain Antisense primer designed to amplify the DNA to be sequenced. SEQ ID NO: 13—Description of Artificial Sequence: Amplify DNA that codes for the hinge and part of the CH2 region of the IgG1 heavy chain. Sense primer designed as follows

SEQ ID NO: 14 Description of artificial sequence: Antisense primer designed to amplify DNA encoding the hinge and part of the CH2 region of IgG1 heavy chain SEQ ID NO: 15-Artificial sequence Description: Sense primer designed to amplify DNA encoding part of the alpha chain of the IL-5 receptor, and part of the hinge and CH2 regions of the IgG4 heavy chain

SEQ ID NO: 16—Description of Artificial Sequence: Designed to amplify DNA encoding part of the _a- chain of the IL-5 receptor, and part of the hinge and CH2 regions of the IgG4 heavy chain Antisense primer

SEQ ID NO: 17—Description of Artificial Sequence: Designed to amplify DNA encoding a portion of the IL-5 receptor's << chain, and part of the hinge and CH2 regions of the IgG4 heavy chain. Antisense primer

SEQ ID NO: 18—Description of Artificial Sequence: Designed to amplify DNA encoding part of the heavy chain of the IL-15 receptor, and part of the hinge and CH2 regions of the IgG4 heavy chain Antisense primer

Claims

The scope of the claims

 1. Chimeric polypeptide in which closoprotein and immunoglobulin are linked.

2. Klotho protein ^force?, Region chosen signal sequence and the extracellular domain of Klotho protein, a signal sequence Contact Yopi KL 1 region of click port Seo protein, the extracellular domain of click port source proteins, as well as from KL 1 of Klotho protein The chimeric polypeptide according to claim 1, which is a closoprotein having the following formula:

 3. The chimeric polypeptide according to claim 1 or 2, wherein the closoprotein is a closoprotein derived from human or mouse.

4. Immune ^Guropurinka? Is an immune globulin with only constant region of immune globulin, Kimerapori base peptide of claim 1, wherein.

 5. The chimeric polypeptide according to claim 4, wherein the constant region of the immunoglobulin is the Fc region of human IgG (hinge-CH2-CH3).

 6. The chimeric polypeptide according to claim 1, 4 or 5, wherein the immunoglobulin is a human or mouse-derived immunoglobulin.

 7. A chimeric polypeptide in which a human IgG1 Fc region or a human IgG4 Fc region is linked to the C-terminus of a human closoprotein having a signal sequence and a KL1 region. The chimeric polypeptide according to claim 1, which is present.

 8. The chimeric polypeptide according to any one of claims 1 to 7, wherein the chimeric polypeptide is an immunoglobulin capable of binding to the C-terminal side of the chimeric polypeptide caclosoprotein via a linker peptide.

 9. A polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the polypeptide, and A polypeptide having the activity of a protein.

 10. DN encoding the polypeptide according to any one of claims 1 to 9

A o

1 1. A DNA consisting of a base sequence selected from the base sequences shown in SEQ ID NOs: 1, 3, 5, and 6, or a DNA that hybridizes with the DNA under stringent conditions and has an activity possessed by a closoprotein DNA that encodes a polypeptide.

 12. A recombinant DNA obtained by incorporating the DNA of claim 10 or 11 into a vector.

 13. The recombinant DNA according to claim 2, which is a recombinant DNA force pKANTEXKLL γ1 (FERM BP-6526) or pKANTEXKLL 74 (FERM BP-6527).

 14. A transformant having the recombinant DNA according to claim 12 or 13.

15. The transformant according to claim 14, wherein the transformant is a transformant selected from a microorganism, an animal cell, a plant cell, an insect cell, a non-human transgenic animal, and a transgenic plant.

 16. The transformant according to claim 15, wherein the animal cell is a CHO cell or a YB2 / 0 cell.

 17.A transformant having a recombinant DNA obtained by incorporating a DNA encoding the polypeptide according to any one of claims 1 to 9 into a vector is cultured in a culture medium, and the polypeptide is purified. A method for producing the polypeptide, comprising producing and accumulating the polypeptide in the culture, and collecting the polypeptide from the culture.

 18. breeding a non-human transgenic animal having a recombinant DNA obtained by incorporating a DNA encoding the polypeptide according to any one of claims 1 to 9 into a vector; A method for producing the polypeptide, comprising producing and accumulating the polypeptide in the animal, and collecting the polypeptide from the animal.

 1. A therapeutic agent for a disease selected from renal disease, cachexia and osteoarthritis, comprising the polypeptide according to any one of claims 1 to 9.

20. A preventive agent for a disease selected from renal disease, cachexia and osteoarthritis, comprising the polypeptide according to any one of claims 1 to 9.

2 1. A preventive agent for aging symptoms, comprising the polypeptide according to any one of claims 1 to 9.

 22. A therapeutic agent for aging symptoms, comprising the polypeptide according to any one of claims 1 to 9.

 23. A vector for gene therapy containing the DNA according to claim 10 or 11.

24. A method for detecting a molecule that interacts with a closoprotein, using the polypeptide of claim 1.

 25. A method for cloning a gene encoding a protein that interacts with a closoprotein, using the polypeptide of claim 1.

 26. A method for quantifying a molecule that interacts with a closoprotein, using the polypeptide of claim 1.

 27. A method for producing an antibody recognizing a lipoprotein, which comprises immunizing a non-human animal using the polypeptide of claims 1 to 9 as an antigen.

 28. An antibody that recognizes the polypeptide of claims 1-9.