WO2001009349A1 - Gene encoding novel serine protease-like protein - Google Patents

Abstract

By an oligocap method originally developed for isolating full-length cDNA, a plural number of full-length cDNAs are isolated from a human placental tissue cDNA library. Among these cDNAs, a clone (hC-PLACE1009992) encoding a novel serine protease-like protein is isolated. Further, a mouse cDNA (mC-PLACE1009992) corresponding to this human cDNA is successfully isolated too. C-PLACE1009992 shows a change in the expression in the brain of a patient with Alzheimer's disease compared with the brain of a normal subject, which suggests that it might relate to Alzheimer's disease.

Description

 Description Genes encoding novel serine protease-like proteins

 The present invention relates to novel serine protease-like proteins, their genes, and their production and use. Background art

It has recently been shown that proteases present in the body are involved not only in blood coagulation and fibrinolysis, but also in various diseases such as cancer, inflammation, allergies, immune diseases, and Alzheimer's disease. In particular, serine proteases having a serine at the active center are the best-studied proteases for their structure and function, and many studies have been conducted on the relationship between their physiological actions and diseases ( Barrett AJ, Salvesen G, et al (eds): Proteinase Inhibitors. Amsterdam, Elsevier, 1986) _c Abnormalities in tribcine, chymotrypsin, and elasase, which are responsible for food digestion, cause tongitis. Blood coagulationThrombin, which controls the fibrinolysis system, Xlla factor, XIa factor, Xa factor, IXa factor, VI la factor, and abnormalities such as plasmin, tissue plasminogen activator, perkinase, hemorrhage, thrombosis DIC, DIC, and myocardial infarction. In addition, abnormalities such as elastase, cathepsin, mast cell-derived chimase and trypase, which are involved in phagocytosis, induce inflammation, emphysema and rheumatism. Currently thought to be the cause of Alzheimer's disease /? — It is thought that serine protease is also involved in the accumulation of amyloid protein (Shunaga Iwata, Satoshi Tsubuki, Takaomi Nishimichi, Ayumi, 189, 9-14, 1999). In addition, serine oral protease is also present in the complement reaction system, and the classical pathway and the alternative pathway, Clr, Cls, factor B and factor D, have been implicated in inflammation, rheumatism and allergy. More recently found MASP-1 and MASP-2, which are involved in the third lectin pathway (Yuichi Endo, Teizou Fujita, Protein Nuclease, 45, 671-678, 2000) It has been shown to have a serine protease domain.

 On the other hand, some domains have a serine protease-like structure but no proteolytic activity. The hepatocyte growth factor (HGF), which is responsible for the proliferation of hepatocytes, is secreted extracellularly as Pro-HGF after removal of the signal peptide, and then cleaved by the HGF activator, a serine protease. As a result, mature HGF in which the heavy and light chains are linked by an SS bond is formed. The light chain region has a structure homologous to the functional domain of serine protease, but no protease activity is detected. However, it is an important domain in the expression of HGF's hepatocellular proliferative and tumor suppressor activities (Tomokazu Ohnishi, Yasushi Daikohara, Hachiburu, 38, 613-621, 1999). As described above, serine proteases and serine protease-like domains play important roles in living organisms, and are considered to be important drug discovery evenings in developing drugs. Disclosure of the invention

 The present invention has been made in view of such circumstances, and an object of the present invention is to provide a novel serine protease-like protein, a gene thereof, and a method for producing the same and use thereof.

In order to solve the above-mentioned problems, the present inventors first isolated multiple full-length cDNAs from a human placental tissue cDNA library using the oligocap method originally developed to isolate full-length cDNAs. did. The nucleotide sequence of one of the isolated cDNAs was determined and its structural analysis was performed. As a result, it was confirmed that the sequence retained in a known serine protease was retained. (This clone was named “hC-PLACE1009992”). The present inventors have also succeeded in isolating a mouse cDNA corresponding to the human cDNA. (This clone was named "mC-PLACE1009992". Human and mouse clones were collectively called "C-PLACE1009992"). The expression of C-PLACE1009992 was fluctuated in the brain of Alzheimer's patients compared to the brain of normal humans, suggesting an association with Alheimer's disease. In addition, the expression is decreased by ultraviolet irradiation, which may be related to skin cancer.

 The present invention relates to a novel serine protease-like protein C-PLACE1009992, DNA encoding the protein, and their production and use.

 (1) DNA described in any of (a) to (d) below,

 (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4.

 (b) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 1 or 3.

 (c) an amino acid sequence represented by SEQ ID NO: 2 or 4, wherein one or more amino acids have a substituted, deleted, inserted, and / or added amino acid sequence; DNA that encodes a protein that is functionally equivalent to a protein consisting of an amino acid sequence.

 (d) hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 under stringent conditions, and is functionally equivalent to a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4 DNA that codes for a protein.

 (2) DNA encoding a partial peptide of a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4,

 (3) a protein or peptide encoded by the DNA according to (1) or (2),

 (4) A vector into which the DNA according to (1) or (2) has been inserted,

 (5) a transformant carrying the DNA of (1) or (2) or the vector of (4),

(6) The protein or peptide is expressed using the transformant according to (5). A method for producing a protein or peptide according to (3), comprising the steps of:

 (7) an antibody that binds to the protein or peptide according to (3),

 (8) a polynucleotide comprising at least 15 nucleotides complementary to DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary strand thereof,

 (9) A method for screening a compound that binds to the protein according to (3),

 (a) contacting a test sample with the protein or a partial peptide thereof,

 (b) a step of detecting a binding activity between the protein or a partial peptide thereof and a test sample

(c) selecting a compound having an activity of binding to the protein or a partial peptide thereof, and

 (10) A pharmaceutical composition containing the DNA according to (1) or (2), the protein or peptide according to (3), or the vector according to (4).

The present invention provides a novel protein “C-PLACE1009992”. The amino acid sequence of the human C-PLACE1009992 (hereinafter referred to as “hC-PLACE1009992”) protein contained in the protein of the present invention is represented by SEQ ID NO: 2, and the nucleotide sequence of the cDNA encoding the protein is represented by SEQ ID NO: 1. Shown in The amino acid sequence of a mouse-derived C-PLACE1009992 (hereinafter referred to as “mC-PLACE1009992”) protein is shown in SEQ ID NO: 4, and the nucleotide sequence of cDNA encoding the protein is shown in SEQ ID NO: 3. The C-PLACE1009992 gene encodes a protein consisting of 737 amino acids (derived from human) or 720 amino acids (derived from mouse) having characteristics of serine protease. The expression of the C-PLACE1009992 gene was significantly reduced in the hippocampus of patients with Alzheimer's disease compared to normal individuals, suggesting that it is related to Alzheimer's disease. For this reason, the diagnosis of Alzheimer's disease by measuring the expression level of the gene of the present invention and the screening or treatment of Alzheimer's disease as a preventive or therapeutic agent are described. It is conceivable to carry out such operations as In addition, by using the mouse-derived gene of the present invention, preparation of knockout mice, preparation of Alzheimer's disease model animals, and screening of prophylactic and therapeutic agents for Alzheimer's disease using the model animals can be performed. Is also conceivable.

 C-PLACE1009992 protein can be prepared as a recombinant protein or as a natural protein. The recombinant protein is prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention has been inserted into an appropriate host cell as described below, and purifying the protein expressed in the transformant. Is possible. On the other hand, a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987)). Publish. John Wiley & Sons Section 16. 16.19). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. In addition, in vitro translation

(For example, ^r 0n the fidelity of mRNA translation in the nuclease-treated rabbit reticulocyte lysate system.Dasso, MC, Jackson, RJ (1989) Nucleic

Acids Res. 17: 3129-3144 ”) can be used to prepare the protein of the present invention.

The present invention includes proteins functionally equivalent to the human-derived C-PLACE1009992 protein identified in this example. Such proteins include, for example, mutants, homologs, and variants of the C-PLACE1009992 protein described in SEQ ID NO: 2 or 4. Here, “functionally equivalent” means that the target protein has the same biological function or biochemical function as the C-PLACE1009992 protein. Such a function includes a function as a serine protease or a function related to Alzheimer's disease. Serine protease functions as, for example, a protein that plays a role in the cascade system found in blood coagulation and fibrinolysis and complement activation, and controls the reaction system by cleaving other proteins. Functions and Or the ability to proliferate cells found in the serine protease-like structure of HGF. Serine protease abnormalities can, for example, disrupt blood coagulation 'regulation of the fibrinolytic system' and the regulating system, which in turn causes diseases such as bleeding, thrombosis and myocardial infarction. Functions related to Alzheimer's disease are thought to be the cause of abnormalities in the complement system (Rogers, J. et al. Pro Natl. Acad. Sci. USA, 89, 10016-10020, 1992) and the onset of Alzheimer's disease / -A function related to the accumulation of amyloid protein (Shunaga Iwata, Sosatsu Tsubuki, Takaomi Nishimichi, History of Medicine, 189, 9-14, 1999).

 Those skilled in the art can use a protein functionally equivalent to the protein identified in this example, for example, by introducing a mutation into an amino acid sequence in the protein (for example, a Hi-specific mutagenesis method (Current Protocols). (1987) Publish. John Wiley & Sons Section 8.1-8.5)). Such proteins may also be generated by amino acid mutations in nature. In the present invention, one or more amino acids in the amino acid sequence (SEQ ID NO: 2 or 4) may be substituted, deleted, inserted and / or substituted, as long as it has a function equivalent to the protein identified in this example. Alternatively, different proteins are included due to addition or the like.

 The number and location of amino acid mutations in proteins are not limited as long as their functions are maintained. The number of mutations is typically within 30 amino acids, preferably within 10 amino acids, and more preferably within 5 amino acids (eg, within 3 amino acids). The substituted amino acid is preferably an amino acid having properties similar to the amino acid before substitution from the viewpoint of maintaining the function of the protein. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as non-polar amino acids, and are considered to have similar properties to each other. In addition, examples of the non-charger include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin. In addition, acidic amino acids include Asp and Glu, and basic amino acids include Lys, Arg, and His.

Proteins functionally equivalent to the proteins identified in this example are well known to those of skill in the art. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4) or gene amplification technology (PCR) (Current protocols in Molecular Biology edit. (1987) Publish. John Wiley & Sons Section 6.1-6.4). That is, those skilled in the art will understand that a DNA encoding the protein identified in this example (SEQ ID NO: 1 or 3) or a part thereof may be used as a probe or an oligonucleotide that specifically hybridizes with the DNA. Using this as a primer, a DNA hybridizing with the DNA can be isolated. Further, based on the isolated DNA, a protein encoded by the DNA can be prepared. The present invention includes proteins encoded by DNAs that hybridize to MAs encoding these proteins, as long as they have the same function as the proteins identified in the Examples. Examples of organisms for isolating functionally equivalent proteins include, but are not limited to, vertebrates such as humans, mice, rats, magpies, bushes, and magpies.

 The stringent conditions for hybridization to isolate DNA encoding a functionally equivalent protein are usually `` lxSSC, 0.1% SDS, 37 ° C '', and more stringent conditions. The condition is about 0.5xSSC, 0.1% SDS, 42 ° C, and the more severe condition is about 0.1xSSC, 0.1¾SDS, 65 ° C. It can be expected that DNA with higher homology to the probe sequence will be isolated as the stringency becomes higher. However, the combination of the above SSC, SDS and temperature conditions is merely an example, and those skilled in the art will appreciate that the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe concentration, etc.). The same stringency as described above can be realized by appropriately combining the length and the hybridization reaction time.

The protein isolated using the hybridization technique or the gene amplification technique is compared with the protein of the present invention described in SEQ ID NO: 2 or 4, Usually, they have high homology in their amino acid sequences. High homology refers to sequence identity of at least 50% or more, more preferably 70% or more, even more preferably 90% or more (eg, 95% or more). Amino acid sequence homology can be determined by homology search using BLAST X.

 The present invention also provides a partial peptide of the protein of the present invention. The partial peptide of the protein of the present invention can be used, for example, for preparing an antibody that binds to the protein of the present invention. The partial peptide of the present invention comprises an amino acid sequence of at least 7 amino acids, preferably 9 amino acids or more, more preferably 12 amino acids or more, more preferably 15 amino acids or more. The partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptide.

 The present invention also provides a DNA encoding the protein of the present invention. The form of the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized MA, and the like, in addition to cDNA. In addition, as long as it can encode the protein of the present invention, a DNA having an arbitrary nucleotide sequence based on the degeneracy of the genetic code is included. As described above, the DNA encoding the protein of the present invention can be obtained by a hybridization method using the DNA sequence of SEQ ID NO: 1 or 3 or a part thereof as a probe, or a primer designed based on the information of these DNA sequences. It can be isolated by a conventional method such as the gene amplification method (PCR) used.

The present invention also provides a vector into which a DNA encoding the protein of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it can stably maintain the inserted DNA. For example, if Escherichia coli is used as a host, a pBluescript vector ( Stratagene) is preferred. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. Examples of the expression vector include a pBEST vector (promega) for expression in a test tube and a pET vector (Novagen) for expression in E. coli. PME18S-FL3 vector (GenBank Accession No. AB009864) for expression in cultured cells, and pME18S vector (Mol Cell Bio 1.8: 466-472 (1988)) for expression in living organisms. Can be used. Insertion of the DNA encoding the protein of the present invention into a vector is performed by a conventional method, for example, ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Pub. John Wiley & Sons). Section 11.4 to 11.11). The present invention also provides a transformant having a DNA encoding the protein of the present invention or a vector into which the DNA has been inserted. The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells can be used depending on the purpose. Host cells can be used, for example, for the production of the proteins of the invention. Production systems for protein production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells. When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts. The host cells of the present invention also include cells of interest for use in analyzing the function of the C-PLACE1009992 protein and screening for function inhibitors and function promoters using the C-PLACE1009992 protein. Vector transfer into host cells can be performed by, for example, calcium phosphate precipitation, electropulse perforation (Current protocol s in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), ribofect It can be performed by the Yumin method (GIBC0-BRL) or the microinjection method. Preparation of the C-PLAC E1009992 protein from the transformant can be carried out by using a protein separation / purification method known to those skilled in the art.

The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary strand thereof. Here, the “complementary strand” refers to one strand of a double-stranded DNA consisting of A: T and G: C base pairs with respect to the other strand. Also, “complementary” means at least 15 contiguous nucleosides The homology is not limited to the case where the nucleotide sequence is completely complementary to the nucleotide sequence, and it is sufficient that the nucleotide sequence has homology of at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or more. The algorithm described in this specification may be used as an algorithm for determining homology.

 Such a polynucleotide can be used as a probe for detecting and isolating a DNA encoding the protein of the present invention, or as a primer for amplifying the DNA of the present invention. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, a DNA having at least a part or the entire sequence of the DNA of the present invention and having a chain length of at least 15 bp is used. When used as a primer, the 3'-side region must be complementary, but a restriction enzyme recognition sequence or evening fragment can be added to the 5'-side.

 Further, the polynucleotide of the present invention includes an antisense for suppressing the expression of the C-PLACE1009992 protein of the present invention. The antisense has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and preferably has a chain length of 2000 bp or less in order to cause an antisense effect. Such an antisense is, for example, prepared based on the DNA sequence information of SEQ ID NO: 1 or 3, based on the information of the phospholipids, <£ (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 16, 3209-21 (1988)).

The DNA of the present invention and its antisense can be applied to, for example, gene therapy. As a disease targeted for gene therapy using the DNA of the present invention, for example, Alheimer's disease can be considered. When these molecules are used for gene therapy, for example, a non-viral vector such as a retroviral vector, an adenovirus vector, a viral vector such as an adeno-associated virus vector, or a non-viral vector such as a liposome is used. It may be administered to a patient by a method or an in vivo method. The present invention also provides an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. It also includes all classes of antibodies. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

 In the case of a polyclonal antibody, the antibody of the present invention can be obtained by synthesizing an oligopeptide corresponding to an amino acid sequence and immunizing a rabbit according to a conventional method (Current protocols in Molecular Biology edit). Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.12 to 11.13) On the other hand, in the case of monoclonal antibodies, mice are immunized using proteins expressed and purified in E. coli according to a conventional method. It can be obtained from hybridoma cells obtained by fusing spleen cells and myeloma cells. (Current protocols in Molecular Biology edit. Ausubel et al • (1987) Publish. John Wiley & Sons. Section 11.4-11 . 11).

 Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of these proteins, in addition to the purification of the protein of the present invention. Specifically, proteins are extracted from, for example, tissues, blood, or cells, and the presence or absence of abnormal expression or structure is examined through detection of the protein of the present invention by Western blotting, immunoprecipitation, or ELISA. Can be diagnosed.

Antibodies that bind to the protein of the present invention may be used for the purpose of treating diseases related to the protein of the present invention. When an antibody is used for the purpose of treating a patient, a human antibody or a humanized antibody is preferred because of its low immunogenicity. Human antibodies can be obtained by introducing a human antibody gene into the chromosome of a mouse (eg, “Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, Mendez, MJ et al. (1997) Nat. Genet. 15: 146- 156 ”). Humanized antibodies can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121). (1991)). The present invention also provides a method for screening for a compound that binds to the protein of the present invention. This screening method comprises: (a) a step of bringing a test sample into contact with the protein of the present invention or a partial peptide thereof; (b) a step of detecting a binding activity between the protein or a partial peptide thereof and the test sample; (C) selecting a compound having an activity of binding to the protein or a partial peptide thereof.

 Specific methods include, for example, a method of contacting and purifying a test sample with an affinity column for the protein of the present invention, a method using a two-hybrid system, a western blotting method, and a method using high-throughput screening. Many known methods can be used. Screening can also be performed by evaluating the binding between the protein of the present invention and a test compound using a measuring device such as BIACORE (Pharmacia). The test sample used for screening is not limited to these, but includes, for example, a cell extract, an expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, a natural compound, and the like. The compound isolated by this screening is a candidate for a compound that promotes or inhibits the activity of the protein of the present invention. In addition, in a living body, the protein of the present invention is a candidate for a compound that inhibits the interaction between the protein and a molecule that interacts with the protein.

When the gene of the present invention, its protein, a compound that regulates the expression of the gene, or a compound that regulates the activity of the protein is used as a drug, the drug itself can be used as a drug, It is also possible to formulate and use it by a pharmaceutical method. For example, it may be used in the form of a formulation by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent and the like. Administration to a patient can be performed by a method known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. When DNA is used as a therapeutic drug, the DM may be incorporated into a vector for gene therapy and administered to a patient. Dosage, injection The administration method varies depending on the patient's weight, age, symptoms, and the like, but those skilled in the art will be able to appropriately select the method. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the alignment of the amino acid sequences for the serine protease 'domain of C-PLACE1009992 with other serine proteases.

 FIG. 2 is a diagram showing the structure of pcDNA3.l (-) / C-PLACE1009992 / MycHis.

 FIG. 3 shows the structure of pENTRIIA / C-PLACE1009992 / MycHis.

 FIG. 4 shows the structure of PDEST8 / C-PLACE1009992 / MycHis.

 FIG. 5 is a view in which the amino acid sequences of human and mouse-derived C-PLACE1009992 are aligned.

 FIG. 6 is a continuation of FIG.

 FIG. 7 is a diagram in which human-derived C-PLACE1009992 and known mouse EST sequences are aligned. FIG. 8 is a continuation of FIG.

 FIG. 9 is a continuation of FIG.

 FIG. 10 is a continuation of FIG. 9. BEST MODE FOR CARRYING OUT THE INVENTION

 Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. Unless otherwise specified, it can be carried out according to a known method (Maniat is, T. at al. (1982): "Molecular Cloning-A Laboratory Manual" Cold Spring Harbor Laboratory, NY).

 [Example 1] Preparation of cDNA library from human placental tissue by oligocap method

Extract mRNA from human placental tissue by the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) did. Further, poly (A) + RNA was purified using an oligo (dT) cellulose column (Collaborative labs) according to the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Co Id Spring Harbor Laboratory Press (1989). . From the poly (A) + RNA, cDNA libraries were prepared by the oligocap method [M. Maruyama and S. Sugano, Gene, 138: 171-174 (1994)]. Using an oligocaplinker consisting of the sequence represented by SEQ ID NO: 7 (synthetic MA) and an oligo (dT) adapter consisting of the sequence represented by SEQ ID NO: 8, the literature [Suzuki Sugano, Protein Nucleic Acid Enzyme] BAP (Bacterial Alkaline Phosphatase) treatment, TAP (T obacco Acid Pyrophosphatase) as described in J. Suzuki et al., Gene, 200: 149-156 (1997)], 41: 197-201 (1996). ) Treatment, RNA ligation, synthesis of first strand cDNA and removal of RNA. Next, the 5 'terminal side represented by SEQ ID NO: 9 and SEQ ID NO:

Using the 3, terminal PCR primer represented by 10, the DNA was converted to double-stranded cDNA by PCR (polymerase chain react ion), and the obtained DNA fragment was digested with Sfil. Next, the direction of the cDNA was determined and cloned into the vector pME18SFL3 (GenBank AB009864) cut with Dralii to prepare a cDNA library. Since the cloning site of pME18SFL3 is an asymmetric Drall site, and a complementary Sfil site is added to the end of the cDNA fragment, the cloned cDNA fragment is It is inserted one way downstream of the evening.

 [Example 2] Analysis of cDNA clone derived from cDNA library prepared from human placental tissue

 (1) Isolation of cDNA clone

A part of the cDNA library prepared in Example 1 was introduced into E. coli DH10B by electroporation using Gene Pulser (manufactured by Biorad). Transformants were selected by culturing on LB agar medium containing 50 μg / ml of ampicillin. These transformants were cultured overnight in an LB medium containing 50 / g / ml of ampicillin, and plasmid was extracted using a plasmid automatic extractor PI100 (manufactured by Kurabo Industries, Ltd.). (2) Analysis of the nucleotide sequence of the isolated cDNA clone

 Plasmid DNA of clones obtained from these transformants was subjected to a sequencing reaction using a MA sequencing reagent (BigDye Terminator Cycle Sequencing FS Ready Reaction Kit, manufactured by PE Biosystems) according to the manual, followed by a DNA sequencer ( ABI PRISM 377, manufactured by PE Biosystems) was used to analyze the nucleotide sequence from the 5, 5 or 3 'end of each cDNA clone.

 For determination of the base sequence from the 5 'end, ME761FW represented by SEQ ID NO: 11 is used. For determination of the base sequence from the 3' end, ME1250RV represented by SEQ ID NO: 12 is used for sequencing. Used as primer.

 (3) Clustering of 5 'and 3' end sequences of cDNA clones

 The 5′-end sequence and the 3′-end sequence of the cDNA clone determined in (2) were separately classi? Ed. In other words, for single-pass sequence analysis from the 5 'end and 3' end determined for cDNA clones, BLAST analysis was performed between each sequence and the clones considered to be derived from the same gene. Was done. If the consensus sequence with a homology of 95% or more is 300 base pairs or more at the 5 'end sequence, and the consensus sequence with a homology of 90% or more is 200 base pairs or more at the 3' end sequence, the same group is considered. The 'terminal sequence group' was further grouped (clustered) so that the 5'-terminal sequence and the 3'-terminal sequence of the same clone belong to the same group (class 1).

 (4) Characterization of cDNA clone sequence

 5 ′ terminal sequence data of the clone sequence was characterized based on the following method.

 (1) By homology search using GenBank with BlastN, mRNA sequences of humans and other organisms (including proprietary sequences) 確認 Confirm that they are identical to human EST sequences.

(2) Confirm that the 5 'end of the human mRNA sequence is longer than the human EST sequence.

(3) ATGpr program for predicting full length [A. Salamov, T. Nishikawa, MB Swin dells. Assessing protein coding region integrity in cDNA sequencing proj ects. Bioinformatics 14: 384-390 (1998)], 5. Determine ATGprl and ATGpr2 values from all start codons in the terminal sequence.

 (4) Determine the number of identical human EST sequences by homology search using BlastN for GenBank.

 In addition, the characterization of the 3′-end sequence of the clone sequence was performed for (1) and (4) above.

 Based on the clone sequence data thus characterized, a new and likely full-length cDNA clone was selected.

 (5) Human mRNA sequence ゃ identity to human EST sequence Comparison of 5'-end length Identity of 5'-end and 3'-end sequences of clone sequences to mRNA sequences of human and other organisms Was considered identical when the length of the comparison sequence portion with each sequence was 200 bases or more and 94 or more matches. The identity to the human EST sequence was considered to be identical if the length of the comparison sequence portion with the 5 'terminal sequence was 200 bases or more and 90% or more matches.

 When comparing the length of the 5 'end with the human mRNA sequence as a comparison sequence, when the length of the 5' end sequence is longer than the human mRNA sequence, or when the 5 'end sequence contains a translation initiation codon, did. When the comparison target sequence is EST, it is convenient if the 5 'end is longer than the human EST sequence in the database, or if the difference between the two is within 50 bases even for a clone with a shorter 5' end. The total length was taken as the total length, and the shorter length was taken as the non-full length.

(6) Prediction of full length by ATGpr

The results of ATGpr [A. Salamov, T. Nishikawa, Μ. Β. Swindells. Asse ssing protein coding region integrity in cDNA sequencing projects. Bioin formatics 14: 384-390 (1998)] were used to predict the full length. . The ATGprl value is a value that predicts the possibility of the full length from the calculated value. The higher the ATGprl value, the higher the possibility of the full length. The maximum ATGprl value and maximum ATGpr2 value indicate the maximum ATGprl value and ATGpr2 value predicted from all start codons contained in the 5 'end sequence of the clone sequence, and these values were used for characterization. . (7) Prediction of novelty from the number of identical EST sequences by homology search

 5′-end sequence The 3′-end sequence was determined by homology search using GenBank. For the human EST sequence, the sequence was determined to be identical when the length of the comparison sequence portion with the 5 'terminal sequence was 90% or more over 200 bases or more. The number of EST sequences was directly used for characterization and used as an index of novelty. Clones having 5′-terminal sequences and 3′-terminal sequences that are not identical to not only mRNA sequences but also EST sequences are genes encoding novel sequences. Similarly, clones with a small number of 5'-terminal sequences or 3'-terminal sequences with a small number of identical EST sequences were also determined to be cDNA clones encoding the novel sequences.

(8) Characterization of class Yuichi

 5'-end sequence The class consisting of 3'-end sequences was characterized based on the following viewpoints.

 (1) By homology search using BlastN for GenBank, humans ゃ mRNA sequences of other organisms (including licensed sequences) か Are they identical to human EST sequences?

 All 5 'end sequences included in class 1 If at least one of the 3' end sequences is identical to the mRNA sequence, that class 1 is identical to the class 1 that is identical to the mRNA sequence. did.

 (2) Human ubiquitous sequence 5 Is the terminal longer than the human EST sequence?

 If all of the 5 'terminal sequences included in class 1 are non-full length with respect to the mRNA sequence or human EST sequence, the class 1 is non-full length with respect to the mRNA sequence / human EST sequence. And

 (3) ATGprl value and ATGpr2 value derived from all start codons in the 5 'terminal sequence by the ATGpr program for predicting full length.

ATGpr program [A. Salamov, T. Nishikaa, MB Swindells. Assessing protein coding region integrity in cDNA sequencing proects. Bioinformatics 14: 384-390 (1998)] All start Regarding the ATGprl value derived from the codon, the maximum value of the ATGprl value for all the 5 'terminal sequences included in the class was defined as the ATGprl value in the class. ATGpr2 values were similar

(4) The number of human EST sequences determined to be identical by homology search using BlastN in GenBank.

 The maximum value of the number of EST sequences is calculated for each 5'-terminal sequence and 3'-terminal sequence included in the cluster, and the number of identical EST sequences of the 5'-terminal sequence and the number of identical EST sequences of the 3'-terminal sequence in the class did.

 (9) Cluster selection method from characterization

 From the data obtained by the characterization, first, a class identical to the mRNA sequence of humans and other organisms (including the licensed sequences) and non-full-length clusters were removed. From those classes, those that met any of the following conditions were selected.

 (a) Classes in which the number of identical EST sequences in the 5'-end sequence in class is less than 20, and ATGprl value in class is more than 0.3.

 (b) Even if the ATGprl value of cluster 1 is less than 0.3, the number of identical EST sequences in the 5 'end sequence in class 1 is 5 or less, and the 3' terminal sequence in class 1 A cluster with less than 5 identical EST sequences and multiple clones within the class.

 (c) Even if the ATGprl value in class 1 is less than 0.3, the number of identical EST sequences in the 5 'end sequence in class 1 is 0, and the 3'-terminal sequence in class 1 Class class with one or more EST sequences.

 (d) Even in a cluster with an ATGprl value of 0.3 or less in cluster 1, the number of identical EST sequences in the 5'-terminal sequence in class 1 is 1 or more and 5 or less, and the same EST sequence in the 3'-terminal sequence in the cluster Cluster with zero count.

In the class selected in (a), at least one clone contains a clone with high novelty and full length. In the class selected in (b), (c) and (d), the total length is Are still full-length, but contain new clones.

 (10) How to select clones from class

 If the clone contained only one clone in the same class, the clone was selected. When a plurality of clones were included in the same cluster and there were a plurality of clones having an ATGprl value of more than 0.3, a clone having a larger ATGprl value was selected. When multiple clones are included in the same cluster and there are multiple clones with an ATGprl value of 0.3 or less, if the dog has an ATGpr2 value of more than 0.3, the clone with the larger ATGpr2 value was selected. . In addition, when multiple clones are included in the same cluster, even if the ATGprl value and ATGpr2 value are both 0.3 or less, if there is a clone in which the ATGprl value and ATGpr2 value both have the maximum values within the class, the clone is selected. Selected. If multiple clones are included in the same class and selection cannot be performed based on the ATGpr value as described above, assemble them using the 5 'end sequence and the 3' end sequence and the human EST sequence. A longer clone was selected on the terminal side. Sequencher (Gene Codes) was used for assembling, and if it could not be determined by assembling a part, all the target clones were judged to be full length.

 (11) Analysis of full-length sequence of cDNA clone

The nucleotide sequence of the full-length cDNA was determined for the cDNA clones selected from (1) to (10) and derived from human placental tissue, which were determined to be highly likely to be novel, as described in (1) to (10). The nucleotide sequence is mainly primer walking by dideoxy and mine using custom synthesized DNA primers (sequencing is performed according to the manual using a DNA sequencing reagent manufactured by PE Biosystems using custom synthesized DNA primers). After the reaction, the DNA sequence was analyzed using the company's sequencer). The full-length nucleotide sequence was finally determined by completely overlapping the partial nucleotide sequence determined by the above method. Next, a deduced amino acid sequence was determined from the determined nucleotide sequence of the full-length cDNA. The nucleotide sequence of cDNA clone C-PLACE1009992, one of which is shown in SEQ ID NO: 1. The cDNA clone C-PLACE1009992 estimated from the full-length nucleotide sequence The amino acid sequence of the gene product to be loaded is shown in SEQ ID NO: 2.

 (12) Evaluation of the full-length ratio of 5, -terminal of cDNA in ATGpr and ESTiMateFL

 ATGpr is a program developed by AA Salamov, T. Nishikawa, and MB Swindells of the Helix Research Institute to predict whether a translation initiation codon is based on the characteristics of the sequence around the ATG codon [AA Salamov , T. Nishikawa, MB Swindells, Bio informatics, 14: 384-390 (1998); http://ww.hri.co.jp/atgpr/]. The results were expressed as the expected value of the ATG being the true start codon (hereinafter sometimes referred to as ATGprl) (0.05-0.94). When this program was applied to the 5'-end sequence of a cDNA clone from a library prepared by the oligocap method with a total length of 65% and a clone was selected with an ATGpr 1 value of 0.6 or more, the full-length clone (0RF N- The sensitivity and specificity of the evaluation both increased to 82-83%. The maximum ATGprl value of C-PLACE1009992 was 0.89.

[Example 3] Analysis of UV damage-related genes

 Ultraviolet rays are known to have considerable effects on health. In recent years, there has been an increasing number of opportunities to be exposed to UV damage due to the depletion of the ozone layer, and it has been recognized as a risk factor for skin cancer (United States Environmental Protection Age ncy: Ozone Depletion Home Page, http: // www epa.gov/ozone/). Genes whose expression is altered by the action of ultraviolet light on skin epidermal cells are thought to be related to ultraviolet damage to the skin.

Cultures of primary cultured skin-derived fibroblasts irradiated with ultraviolet light were searched for genes whose expression was altered. Primary cultured skin fibroblasts (Cel l Applications Inc.) is cultured Konfuruento a culture dish, a 254 nm UV was irradiated 10,000 〃J / cm ^2.

Extraction of mRNA from cells was performed using FastTrack ™ 2.0 mRNA isolation kit (Invitrogen) on unirradiated cells and cells cultured for 4 or 24 hours after irradiation. The labeling of the probe for hybridization is performed using this mRNA 1 Using .5 g, the procedure was as described below. The data was obtained at n = 3 and the signal value of cells with UV stimulation and the signal value of cells without UV were compared. For comparison, statistical processing of a two-sample t-test was performed, and clones having a significant difference in the distribution of signal values were selected at P <0.05. This analysis can detect differences statistically even in clones with low signal values. Therefore, clones with a signal value of 40 or less were also evaluated.

 Expression of each cDNA in skin-derived fibroblasts not irradiated with ultraviolet light and in skin-derived fibroblasts irradiated with ultraviolet light was measured according to the following procedure.

The mean (M ₁₅ M ₂ ) and the sample variance (s! ² , s ₂ ² ) of the signal values for each gene in each cell were determined, and the composite sample variance s ² was determined from the sample variance of the two cells to be compared. t two _{(M t - M 2) /} s / (l / 3 + l / 3) was determined ^1/2. Compared to the t value of 0.05 and 0.01, which is the probability P of the significance level in the t-distribution table with 4 degrees of freedom, if the value is large, P = 0.05 or P = 0.01 It was determined that there was a difference in expression. The expression of C-PLACE1009992 decreased by ultraviolet irradiation after 4 hours or 24 hours.

 [Example 4] Preparation of DNA array and measurement of expression level

DNA for nylon membrane spots was prepared as follows. In other words, Escherichia coli retaining plasmid is cultured in each well of a .96-well plate (37 ° (:, 16 hours) in LB medium), and a part of the culture solution is dispensed in 10 / L portions of the 96-well plate. After suspending in sterilized water and treating for 10 minutes at 100 ° C, the sample was used as a PCR reaction sample.The PCR was performed using TaKaRa PCR Amplification Kit (Takara) according to the protocol. To amplify the plasmid insert cDNA, primers were used for sequencing primers ME761FW (5, tacggaagtgttacttctgc3, / sequence number: 11) and ME1250RV (5, tgtgggaggttttttctcta3, / sequence number: One pair of 12) or one pair of M13M4 (5, gttttcccagtcacgac3, / SEQ ID NO: 13) and M13RV (5'caggaaacagctatgac 3, / SEQ ID NO: 14) was used.The PCR reaction was GeneAmp System9600. (PE Biosystems) at 95 ° C After 5 minutes treatment, 95 cycles at 10 seconds at 68 ° C for 10 minutes The cycle was further performed at 98 ° C for 20 seconds, 60 ° C for 3 minutes, and 20 cycles at 72 ° C for 10 minutes. After the PCR reaction, 2 L of the reaction solution was subjected to 1% agarose gel electrophoresis, DNA was stained with a bromide reagent, and the amplified cDNA was confirmed. For those that could not be amplified, the plasmid containing the cDNA insert was extracted by alkali extraction (J Sambrook, EF Fritsh, T Mania tis, Molecular Cloning, A laboratory manual / 2nd edition, Cold Spring Harbor Laboratory Press, 1989). Was prepared.

 Preparation of the DNA array was performed as follows. DNA was dispensed into each well of a 384-well plate. DNA spotting on a nylon membrane (manufactured by Behringer) was performed using a Biomek2000 Laboratory Automation System (manufactured by Beckman Coal, Yuichi) using 384-pin pellets. That is, a 384-well plate containing DNA was set. 384 independent pins of a pin tool were simultaneously immersed in the DNA solution, and the needles were covered with DNA. MA stuck to the needle was spotted on the nylon membrane by gently pressing the needle against the nylon membrane. Denaturation of the spotted DNA and fixation to the nylon membrane were performed according to a conventional method (J Sambrook, EF Fritsh, T Maniatis, Molecular Cloning, A laboratory manual / 2nd edition, Cold Spring Harbor Laboratory Press, 1989).

As a probe for hybridization, 1st strand cDNA labeled with a radioisotope was used. The synthesis of 1st strand cDNA was performed using Thermoscript ™ RT-PCR System (GIBC0). That is, a 1.5 〃G of each tissue derived mRNA in human (Clontech Co.), 50 ζΜ Ol igo (dT ) using 20, 50 〃Ci Non ³³ P] 1st strand according to the protocol that came with the addition of dATP cDNA was synthesized. The probe was purified using a ProbeQuant (™) G-50 micro column (manufactured by Amersham Pharmacia Biotech) according to the attached protocol. Next, add 2 units of E. coli RNase H and incubate at room temperature for 10 minutes.Add 100 nl of human COT-1 DNA (GIBC0) and incubate at 97 ° C for 10 minutes. Thereafter, it was left still on ice to obtain a probe for hybridization. Hybridization of the radioisotope-labeled probe to the DNA array was performed according to a standard method (J Sambrook, EF Fritsh, T Maniatis, Molecular Cloning, Laboratory manual / 2nd edition, Cold Spring Harbor Laboratory Press, 1989). Was. Washing is performed by washing the nylon membrane three times in a washing solution 1 (2X SSC, 1% SDS) at room temperature (about 26 ° C) for 20 minutes, and then washing solution 2 (0.1X SSC, 1% SDS) In the chamber, washing was performed three times at 65 ° C for 20 minutes. The autoradiogram was obtained using an image plate of BAS2000 (manufactured by Fuji Photo Film Co., Ltd.). That is, the hybridized nylon film was wrapped in Saran wrap, brought into close contact with the photosensitive surface of the image plate, placed in a cassette for radioisotope exposure, and allowed to stand in a dark place for 4 hours. The radioisotope activity recorded on the image plate was analyzed using BAS2000, and converted and recorded as an autoradiogram image file. The signal intensity of each DNA spot was analyzed using Visage High Density Grid Analysis Systems (manufactured by Genomic Solutions), and the signal intensity was numerically reduced. The data were obtained with Duplicate, and the reproducibility was determined by hybridizing two DNA filters with one probe and comparing the signal intensity of the corresponding spots with both filters. 95% of all spots have signal values within 2 times that of the corresponding spot, and the correlation coefficient is r = 0.97. The reproducibility of the data is sufficient.

 [Example 5]

When the nucleotide sequence of C-PALCE1009992 was compared with a previously reported sequence using an analysis program (BLAST2.0), the sequences shown in Table 1 showed significant homology. Hit Expect homology (%)

S77064 6e- 63 34

 P28175 le-62 34

 P48740 le-22 29 When the amino acid sequence of C-PLACE1009992 was compared with the previously reported sequence using the above analysis program (BLAST2.0), the EST sequence shown in Table 1 showed significant homology. Indicated.

 FIG. 1 shows the results of aligning C-PLACE1009992 and a serine protease containing the clone identified by the above analysis program with respect to the protease domain. C-PLACE1009992 has the @ -marked H (His) at position 521 and the D (Asp) at position 576 as well as other serine proteases, and has a serine protease-like domain. I understand. Since the amino acid at position 682 is D (Asp), it is considered to be a protein which recognizes a peptide having K (Lys) or R (A rg).

 [Example 6]

 Using the amino acid sequence of C-PLACE1009992 as a query, search tool included in Pfam ver3

Search was performed using Pfam HMM Search (HMMPFAM). As a result, it was found to have a CUB domain, a CCP domain, an EGF domain, and a serine protease domain (Table 2). Table 2

Hit Score Expect Q from Q to Method pfam | hmm | trypsin 132.0 4.4e-41 479 732 HMMPFAM (Proteazedomein)

 pfam | hmm | CUB 105.0 1.4e-27 128 233 HMMPFAM

 pfajn | hmm | sushi 45.0 1.6e-09 297 359 HMMPFAM (CCP domain) pfajn | hmm | EGF 35.3 1.4e-06 239 271 HMMPFAM

 pfam | hmm | sushi 21.8 0.016 407 459 HMMPFAM (CCP domain)

In Table 2, “Hit” indicates the name of the putative domain that was exported as a result of the search. The higher the value of “Score”, the higher the reliability. "The closer the value of Expxextj is to 0, the higher the reliability." Q fromj indicates the starting position of the estimated domain. "Q toj indicates the ending position of the estimated domain. "Method" means that the search was performed using the HMMPFAM method. References (Sonnhammer EL, Eddy SR, Birney E, Bateman A, Durbin Pfam: multiple sequence alignments and marauding-profiles of protein domains.Nucleic Acids Res 1 998 Jan 1; 26 (1): 320-322) and the Internet See the address (http: //pfam.wust 1. edu / hmmsearch. Shtml).

 [Example 7]

 When the mouse EST sequence was searched using the sequence of C-PLACE1009992 as type III, a nearly full-length sequence of the mouse county part part gene was obtained as shown in FIGS.

 [Example 8]

Domain analysis of C-PLACE1009992 protein was performed. That is, estimated from cDNA Z6

Homology search using blast2.0 (Nucleic Acids Res., 25 (17) ₃ p3389-3402, 1997) for the amino acid sequence (SEQ ID NO: 2) obtained, and a known amino acid registered in GenBank. Comparison with sequence or domain was performed. Table 3 shows the results

Table 3

[Example 9]

 For the purpose of estimating the function of a novel human placenta-derived cDNA clone C-PLACE1009992 having a protease domain, the expression tissue of the gene corresponding to this clone was analyzed.

Primer TP-S05 (TGTCTGAGGACTGGGAAGTG / SEQ ID NO: 15) and antisense primer TP-A06 (TGCCATGGTCCTCATGCTGC / SEQ ID NO: 16) were used as polymerase chain reaction (PCH) primers for C-PLACE1009992 expression analysis. used. Human RAP ID-SCAN ™ GENE EXPRESSION PANEL used OriGene Technologies, Inc. as cDNA prepared from mA of 24 human organs at 4 different concentrations in a 96-well plate. . The DNA polymerase used was KOD Dash (Toyobo). Amplification of the gene by PCR was performed as follows. First, the RAPID-SCAN plate was moved from 4 ° C to room temperature and allowed to stand. A reaction solution having the following composition (Table 4) was prepared and kept in ice. Table 4

 *: The reagent attached to the enzyme was used.

 **: equimolar mixture of dATP, dGTP, dCTP, dTTP: attached to enzyme

The reaction solution prepared above was dispensed in an amount of 25 L per 1 L of the RAPID-SCAN plate, and an appropriate amount (about 25 L) of mineral oil was overlaid. The plate was covered with a plastic bar sheet and allowed to stand for 15 minutes. The plate was set on a PCR thermal cycler (PCR Thermal Cycler MP: Takara Shuzo) and reacted according to the following operating program (98 ° C for 3 minutes, 1 cycle at 98 ° C for 20 seconds, 60 ° C for 20 seconds, 75 ° C for 3 seconds). 35 cycles per minute → 1 cycle at 75 ° C for 10 minutes).

Subsequently, agarose gel electrophoresis was performed. 1% agarose gel (SeaKem GTG agarose: FMC BioProducts: 1 zg) mixed with 10 L of the PCR product and 1 L of 10X buffer for electrophoresis (attached to Takara Shuzo restriction enzyme) and set in electrophoresis apparatus Mupid (Cosmo Bio) / mL of ethidium mouth mold). Electrophoresis was performed at a constant voltage of 100 V for about 45 minutes. The buffer for electrophoresis was Tris-borate buffer (Takara Shuzo). Electrophoresis images were observed under UV irradiation. As a result, from the electrophoresis image of the PCR product, the PCR product was obtained as a DNA of about 630 bp, which was the size expected from the primer design. It was confirmed that the expression of the gene corresponding to C-PLACE1009992 clone was higher in the order of “brain, testis, prostate”> “heart, placenta, Teng”> skeletal muscle (Table 5). The PCR product was observed only in the concentration series containing 2.5 ng of cDNA in the RAP ID-SCAN plate, and could not be obtained with a concentration series lower than that. Table 5

 Strong band observed

 ++ Moderate band observed

 + Weak band observed

 No bands observed

[Example 10]

The expression level of this clone in each tissue of the human brain was examined. An experiment was performed according to the method of Example 9 using a library of cDNAs derived from mRNAs of the whole brain, amygdala, caudate nucleus, cerebellum, corpus callosum, hippocampus, substantia nigra, and thalamus-(Takara Shuzo). Was. Table 6 shows the results. Table 6

 ++: strong band observed

 +: A weak band was observed

[Example 11]

 For the purpose of estimating the involvement of this clone in the disease state, the expression level in human Alzheimer's diseased brain tissue was compared with that in normal tissue.

 As the primers for polymerase chain reaction (PCR), the ones designed in Example 9 were used for both the C-PLACE1009992 expression analysis primer and the antisense primer.

 This primer set gives a PCR product of 0.63 kb. A commercially available primer set (CL0NTECH) was used for expression analysis of the control gene, human glyceraldehyde 3-phosphate dehydrogenase (G3PDH). This primer set gives a PCR product of 0.45 kb. As brain tissue cDNA, cDNA derived from the frontal lobe and hippocampus of Alzheimer patients and normal adults was purchased from the BioChain Institute and used. TaKaRa Ex Taq ™ (Takara Shuzo) was used as a DNA polymerase.

First, a PCR reaction was performed by the following procedure. After diluting the cDNA stock solution 1 / 2-fold with a TE solution (10 mmol / LTris-HCU 1 mmol / L EDTA, pH 8.0), repeat this procedure four times in succession to obtain a concentration of 1x, 1x. A dilution series of / 2 times, (1/2) ² times, (1/2) ³ times, (1/2) ⁴ times, and (1/2) ⁵ times was prepared. A reaction solution having the following composition (Table 7) was prepared in a 15-mL centrifuge tube, and kept cool on ice. Table 7

 *: The reagent attached to the enzyme was used.

**: ^^ ⁾ , (167 ?, (1 (^, {^? Equimolar mixture: attached to enzyme)

 ***: 25 reactions

1 L of the diluted cDNA solution and 99 / L of the above-prepared solution were added to a 200-µL PCR tube.

 The tubes were set in a thermal cycler (PCR Thermal Cycler MP: Takara Shuzo) and reacted according to the following operating program (1 cycle of 94 ° C for 2 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes). 30 cycles 1 cycle at 72 ° C for 5 minutes).

 Then, agarose gel electrophoresis was performed as follows. After stopping the reaction in 30 cycles, 10 µL of the PCR product obtained was mixed with 2 µL of 5x electrophoresis buffer, and a 1.5% agarose gel (SeaKem) was set on a swimming device Mupid (ADVANCE). GTG agarose: applied to FMC BioProducts). Electrophoresis was performed at a constant voltage of 50 V for about 60 minutes. In addition, a tris-borate buffer was used as the electrophoresis buffer. The gel was immersed in ethidium mouth solution (500 ng / mL) for about 1 hour to stain, and the electrophoresis image was observed under ultraviolet irradiation.

Table 8 shows the results for the frontal lobe and Table 9 shows the results for the hippocampus. Table 8

 +: Band observed

 +/-: Some bands were observed

 1: No band was observed The expression level of G3PDH gene in the frontal lobe was similar in normal adults and Alzheimer's disease patients, but the expression level of C-PLACE1009992 gene was about twice as high in the patient's brain. Table 9

 ++: strong band observed

 +: Band observed

 +/-: Some bands were observed

1: The expression level of the G3PDH gene in the hippocampus where no band was observed was similar between normal adults and Alzheimer's patients, but the expression level of the C-PLACE1009992 gene was about 8 times higher in normal brain. It was suggested that the expression of the C-PLACE1009992 gene was reduced in the hippocampus region of the brain of a patient with Alzheimer's disease. In other words, it was suggested that this gene may be associated with memory and learning ability in normal brain. Further, the gene of the present invention can be expected to be applied to gene therapy for Alzheimer's disease, application to diagnosis, and the like.

 [Example 1 2] Preparation of expression vector for C-PLACE1009992

 The following procedure was performed to prepare an expression vector for mammalian cells in which the MycHis tag sequence was fused and expressed at the C-terminus of hC-PLACE1009992.

 1) A pair of primers for full-length amplification of hC-PLACE1009992 was prepared.

 PCR primer pair ATGGAGCTGGGTTGCTGGACZ SEQ ID NO: 17

 TTTCATATTTCTTTCMTCC / SEQ ID NO: 18

In addition, the N-terminal primer has a 4-base spacer and a recognition sequence for the restriction enzyme Nhel, and the C-terminal primer has a 4-base spacer and a recognition sequence for the restriction enzyme Hindi 11 added to the PCR. Primer pair

 NHTP-S01: GAMGCTAGCATGGAGCTGGGTTGCTGGAC / SEQ ID NO: 19

 HDTP-A01: GAAAAAGCTTTTTCATATTTCTTTCMTCC / SEQ ID NO: 20

Designed and synthesized.

 2) hC-PLACE1009992 full-length cDNA was amplified using a pair of full-length cDNA amplification primers. PCR was performed using NHTP-S01 and HDTP-A01 as primers and a plasmid containing hC-PLACE1009992 as type III, using the heat-resistant DNA polymerase KOD DNA Polymerase (Toyobo) to obtain the desired human full-length cDNA. A possible 2.2 Kbp MA was obtained as the main amplification product. The composition of the PCR reaction solution was in accordance with the protocol attached to KOD DNA Polymerase, and the temperature conditions were mild only for annealing because the Tm of HDTP-A01 was low.

(45 ° C).

3) The full-length cDNA was subcloned into the MycHis tag fusion expression vector. Vector for mammalian cell expression pcDNA3.1 / Myc-His (-) A (Invitrogen) and amplified human full-length cDNA were digested with restriction enzymes Nhel (Takara Shuzo) and Hindlll (Takara Shuzo), and then agarose gel electrophoresed. After electrophoresis, the target DNA fragment was recovered from the gel using the QIA Quick Gel Extraction Kit (QIAGEN) according to the attached protocol. The full-length human cDNA and pcMA3.1 / Myc-His (-) A fragment recovered from the gel were ligated using Ready-To-Go T4 DNA ligase (Amersham 'Pharmacia' Biotech) according to the attached protocol. The ligated plasmid (complex of human full-length cDNA and pcDNA3.1 / Myc-His (-) A) was introduced into DH5-competent cells (Toyobo) to obtain an E. coli transformant. The resulting E. coli transformant was cultured in a Terrific Broth medium containing ampicillin (Nacalai Tesque, Inc.) at a final concentration of 50 μg / mL, and the plasmid containing the DNA was purified using the QIAprep® Spin Miniprep Kit (QIAGEN). Extracted.

 4) Determination of the entire nucleotide sequence

The extracted plasmid sequencing sample was designed from the plasmid vector pcDNA3.1 / Myc-His (-) A.

 pcDNA-Sl: CACTGCTTACTGGCTTATCG / SEQ ID NO: 21

And hC-PLACE1009992 base sequence designed / synthesized primer

 TP-S04: CTTATCMCGGACGCCATGC / SEQ ID NO: 22

 TP-S06: CCTGMGCTCCTAGACAAGG / SEQ ID NO: 23

 TP-A02: GCACTCTGCACAGTAGMCC SEQ ID NO: 24

 TP-A03: AAGGTCCAGCCTTGTCMGG / SEQ ID NO: 25

 TP-A04: CCTTGACTGMCCTGCATCG / SEQ ID NO: 26

 TP-A05: CCAGGTCAGTMCACAGTGG / SEQ ID NO: 27

 TP-A06: TGCCATGGTCCTCATGCTGC / SEQ ID NO: 28

Was prepared using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (PE Biosystems) according to the attached protocol. The prepared sample is a capillary electrophoresis base sequence analyzer AB I PRISM® 310 Genetic Analyzer W

34

 (PE Biosystems), and the base sequence of the prepared plasmid was determined. Of the two clones whose nucleotide sequences were determined, one clone was a plasmid having the amino acid coding region in which the target hC-PLACE1009992 and the MycHis tag were fused.

 A vector that fuses and expresses the MycHis tag sequence at the C-terminus of the prepared hC-PLACE1009992 was named pcDNA3.1 (-) / C-PLACE1009992 / MycHis. The nucleotide sequence of the amino acid code region is shown in SEQ ID NO: 5, and the deduced amino acid sequence expressed therefrom is shown in SEQ ID NO: 6.

[Example 13] Expression of C-PLACE1009992 gene in insect cells

 1) Preparation of recombinant baculovirus vector

C-PLACE1009992 gene animal cell expression vector, cut pcDNA3.1 (-) / C-PLACE1009992 / MycHis with restriction enzymes Nhe I (Takara Shuzo, Code No. 1162Α) and Afl II (Takara Shuzo, Code NO.1003A) Thereafter, the cut ends were blunt-ended with a “DNA Blunting Kit” (Takara Shuzo Code No. 6025). After completion of the reaction, the DNA sample was subjected to agarose gel electrophoresis, and a DNA fragment of about 2.2 Kb consisting of 0 to 1009992 (; 3 fragments) was separated and purified from the agarose gel. Next, a commercially available cloning vector was used. there pENT ^TM lA (GIBCO BRL Co., Code No. 11813-011) restriction enzyme Dra I (Takara Shuzo, Code Νο. 1037Α) and Eco RV (Takara Shuzo, Code NO. 1042A) was cleaved with, Serufuraige one Chillon To prevent the reaction, alkaline phosphatase (Takara Shuzo, Code NO.2120A) was added and reacted at 60 ° C for 30 minutes to dephosphorylate the ends.After completion of the reaction, agarose gel electrophoresis was performed. The DNA fragment of about 2 Kb, which is a vector fragment, was separated and purified from the gel, and both DNA fragments were mixed at a molar ratio of about 1: 3 (= vector: insert), and the DNA Ligation Kit was used. (Takara Shuzo, Code No.6021) Enterobacter constitutive cell DH5 (Takara Shuzo, Code No. 9057) was transformed, and the transformed E. coli was spread on an LB plate containing kanamycin (final concentration 50 mg / L) and cultured at 37 ° C overnight. After arbitrarily selecting several colonies that appeared, inoculating about 2 mL of LB liquid medium (containing 50 mg / L kanamycin), and culturing overnight at 37 ° C, commercially available “Q Each plasmid DNA was extracted and purified using IAprep Spin Miniprep Kitj (QIAGEN, Code No. 27106). Each plasmid DNA was digested with the restriction enzymes Psh BI or Nhe I and Hind III, and the digestion pattern was analyzed by agarose gel electrophoresis to select the desired plasmid, and the resulting plasmid was used as a PENTR1A / C-PLACE1009992 / MycHis (Figure 3).

Next, an in vitro recombination reaction was performed using rsaculovirus Expression Systemj (GIBCO BRL, Code No. 11827-Oil), and the C-PLACE1009992 / MycHis gene subcloned into pENTRlA / C-PLACE1009992 / MycHis was baculo-modified. It was cloned into pDEST ^™ 8, a transfer plasmid for viruses. In accordance with the attached protocol "Baculovir us Expression Systemj, after in vitro recombination reactions pDEST ^TM 8 and pENTRlA / C-PLAC E1009992 / MycHis , E. combi competent cells DH 5 (Takara Shuzo, Code No.9057) transformed The transformed E. coli was spread on an LB plate containing ampicillin (final concentration 50 mg / L) and cultured overnight at 37 ° C. Were suspended in LB liquid medium (containing 50 mg / L ampicillin), and 1 µL of the suspension was used for colony PCR and the primers were used for the two primers that amplify the C-PLACE1009992 gene. (TP-S05, TP-A06; sequence is as described above). One cycle of 98 ° C for 3 minutes at 98 ° C for 20 seconds, 60 ° C for 20 seconds, 75 ° C with commercial amplification enzyme K0D Dash (Toyobo, Code No.) Perform a PCR reaction under the conditions of 1 cycle at 2.5 ° C for 35 minutes>> 75 ° C for 10 minutes. A clone in which a 630 bp DNA amplification band was detected by agarose gel electrophoresis was selected. After inoculating the selected clones into about 2 mL of LB liquid medium (containing 50 mg / L ampicillin) and culturing overnight at 37 ° C, commercially available “QIAprep Spin Miniprep Kitj (QIAGEN, Code No. 27106), each plasmid DNA was extracted and purified, and the nucleotide sequence of C-PLACE1009992 gene was confirmed using the above-mentioned sequence primers. The obtained target transfer plasmid was identified as pDEST8 / C-PLACE1009992. / MycHis (Figure 4). Commercially available Escherichia coli competent cells incorporating Bacmid to produce the C-PLACE1009992 / MycHis gene-expressing virus by recombination of the transfer plasmid PDEST8 / C-PLACE1009992 / MycHis prepared in the previous section and baculovirus DNA. "MAX EFF ICIENCY DH10BAC Competent Cel lsj (GIBCO BRL, Code No. 10361-012) was transformed with pDEST8 / C-PLACE1009992 / MycHis. E. coli after transformation was transformed into LB medium (50 mg / L kanamycin). , 10 mg / L tetracycline, 7 mg / L genyumycin, 100 mg / L X-gal and 40 mg / L IPTG) and cultured overnight at 37 ° C. From a white clone and cultured overnight at 37 ° C in 50 mL of LB liquid medium (containing 50 mg / L kanamycin, 10 mg / L tetracycline, and 7 mg / L genyumycin) `` C0NCERT High Purity Maxiprep Systemj (GIBCO BRL, Code No. 11452-018) From the obtained recombinant Bacmid DNA, two clones were selected as Bacmid / C-PLACE1009992 / MycHis (Clone No.8 and Clone No.10) and used for insect cell transfection. DNA.

2) Expression of C-PLACE1009992 / MycHis protein by recombinant baculovirus BEC Yuichi Bacmid / C-PLACE1009992 / MycHis (Clone No. 8 and Clone No. 10) DNA obtained in the previous section was commercially available using rcELLFECTIN Reagent j (GIBCO Insect cells were transfected into Sf9 cells using Code No. 10362-01 0) manufactured by BRL. The transfusion method was performed according to the standard protocol attached to "CELLFECTIN Reagent;", and the culture supernatant was collected as a recombinant virus solution (about 2 mL) three days after the transfusion. In order to further amplify the virus solution, 3.0 x 10 ⁶ Sf9 cells were attached to a 50 mL culture flask (FLASK50, manufactured by Sumitomo Berkit, Code No. MS-20050), and the culture medium was removed by suction. Thereafter, 0.5 mL of the above-mentioned virus solution was added. After standing at room temperature for 1 hour, 5 mL of medium (Sf-900 II Serum-Free Medium, manufactured by GIBCO BRL, Code No. 10902-096) was added, and the cells were cultured at 28 ° C for 4 days. After completion of the culture, a supernatant (about 5 mL) was collected, and the liquid from which cells had been removed by centrifugation (3000 rpm, 5 minutes, room temperature, KUB0TA5200) was stored at 4 ° C as a “recombinant virus liquid”. The following experiment was performed to examine the expression of C-PLACE1009992 / MycHis protein by the prepared recombinant baculovirus. First, 1.0 x 10 ⁶ Sf9 cells per well are attached to ⁶ Well Tissue Culture Plate (35 mm) (Multiplate 6F, manufactured by Sumitomo Beichik Co., Ltd., Code No.MS-80060). After suction was removed, 0.3 mL of the above-mentioned “recombinant virus solution” was infected. After standing at room temperature for 1 hour, 2 mL of medium (Sf-900 II Serum-Free Medium, manufactured by GIBCO BRL, Code No. 10902-096) was added, and the cells were cultured at 28 ° C. On the 2nd and 3rd days after the start of the culture, remove the plate and use a CELL LIFTER (Costar, Code. No. 3008) to scrape all attached cells and collect them in a centrifuge tube ( The cells and the culture supernatant (about 2 mL) were separated by centrifugation (3000 rpm, 5 minutes, room temperature, KUB0TA5200). The precipitated cells were resuspended in 0.2 mL of TE Buffer (10 mM Tris-HCl, pH = 8.0, 1 mM EDTA).

Next, in order to detect C-PLACE1009992 / MycHis protein in the culture supernatant and the cell suspension, Western blotting analysis using a His-Tag antibody was performed. For 0.1 mL each of the culture supernatant (Extracelular) and cell suspension (Intracellular) prepared in the previous section, use an equal volume (0.1 mL) of SDS Sample Buffer (Tris-SDS-BME Sample Loading). Buffer, Daiichi Kagaku, Code. No. ER33) was added and heated at 100 ° C for 5 minutes. 10 mL of 0.2 mL of each sample was subjected to SDS-polyacrylamide electrophoresis. For SDS-polyacrylamide electrophoresis, commercially available “Multi Gel 4/20” (Daiichi Pure Chemicals, Code. No. 211010) was converted to “force set electrophoresis tank“ Daiichi ”DPE-120, Code No. 130472 ", and the electrophoresis was performed according to the attached standard protocol. The electrophoresis buffer used was Takara Shuzo's `` Tris-Glycine-SDS Powder ヽ Code.No.T901j, and the molecular weight marker was BIO-RAD rprestained SDS -PAGE Standards, Low Range (Co de.No.161-0305, Control; 86580) and High Range (Code. No. 161-1309, Contro 1; 86878) ". After completion of the electrophoresis, the membrane was blotted on a PVDF membrane (TEFC0, Code. No. 03-056) by Electro Transfer using a "Plotting System" (TAITEC, Code. No. TM-6). Electro Transfer is manufactured by Takara Shuzo “Tr This was performed for 1.5 hours at a constant current of 15 OmA in a solution obtained by adding 20% methanol to is-Glycine Powder, Code. No. T902j. The plotted PVDF membrane was subjected to an antibody reaction using “Anti-His (C-term) Antibody, Code No. R930-25j” manufactured by Invitrogen according to the attached standard protocol. The labeled peroxidase was detected using Amershajn LIFE SCIENCE's factory ECL + Plus + Code. No. RPN213j according to the attached standard protocol.

 Analysis of C-PLACE1009992 / MycHis protein by i-His-Tag antibody revealed that both No. 8 and No. 10 clones had a molecular weight of approximately 82 kDa in the cell fraction on day 3 after infection with the recombinant virus. A clear band was detected at the position. This molecular weight was consistent with that expected for the C-PLACE1009992 / MycHis protein. From these results, it was confirmed that C-PLACE1009992 / MycHis protein was expressed in the insect cell expression system.

 [Example 14] Acquisition of cDNA for mouse counterpart of C-PLACE1009992

 The following procedure was performed with the aim of obtaining a mouse counterpart of C-PLACE1009992, a novel human cDNA.

 1) The EST sequence of the mouse counterpart was searched using hC-PLACEl 009992, and a primer pair for amplifying full length cDNA of mC-PLACE1009992 was prepared.

hC-PLACE1009992 Amino acid sequence (SEQ ID NO: 2) was used as a query to search public DBs. Among mouse ESTs, AU 067539 was hit near the 5 'end of the amino acid coding region and hit near the 3' end. PCR primer pairs were used to amplify the cDNA encoding the entire amino acid sequence of the mouse from the nucleotide sequence of AA199196

 mTP-SOl: TCACTCGGTACCGACACAGC / SEQ ID NO: 29

 mTP-AOl: ATGTACAGACGGATGCTAGG / SEQ ID NO: 30

Designed and synthesized.

 2) mC-PLACE1009992 full-length cDNA was amplified by PCR using a pair of full-length cDNA amplification primers and a mouse-derived cDNA source.

Using mTP-S01 and mTP-A01, mouse testis-derived Quick Clone cDNA (Clontech ) As type III, PCR was performed using the thermostable DNA polymerase KOD-PLUS (Toyobo) in accordance with the protocol attached to KOD-PLUS. DNA of approximately 2.3 Kb P, which is considered to be the target mouse cDNA, was mainly used. Obtained as a high amplification product.

 3) Cloning of full-length cDNA

 The amplified DNA solution was electrophoresed on an agarose gel, and a DNA of about 2.3 Kbp was recovered using QIA Quick Gel Extraction Kit (QIAGEN) according to the attached protocol. Using the Zero Blunt ™ TOPO ™ PCR Cloning Kit (Invitrogen), ligate the recovered DNA to the plasmid vector pCR®-Bluntn-T0P0 attached to the kit according to the attached protocol, and then introduce it into the TOP10 competent cells attached to the kit. Then, an E. coli transformant was obtained. The resulting E. coli transformant is cultured in a Terrific Broth medium containing kanamycin sulfate (Nacalai Tesque, Inc.) at a final concentration of 25 µg / mL, and the plasmid containing the DNA is purified using QIAprep® Spin Miniprep Kit (QIAGEN). Extracted.

 4) Determination of the entire nucleotide sequence

 The DNA sequence determination sample contained in the extracted plasmid is a primer that anneals to the plasmid vector pCR®-Blunt 11-TOPO.

 SP6Primer: CTATTTAGGTGACACTATAG / SEQ ID NO: 3 1

 T7Primer: GTAATACGACTCACTATAGGGC / SEQ ID NO: 32

Designed from mouse EST (AU067539, AW323842, AA444868, AI536361, AA833210, AA199196) on public DB hit with hC-PLACE1009992

 mTP-SOl and mTP-A01 are the same as above,

 mTP- -S03: GATTCTACTGCGCAGAGTGC / SEQ ID NO: 33

 mTP- -S04: GTCTTTGAGGAGATCACAGC / SEQ ID NO: 34

 mTP- -S05: CAGAATGGAGAGTGGTCAGG / SEQ ID NO: 35

 mTP- -S06: TCTCCAAAGACCCMGGCAC / SEQ ID NO: 36

 mTP- -S07: GGTTTCTGCTATCATTCTGC / SEQ ID NO: 37

mTP- -S08: ATCTGCACTGCAGAGACAGG / SEQ ID NO: 38 mTP-A03: CATGCAGTCTCCTCCGTACC / SEQ ID NO: 39

 mTP-A04: GAAACAAGGGGATGAGGAGC / SEQ ID NO: 40

 mTP-A05: GGACTCTCCTTCTCACCAGG / SEQ ID NO: 41

 mTP-A06: CATCGTGTACACCACTGGTC / SEQ ID NO: 42

 mTP-A07: TCTGGATGCTCTTCTCATCC SEQ ID NO: 43

 mTP-A08: CATGGTCTTCATGCTGTTCC / SEQ ID NO: 44

Was prepared using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (PE Biosystems) according to the attached protocol. The prepared sample was analyzed with a capillary electrophoresis-based nucleotide sequence analyzer ABI PRI SM®310 Genetic Analyzer (PE Biosystems) to determine the nucleotide sequence of the DNA. The obtained cDNA base sequence of mC-PLA CE1009992 is shown in SEQ ID NO: 3, and the deduced amino acid sequence is shown in SEQ ID NO: 4.

 Comparing those derived from humans with those derived from mice, the homology was about 90% at the amino acid sequence level (Figures 5 and 6). Industrial applicability

 Since the cDNA of the present invention is full-length, it contains a translation initiation point and provides useful information in protein function analysis. The present invention provides a novel serine protease-like protein (C-PLACE1009992), a gene encoding the protein, a vector including the gene, a transformant including the vector, and a method for producing the protein. Was It is suggested that the cDNA of the present invention is related to Alzheimer's. Therefore, the gene or protein of the present invention is particularly effective for the development of a diagnostic marker, a drug that regulates expression or activity, or the development of a drug such as a target for gene therapy, in connection with Alzheimer's disease. is there.

Claims

The scope of the claims 1. The DNA according to any one of (a) to (d) below.  (a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4.  (b) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 1 or 3.  (c) one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or 4 having substitution, deletion, insertion, and / or addition of an amino acid sequence; DNA that encodes a protein that is functionally equivalent to a protein consisting of an amino acid sequence.  (d) It hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 under stringent conditions, and is functionally equivalent to a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4. DNA that codes for a protein.  2. DNA encoding a partial peptide of a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4.  3. A protein or peptide encoded by the DNA according to claim 1 or 2. 4. A vector into which the DNA according to claim 1 or 2 has been inserted.  5. A transformant carrying the DNA according to claim 1 or 2 or the vector according to claim 4.  6. The method for producing a protein or peptide according to claim 3, comprising a step of expressing a protein or peptide using the transformant according to claim 5, and a step of recovering the expressed protein or peptide.  7. An antibody that binds to the protein or peptide according to claim 3.  8. A polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary strand thereof. 9. A method for screening a compound that binds to the protein according to claim 3, (a) contacting a test sample with the protein or a partial peptide thereof,  (b) a step of detecting a binding activity between the protein or a partial peptide thereof and a test sample (c) selecting a compound having an activity of binding to the protein or a partial peptide thereof.  10. A pharmaceutical composition comprising the DNA according to claim 1 or 2, the protein or peptide according to claim 3, or the vector according to claim 4.