JPH1132768A - New acidocyte serine protease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme useful for screening a serine protease inhibitor, diagnosing allergic diseases, infectious diseases, neoplastic diseases, collagen diseases, etc., comprising an acidocyte serine protease shown by a specific amino acid sequence. SOLUTION: This new polypeptide is an acidocyte serine protease composed of an amino acid sequence shown by the formula in a substantially pure form, its homolog, its fragment or the homolog of the fragment and is useful for a method for screening a serine protease inhibitor and for diagnosing diseases such as allergic diseases, infectious diseases, neoplastic diseases, granulomatous diseases, collagen diseases, angiitis, etc. This serine protease is obtained by extracting the whole RNA from the blood of a patient of eosinophilic leucocytosis, cloning the RNA by RT-PCR to give a cDNA encoding a serine protease, linking the serine protease to a vector, inserting the vector into Escherichia coli and culturing the prepared transformant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel serine protease. More specifically, a novel serine protease produced by human eosinophils, a method for producing the same, a cDNA encoding the serine protease, a cDN
A, a host cell transformed with the vector, an antibody to the serine protease, and a pharmaceutical composition containing the serine protease or antibody.
The present invention relates to a method for screening a serine protease inhibitor, a method for diagnosing an allergic disease characterized by measuring the enzyme activity of a serine protease, and a method for diagnosing an allergic disease using an antibody against the serine protease.

[0002]

BACKGROUND OF THE INVENTION Eosinophils are a type of leukocyte and are named because they are stained with eosin. Eosinophils are similar to neutrophils in their morphology, source, and phagocytosis, but differ in their staining, reactivity to pathogenic microorganisms, chemicals, hormones, and the like. Eosinophils make up about 1-5% of peripheral blood leukocytes of healthy individuals. Known diseases associated with eosinophilia include allergic diseases, infectious diseases, neoplastic diseases, granulomatous diseases, collagen diseases, vasculitis and the like. Furthermore, eosinophils infiltrate not only peripheral blood but also allergic granulomatous vasculitis and eosinophilic pneumonia tissues associated with asthma in the airways of patients with asthma. In the case of pulmonary infiltration and pleural effusion, activated low-density eosinophils are frequently found in those areas, and eosinophils in peripheral blood of patients with allergic rhinitis and dermatitis are activated It has been reported that the disease is in a state of inflammation, and it is considered that the onset of the disease state and the activation of eosinophils or infiltration into tissues are closely related (Therapeutics, Vol. 25, No. 4, p.
p.384-387, 1991).

[0003] Eosinophils play a role in attacking parasites in parasitic infections. However, in allergic reactions, it is thought that they damage self tissues and promote allergic inflammation. Mediators of eosinophils include granule proteins (eosinophil cationic proteins (eosinophil
ic cationic protein), major basic protein (major basic protein)
protein), cell membrane phospholipid metabolites (prostaglandin, leukotriene, thromboxane, platelet activating factor, etc.), active oxygen, cytokines, etc., and eosinophils have been reported. It is considered that the function is exerted through the mediator. However, the mediators identified so far cannot completely explain the role of eosinophils, suggesting that some mediators have not yet been isolated.

[0004] In the allergic reaction, mast cells are considered to be involved in the early stage, but the involvement of eosinophils in the chronic stage is strongly suggested. Eosinophils
Degranulation is thought to attack self-tissues, and it has been shown that proteases, particularly serine proteases, are involved in this degranulation (Archives
of Biochemistry and Biophysics Vol. 312, No. 1, Jul
y, pp.67-74, 1994)

[0005]

DISCLOSURE OF THE INVENTION The present inventors have paid close attention to this point, and have conducted intensive studies to find a novel factor (polypeptide) produced by human eosinophils, particularly serine protease. Was.

Conventionally, when obtaining a specific polypeptide or cDNA encoding the same, the desired biological activity is confirmed in a tissue or cell culture, and then the polypeptide is isolated and purified to obtain the gene. Or a method of expressing and cloning a gene using its biological activity as an index has been generally used. However, bioactive polypeptides in vivo often have a variety of biological activities, and as a result of cloning a gene using a certain activity as an index, it may be identical to a known polypeptide. The number of cases that will be found later is increasing. In addition, most of the factors produced by specific cells produce only trace amounts, which makes isolation, purification and confirmation of biological activity difficult.

[0007] In recent years, techniques for preparing and sequencing cDNA have rapidly developed, and it has become possible to rapidly sequence a large amount of cDNA. Therefore, using these techniques, a cDNA library is prepared from various cells and tissues, the cDNA is randomly cloned, the nucleotide sequence is determined, the corresponding polypeptide is expressed, and its physiological functions are analyzed. The method of doing it is developing. As part of such a technique, PCR using sense and antisense primers for RNA having a characteristic partial sequence (Polymerase chain reactant)
ion) has made it possible to find out unknown polypeptides with characteristic partial sequences. By using this method, the present inventors have developed a novel factor (polypeptide) produced by eosinophils and a cDNA encoding the same.
And completed the present invention.

The novelness of the sequence of the present invention was confirmed by homology search. Among several clones obtained by the method for producing the sequence of the present invention described in detail below, only the sequence of the present invention is novel. The deduced amino acid sequence of the present invention had a serine protease profile, and had a hydrophobic amino acid sequence at the C-terminus. Also, GPI
(Glycosylphosphatidylinositol) It has ω, ω1, and ω2 sites for uncurd membrane protein, and has both characteristics as a membrane protein and characteristics as a secretory protein. By comparison with the known sequence, the present serine protease is considered to be produced in the form of a proform, and it is considered that the upstream portion is cut off to become a mature protein.

[0009]

The present invention relates to (1) a serine protease having the amino acid sequence represented by SEQ ID NO: 1, and (2) a cD encoding the serine protease described in (1) above.
NA, (3) c having a base sequence represented by SEQ ID NO: 2
DNA, (4) a cDNA having the nucleotide sequence of SEQ ID NO: 3.

More specifically, the present invention relates to a polypeptide which is a serine protease consisting of the amino acid sequence shown in SEQ ID NO: 1 in a substantially pure form, a homolog thereof, a fragment of the sequence and a homolog thereof. The invention further relates to cDNAs encoding these serine proteases. More specifically, a cDN having the nucleotide sequence represented by SEQ ID NO: 2 or 3
A and a cDN having a fragment that selectively hybridizes to the nucleotide sequence of SEQ ID NO: 2 or 3.
About A.

The serine protease of the present invention having the amino acid sequence shown in SEQ ID NO: 1 in a substantially pure form generally means that at least 90%, for example, 95, 98 or 99% of the polypeptide at the time of production is a sequence. It means a serine protease having the amino acid sequence represented by No. 1.

The homologue of the serine protease of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 1 is generally at least 20, preferably at least 30, for example, 40, 60 or 100 contiguous amino acid regions and at least 70 %, Preferably at least 80 or 90%, more preferably 95% or more homologous, and such homologs are hereinafter referred to as serine proteases of the present invention.

Further, the fragment of the serine protease of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 1 or a homologous fragment thereof is at least 10 amino acids, preferably at least 15 amino acids, for example, 20, 25, 30, 40. , 50 or 60
Means the amino acid portion.

A cDNA which selectively hybridizes to a cDNA having the nucleotide sequence of SEQ ID NO: 2 or 3
Generally means at least 70%, preferably at least 80 or 90%, more preferably at least 95% homology in at least 20, preferably at least 30, for example 40, 60 or 100 contiguous base sequence regions. Such a cDNA is hereinafter referred to as cDNA of the present invention.

A cDNA fragment having the nucleotide sequence of SEQ ID NO: 2 or 3 is at least 10 bases, preferably at least 15 bases,
It means a 5, 30 or 40 base portion, and such a fragment is also included in the cDNA of the present invention.

Further, the present invention includes a replication or expression vector into which the cDNA of the present invention has been incorporated. Examples of the vector include a plasmid, virus or phage vector comprising an ori region and, if necessary, a promoter for expression of the above-mentioned cDNA, a regulator of the promoter, and the like. The vector may contain one or more selectable marker genes, for example, an ampicillin resistance gene. The vector is used in vitro, for example, for the R corresponding to cDNA.
It can be used for production of NA and transformation of host cells.

Further, the present invention relates to SEQ ID NO: 2 or 3
Or a cDNA of the present invention comprising a cDNA having an open reading frame thereof,
A host cell transformed with a vector for replicating or expressing E. coli is also included. Cells include, for example, bacteria, yeast, insect cells or mammalian cells.

Furthermore, the present invention includes a method for producing the serine protease of the present invention, which comprises culturing the host cell of the present invention under conditions for expressing the serine protease of the present invention. The cultivation is preferably performed under conditions in which the serine protease of the present invention is expressed and produced from host cells.

The antisense can also be produced by inserting the cDNA of the present invention into the antisense region of a vector as described above. Such antisense can also be used to control the level of a polypeptide of the present invention in cells.

The present invention includes monoclonal or polyclonal antibodies against the serine protease of the present invention. Furthermore, the present invention also includes a method for producing a monoclonal or polyclonal antibody against serine protease according to the present invention. A monoclonal antibody can be produced by a usual hybridoma technique using the serine protease of the present invention or a fragment thereof as an antigen. The polyclonal antibody can be produced by a usual method in which a host animal (for example, rat or rabbit) is inoculated with the serine protease of the present invention and immune serum is collected.

The present invention also includes a pharmaceutical composition comprising the serine protease of the present invention, its antibody, and a pharmaceutically acceptable excipient and / or carrier. The present invention further includes a method for screening for a serine protease inhibitor using the serine protease of the present invention.
The screening is carried out, for example, by coexisting the serine protease of the present invention with the compound to be screened in a buffer, and measuring the enzyme activity from the absorbance or the like using, for example, a synthetic substrate with a coloring agent. Further, by applying this method, it can be used for diagnosis of allergic diseases and the like. Furthermore, the present invention includes a method for diagnosing an allergic disease or the like using an antibody against the serine protease of the present application. Diagnosis using antibodies is known in the art, and is performed, for example, by measuring the amount of an enzyme using an antigen-antibody reaction by an enzyme immunoassay (EIA) method or the like.

As the serine protease of the present invention (1), in addition to the one having the amino acid sequence represented by SEQ ID NO: 1, a part thereof is deleted (for example, SEQ ID NO: 1).
Medium, a polypeptide consisting only of a part essential for the expression of biological activity, etc.), a part of which is substituted with another amino acid (for example, one substituted with an amino acid having similar physical properties), and a part of which is substituted with another amino acid. Also included are those in which amino acids are added or inserted. The codon for one amino acid is 1
-6 types (for example, Met is 1 type, Leu is 6 types)
Yes, without changing the amino acid sequence of the polypeptide
The base sequence of DNA can be changed.

The cDNA of the present invention specified in (2) includes all the base sequence groups encoding the serine protease represented by SEQ ID NO: 1 in (1). By changing the nucleotide sequence, the productivity of serine protease may be improved. The cDNA specified in (3) is
One embodiment of the cDNA shown in (2), which represents a natural sequence. The cDNA shown in (4) shows a sequence obtained by adding a natural untranslated portion to the cDNA specified in (3).

C having the base sequence of SEQ ID NO: 3
DNA is prepared according to the following method. 1) Extraction of Total RNA (Total RNA) The blood of a patient with eosinophilia is centrifuged to separate the eosinophil fraction. It is preferable to confirm the purity of eosinophils by a method such as FACS analysis. From the obtained fraction, a known method, guanidine-phenol method (Cu
rrent Protocol in Molecular Biology, WILEY inter sc
ience) to extract total RNA. 2) Synthesis of cDNA cDNA is synthesized using the obtained total RNA as a template and a primer and a reverse transcriptase. Examples of the primer to be used include oligo (Oligo) NotI (dT18) V and oligo (dT18). As a reverse transcriptase,
Super Script II, MMLV
Reverse transcriptase (MMLV reverse transcriptase), AMV reverse transcriptase (AMV reverse transcriptase) and the like can be used.

3) Synthesis of Primers for RT-PCR Using the active site of serine protease as a motif, sense primers and antisense primers are chemically synthesized. These are combined to prepare a primer mix. The sense primer and the antisense primer may be any as long as they encode the target amino acid, but need to be prepared and combined in consideration of the ease of hybridization. 4) Selective PCR PCR is performed using the cDNA obtained in 2) as a template and the primer mix obtained in 3). By PCR, a partial sequence of a serine protease having an active site is amplified. The obtained product is subjected to electrophoresis, a band is cut out in consideration of the length of the serine protease known so far, and a cDNA fragment is recovered. This time, 4
Those having a size of 00 to 600 bp were selected.

5) Transformation into Escherichia coli The cDNA fragment obtained in 4) is ligated to a T vector, and the resulting plasmid is transformed into cells. Preferred cells are XL-2Blue, DH5α, HB10
1, HB109, XL-1 and the like. The cells are cultured and the plasmid is extracted. Plasmid is replaced with a restriction enzyme (E
coRI, etc.), and electrophoreses for 400 to 60
A 0 bp band is cut out, collected, and the insert is confirmed. 6) Sequencing The clone obtained in 5) was obtained by a known method.
Perform sequencing and determine the sequence of each clone. A homology search is performed to search for a novel partial sequence that has the sequence of the active site of serine protease and that is novel.

7) Identification of 5 'end In order to obtain the full length of the sequence obtained in 6), 5' RACE
Identify by method. For example, Cap Finder (CA
Using the modified method (Clontec), 5'RACE system (Gibco BRL), etc., the 5 'end of the partial sequence obtained in 6) is identified. As a template for this, R
Create cDNA for ACE. Specifically, i) cDNA is prepared by using primers and reverse transcriptase using the total RNA obtained in 2). As the primer, a cap switch (CAP switch) primer, an oligo NotI (dT18) V primer, or the like is used. The same reverse transcriptase as described above can be used. ii) PCR is performed using the cDNA obtained in i) as a template and the primer and the partial sequence obtained in 6) as a primer. iii) To increase the certainty and amount of PCR product,
Nested PCR is performed using the same primer and another partial sequence of the partial sequence obtained in 6). iv) The PCR product thus obtained is subjected to electrophoresis, a band is cut out, and cDNA is recovered. v) The recovered cDNA is subcloned into a T vector, and the resulting plasmid is transformed into cells and cultured. The plasmid is recovered from the cells, cut with restriction enzymes, and the insert is confirmed by electrophoresis. vi) Sequencing of clones to identify the sequence at the 5 'end.

8) Identification of 3 'end Using the 3' RACE method, the sequence at the 3 'end of the sequence obtained in 6) is obtained. The method is the same as the 5′RACE method, but for the partial sequence in 6), a sequence on the downstream side is used. The template was the same cD as used in the 5'RACE method.
NA is used. 9) Confirmation of full length In order to confirm that the full length sequence obtained by the operations of 6), 7) and 8) really exists, partial sequences at the 3 'end and 5' end of the obtained sequence were Using PC
After performing R, the product is subjected to electrophoresis, a band is cut out, collected, ligated to a T vector to obtain a plasmid, the resulting plasmid is transformed into cells, and the cells are cultured.
The plasmid is recovered and subjected to sequencing. 10) Confirmation of this sequence in neutrophils The neutrophil fraction was subjected to PCR using partial sequences,
It was confirmed that the sequence of the present invention was not present.

It is necessary to confirm that the cDNA thus obtained is full length or almost full length. Comparison with the known signal sequence length and sequence confirmed that the polypeptide of the present invention was almost full-length. Once the nucleotide sequences shown in SEQ ID NOs: 2 and 3 have been determined, the nucleotide sequence of the present invention is subsequently determined by chemical synthesis or by chemically synthesizing a fragment of the nucleotide sequence of the present invention and hybridizing it with a probe. cDNA can be obtained. Furthermore, a required amount of the desired cDNA can be obtained by introducing a vector cDNA containing the present cDNA into an appropriate host and growing the vector.

The method of obtaining the polypeptide of the present invention includes: (1) a method of purifying and isolating from a living body or cultured cells, (2) a method of synthesizing a peptide, or (3) a method of producing using a gene recombination technique. And the like, and the method described in (3) is industrially preferable.

Examples of an expression system (host-vector system) for producing a polypeptide using a genetic recombination technique include bacterial, yeast, insect cell and mammalian cell expression systems. For example, when expressed in Escherichia coli, an initiation codon (ATG) is added to the 5 'end of the cDNA encoding the mature protein portion or the proform protein portion, and the resulting cDNA is converted to an appropriate promoter (for example, trp promoter). , Lac promoter, λP
A vector (eg, pBR32) that is connected downstream of the L promoter and T7 promoter and functions in E. coli.
2, pUC18, pUC19, etc.) to prepare an expression vector.

Next, E. coli transformed with this expression vector (for example, E. coli DH5α, E. coli JM10)
9, E. Coli HB101 strain, etc.) in an appropriate medium to obtain the desired polypeptide from the cells. In addition, if a bacterial signal peptide (eg, pelB signal peptide) is used, the target polypeptide can be secreted into the periplasm. In addition, fusion with other polypeptides
It can also produce protein (fusion protein).

For expression in mammalian cells, for example, a cDNA encoding the nucleotide sequence of SEQ ID NO: 3 may be replaced with an appropriate vector (for example, a retrovirus vector, a papilloma virus vector, a vaccinia virus vector, or an SV40 system). The vector is inserted downstream of an appropriate promoter (eg, SV40 promoter, SRα promoter, LTR promoter, metallothionein promoter, etc.) in the vector or the like to prepare an expression vector. Next, appropriate mammalian cells (for example, human undifferentiated eosinophil leukemia EOL cells, COS
-7 cells, CHO cells, mouse L cells, etc.) and culturing the transformant in an appropriate medium.
The desired polypeptide is secreted into the culture solution.
The polypeptide obtained as described above can be isolated and purified by a general biochemical method.

[0034]

The polypeptide of the present invention is a serine protease produced by eosinophils and is involved in degranulation. As described above, the increase in eosinophils is associated with allergic diseases, infectious diseases, It is thought to be involved in these diseases because it occurs with oncological diseases, granulomatous diseases, collagen diseases, vasculitis and the like. Therefore, inhibiting the serine protease of the present invention is considered to be useful for preventing and / or treating the above-mentioned diseases. Serine protease of the present invention, for screening to obtain its inhibitor,
Required. In addition, by applying this method, it can be used for diagnosis of allergic diseases.

The serine protease antibody of the present invention can be used for diagnosis of the above diseases. That is,
The serine protease of the present invention can be quantified in a living body using the polyclonal antibody or the monoclonal antibody of the serine protease of the present invention, whereby it can be used for studying the relationship between the serine protease of the present invention and a disease or diagnosing a disease. it can. Polyclonal and monoclonal antibodies can be prepared by a conventional method using the polypeptide of the present invention or a fragment thereof as an antigen. Further, by using the serine protease of the present invention, a protein (receptor) that binds to the polypeptide of the present invention can be purified or a gene can be cloned. It can also be used to search for agonists and antagonists of the serine protease of the present invention.

The cDNA of the present invention is not only an important and indispensable template for producing the serine protease of the present invention, which is expected to have tremendous utility, but also the diagnosis and treatment of genetic diseases (treatment of genetic deficiency or treatment of genetic deficiency). Antisense DNA (RN
A) can be used for treatment by stopping the expression of serine protease, etc.). In addition, the cD of the present invention
Genomic DNA using NA as a probe
Can be separated. Similarly, it is possible to isolate a gene of a human-related polypeptide highly homologous to the cDNA of the present invention and a gene of a polypeptide highly homologous to the serine protease of the present invention in a non-human organism.

[0037]

[Application to pharmaceuticals] The serine protease of the present invention
It is useful for preventing and / or treating allergic diseases, infectious diseases, neoplastic diseases, granulomatous diseases, collagen diseases, vasculitis and the like. It is also useful for diagnosis of the above diseases. The serine protease of the present invention, or an antibody against the serine protease of the present invention, is usually administered systemically or locally, generally orally or parenterally. Preferably, oral administration and intravenous administration.

When administering the compound, antibody or antisense of the present invention, solid compositions, liquid compositions and other compositions for oral administration, injections, external preparations and suppositories for parenteral administration Used as etc. Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. Capsules include soft capsules and hard capsules.

The dosage is determined by the age, body weight, symptoms, therapeutic effect,
Depending on the administration method, treatment time, etc., it is usually orally administered once to several times a day, per adult, in a range of 100 μg to 100 mg per time, or 10 μg per time per adult, per time Parenteral doses ranging from 1 to 100 mg once to several times a day. Of course, as described above, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or may be required beyond the range.

[0040]

The present invention will be described in more detail with reference to the following Examples and Reference Examples, which do not limit the scope of the present invention.

Example 1 Extraction of Total RNA The granulocyte fraction was separated from the blood of a patient with eosinophilia by Percoll (Sigma) density centrifugation, and negative selection was performed using an anti-CD16 antibody. Was. It was confirmed by FACS analysis (Becton Dickinson) that this fraction had an eosinophil purity of 98% or more. Guanidine-phenol method (Current Protocol in Mol)
ecular Biology, WILEY inter science)
Total RNA was extracted.

Example 2 Synthesis of Single-Stranded cDNA Oligo NotI (dT18) V Primer: 5'-AA
CTGGAAGAATTCGCGGGCCGCAGGAA
TTTTTTTTTTTTTTTTTTTTV-3 ′ (SEQ ID NO: 5) was synthesized. Total RNA (1 μl) obtained in Example 1
(1 μg / 1 μl)) and the bifunctional primer (1 μl, 100 μM) synthesized above, and mixed in a 0.5 ml RNAase free tube (RNAase free t
ube) for 2 minutes at 72 ° C. and 5 minutes on ice. RNAsin (1 μl), Super Script II (1 μl, Gibco BRL),
Dithiothreitol (DTT, 1 μl), 5 × Super Script II Buffer
(4 μl) and DEPE-treated water (11 μl)
The reaction was carried out at 2 ° C for 60 minutes and at 94 ° C for 5 minutes.

Example 3 Synthesis of Primers for RT-PCR The following four types of sense primers and antisense primers using the plasmin active site as a motif were chemically synthesized, and these were combined to prepare 16 types of primer mixes. Obtained.

[0044]

Embedded image

[0045]

Embedded image

[0046]

[Table 1]

Example 4: Selective PCR 10 × EX tack buffer (EX Taq Buffer, 40 μm)
l, Takara Shuzo, dNTP mixture (dNTP Mix, 32)
μl, Takara Shuzo), EX tack polymerase (EX Taq p
Olymase, 2 μl, Takara Shuzo), cDNA (10 μl) obtained in Example 2 and dH ₂ O (296 μl) were mixed to prepare a PCR mix. 1 obtained in Example 3
Six kinds of primer mix (1 μl) and PCR mix (19 μl each) were dispensed into a PCR tube, and the gene amplification PCR system 2400 (Gene Amp PCR system 24) was used.
(00, Perkin Elmer) for 35 cycles of PCR. PCR was carried out at 94 ° C for 1.5 minutes and then at 94 ° C.
After 35 cycles of 0.5 minutes, 1.0 minute at 66 ° C. and 2.0 minutes at 72 ° C., 7.0 minutes at 72 ° C. After the reaction, the product in each tube was subjected to electrophoresis using agarose gel (1 × TAE), and then ethidium bromide (Ethidium bromide) was used.
de) A solution treatment was performed, and a band of 400 to 600 bp was collected. The combination that resulted in a band of this length was
No. 3, 9, 11, and 13.

Example 5: Transformation into E. coli The cDNA (1 μl) recovered in Example 4 was
Ligase I (Ligas) to CR2.1 (1 μl, Invitrogen)
eI, 2 μl, Takara Shuzo). The resulting plasmid was transformed into XL-2Blue (Stratagen) to obtain a clone. Twenty-four clones were randomly selected and shaken in an LB culture solution containing ampicillin (Difco) at 37 ° C. overnight. Plasmids were extracted using a Wizard miniprep kit (Promega). The plasmid was cut with EcoRI restriction enzyme (New England Biolab), subjected to agarose electrophoresis, and treated with an ethidium bromide solution. As a result, bands of 400 to 600 bp were obtained in five clones.

Example 6: Sequencing Five clones obtained in Example 5 were subjected to a cycle sequence kit (Cycle Sequence kit, Applied Biosystems).
Was used to obtain each nucleic acid sequence. As a result of homology search (BLASTX, NCBI), three clones had a serine protease sequence. One of them had a novel sequence.

Example 7: Identification of 5 'end In order to obtain the full length of the clone obtained in Example 6, first, single-stranded cDNA was obtained by using a cap finder.
The modified 5′RACE method was performed using this as a template. That is, the total RNA obtained in Example 1 (1 μm
l) as a template, Cap Switch II
Oligonucleotide (1 μl, 5 μM): 5′-AAG
CAGTGGTATCAACGCAGAGTACGCG
GG-3 '(SEQ ID NO: 6) and the oligo NotI (dT18) V primer synthesized in Example 3 (1 μl, 10 μl)
μM) and dH ₂ O (2 μl) were placed in an RNAase-free tube, reacted at 72 ° C. for 2 minutes, left on ice for 5 minutes, and then placed in a 5 × Superscript II buffer (Super Scri
pt II Buffer) (2 μl), DTT (1 μl, 20 m
M), dNTPs (1 μl, 10 mM) and Super Script II (1 μl) were added, and the mixture was reacted at 42 ° C. for 1 hour and denatured at 95 ° C. for 2 minutes. The obtained single-stranded cDNA was used as a QIA quick PCR purification kit (QIA quick PCR Purification K).
It, using QIAGEN, was purified and 30 μl of Tris (Tr
is, 10 mM) and resuspended in EDTA (1 mM).
end)

Using this as a template, a cap (CAP)
PCR primer: 5'-AAGCAGTGGTATC
AACGCAGGT-3 '(SEQ ID NO: 7) and a partial sequence ESP-1 (68) of the sequence obtained in Example 6:
5'-CCAAACTGGACCATCCACC-3 '
PCR (First PCR) was performed for 35 cycles using (SEQ ID NO: 8), and after purification, the CAP PCR primer and the partial sequence ES of the sequence obtained in Example 6 were further added.
P-1 (49): 5'-CGGAGGGATCACTA
PCR using AGGTCAC-3 '(SEQ ID NO: 9)
(Second PCR) was performed for 20 cycles. The product was subjected to agarose electrophoresis and treated with an ethidium bromide solution to obtain a band of about 300 bp. The obtained band was cut out, and the recovered cDNA was subcloned into a T vector by the same operation as in Example 5 to obtain XL-2Bl.
ue. The plate was shaken in an LB culture solution containing ampicillin (Difco) at 37 ° C. overnight. Wizard miniprep kit, Pro
mega) was used to extract the plasmid. The plasmid was cut with EcoRI restriction enzyme (New England Biolab), subjected to agarose electrophoresis, treated with an ethidium bromide solution, and a 300 bp insert was confirmed. The obtained 5 clones were subjected to sequencing in the same manner as in Example 6. As a result, two clones were identical to a part of the sequence obtained in Example 6.

Example 8: Identification of 3 'end NotI (d) was obtained in the same manner as in Example 7 (modified 3' RACE) using the cDNA obtained in Example 7 as a template.
T) Primer: 5'-CTGGAAGAATTCGC
GGCCGCAGG-3 ′ (SEQ ID NO: 10) and a partial sequence ESP-1 (424) of the sequence obtained in Example 6:
5'-GGAGACATGGTTTTGTGCTGGC-
PCR (First PCR) was performed for 35 cycles using 3 '(SEQ ID NO: 11), and after purification, the above NotI
(DT) primer and partial sequence ESP-1 (456) of the sequence obtained in Example 6: 5'-CGGGAAGG
20 cycles of PCR (Second PCR) were performed using ATGCCTGCTTTCG-3 '(SEQ ID NO: 12). The product was subjected to agarose electrophoresis and treated with an ethidium bromide solution to obtain a band of about 400 bp. The obtained band was cut out, and the recovered cDNA was subcloned into a T vector by the same operation as in Example 5, and
L-2Blue was transformed. L containing ampicillin
The mixture was shaken in a B culture solution at 37 ° C. overnight. Wizard miniprepkit, Promega
Was used to extract the plasmid. Plasmid E
Cleavage with coRI restriction enzyme, agarose electrophoresis, treatment with ethidium bromide solution, 400b
The p insert was confirmed. Sequencing was performed on the two clones obtained in the same manner as in Example 6. As a result, two clones were identical to a part of the sequence obtained in Example 6.

Example 9: Confirmation of full length Partial sequence ES of the sequence obtained from Examples 6, 7 and 8
P-1 (-9): 5'-GAGGAGGCCATGGGG
CGCGC-3 '(SEQ ID NO: 13) and partial sequence ES
P-1 (1058): 5'-CCTGCAAGGCAT
The same operation as in Example 7 was performed using CAACTGGAATTGTG-3 '(SEQ ID NO: 14) as a primer and the cDNA obtained in Example 2 as a template, and out of 8 clones, a sequence having a total length of 1058 bp was obtained in 3 clones. Was. The sequence shown in SEQ ID NO: 3 was obtained by referring to the sequences of four clones including one clone having a deletion. The amino acid sequence deduced from this nucleic acid sequence is shown in SEQ ID NO: 1, the sequence of the translated part is shown in SEQ ID NO: 2, and the amino acid and nucleic acid together are shown in SEQ ID NO: 4.

The deduced amino acid sequence of the present invention consists of a total length of 315 amino acids including a signal peptide (Met ^{-26 to} Ala ^-1 ), and has a serine protease profile (active center, His ⁵⁶ , Asp ¹¹¹ , Ser ²¹³ ). And 18 hydrophobic regions (Trp ^{272 to} Va) at the C-terminus.
l ²⁸⁹ ). On the other hand, Ala ^{261 to} Ser ²⁶³ or Gly ^{264 to} Ser ²⁶⁶ have ω, ω1, and ω2 sites for GPI uncarded membrane protein, and have both characteristics as a membrane protein and characteristics as a secretory protein. . By comparison with the known sequence, the serine protease is considered to be produced in the form of a proform (Ala
^{1 ~Val 289),} as a mature protein, believed to be from Ile ^16.

Example 10: Confirmation that the sequence of the present invention is not expressed in neutrophils In the same manner as in Example 1, the neutrophil fraction obtained by performing a positive selection with an anti-CD16 antibody was used. Then, the same operation as in Example 2 was carried out to obtain cDNA, and the partial sequence ESP-1 (68) used in Example 7 and the partial sequence ESP-1 (−) used in Example 9 were used as PCR primers. Example 6 was performed by PCR using 9).
As a result, a band of about 300 bp was not obtained, and it was confirmed that neutrophils did not express RNA encoding the amino acid sequence of the present invention.

[0056]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 315 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu Ala Arg Ala -26 -25 -20 -15 Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala Pro Leu Ser Gly Pro -10 -5 1 5 Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly Glu Asp Ala 10 15 20 Glu Leu Gly Arg Trp Pro Trp Gln Gly Ser Leu Arg Leu Trp Asp Ser 25 30 35 His Val Cys Gly Val Ser Leu Leu Ser His Arg Trp Ala Leu Thr Ala 40 45 50 Ala His Cys Phe Glu Thr Tyr Ser Asp Leu Ser Asp Pro Ser Gly Trp 55 60 65 70 Met Val Gln Phe Gly Gln Leu Thr Ser Met Pro Ser Phe Trp Ser Leu 75 80 85 Gln Ala Tyr Tyr Thr Arg Tyr Phe Val Ser Asn Ile Tyr Leu Ser Pro 90 95 100 Arg Tyr Leu Gly Asn Ser Pro Tyr Asp Ile Ala Leu Val Lys Leu Ser 105 110 115 Ala Pro Val Thr Tyr Thr Thr Lys His Ile Gln Pro Ile Cys Leu Gln 120 125 130 Ala Ser Thr Phe Glu Phe Glu Asn Arg Thr Asp Cys Trp Val Thr Gly 135 140 145 150 Trp Gly Tyr Ile Lys Glu Asp Glu A la Leu Pro Ser Pro His Thr Leu 155 160 165 Gln Glu Val Gln Val Ala Ile Ile Asn Asn Ser Met Cys Asn His Leu 170 175 180 Phe Leu Lys Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp Met Val Cys 185 190 195 Ala Gly Asn Ala Gln Gly Gly Lys Asp Ala Cys Phe Gly Asp Ser Gly 200 205 210 Gly Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp Tyr Gln Ile Gly Val 215 220 225 230 Val Ser Trp Gly Val Gly Cys Gly Arg Pro Asn Arg Pro Gly Val Tyr 235 240 245 Thr Asn Ile Ser His His Phe Glu Trp Ile Gln Lys Leu Met Ala Gln 250 255 260 Ser Gly Met Ser Gln Pro Asp Pro Ser Trp Pro Leu Leu Phe Phe Pro 265 270 275 Leu Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 280 285

SEQ ID NO: 2 Sequence length: 945 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA sequence ATGGGCGCGC GCGGGGCGCT GCTGCTGGCG CTGCTGCTGG CTCGGGCTGG ACTCAGGAAG 60 CCGGAGTCGC AGGAGGCGGC GCCGTTATCA GGACCATCG GC CATCACGTCG 120 CGCATCGTGG GTGGAGAGGA CGCCGAACTC GGGCGTTGGC CGTGGCAGGG GAGCCTGCGC 180 CTGTGGGATT CCCACGTATG CGGAGTGAGC CTGCTCAGCC ACCGCTGGGC ACTCACGGCG 240 GCGCACTGCT TTGAAACCTA TAGTGACCTT AGTGATCCCT CCGGGTGGAT GGTCCAGTTT 300 GGCCAGCTGA CTTCCATGCC ATCCTTCTGG AGCCTGCAGG CCTACTACAC CCGTTACTTC 360 GTATCGAATA TCTATCTGAG CCCTCGCTAC CTGGGGAATT CACCCTATGA CATTGCCTTG 420 GTGAAGCTGT CTGCACCTGT CACCTACACT ACTAAACACA TCCAGCCCAT CTGTCTCCAG 480 GCCTCCACAT TTGAGTTTGA GAACCGGACA GACTGCTGGG TGACTGGCTG GGGGTACATC 540 AAAGAGGATG AGGCACTGCC ATCTCCCCAC ACCCTCCAGG AAGTTCAGGT CGCCATCATA 600 AACAACTCTA TGTGCAACCA CCTCTTCCTC AAGTACAGTT TCCGCAAGGA CATCTTTGGA 660 GACATGGTTT GTGCTGGCGCAA TGCCCAAGGC GGGAAGGATG CCTGCTTCGG T GACTCAGGT 720 GGACCCTTGG CCTGTAACAA GAATGGACTG TGGTATCAGA TTGGAGTCGT GAGCTGGGGA 780 GTGGGCTGTG GTCGGCCCAA TCGGCCCGGT GTCTACACCA ATATCAGCCA CCACTTTGAG 840 TGGATCCAAGAGCTGATGGC CCAGAGCTGCCTCCTCCCCCGACCATCCCCTCCCCCACT

SEQ ID NO: 3 Sequence length: 1085 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA to mRNA sequence GAGGAGGCCA TGGGCGCGCG CGGGGCGCTG CTGCTGGCGC TGCTGCTGGC TCGGGCTGGA60 CTCAGGAAGC CGGAGTGGCACGGAGGTCGGCGCT CCGACGGGTC 120 ATCACGTCGC GCATCGTGGG TGGAGAGGAC GCCGAACTCG GGCGTTGGCC GTGGCAGGGG 180 AGCCTGCGCC TGTGGGATTC CCACGTATGC GGAGTGAGCC TGCTCAGCCA CCGCTGGGCA 240 CTCACGGCGG CGCACTGCTT TGAAACCTAT AGTGACCTTA GTGATCCCTC CGGGTGGATG 300 GTCCAGTTTG GCCAGCTGAC TTCCATGCCA TCCTTCTGGA GCCTGCAGGC CTACTACACC 360 CGTTACTTCG TATCGAATAT CTATCTGAGC CCTCGCTACC TGGGGAATTC ACCCTATGAC 420 ATTGCCTTGG TGAAGCTGTC TGCACCTGTC ACCTACACTA CTAAACACAT CCAGCCCATC 480 TGTCTCCAGG CCTCCACATT TGAGTTTGAG AACCGGACAG ACTGCTGGGT GACTGGCTGG 540 GGGTACATCA AAGAGGATGA GGCACTGCCA TCTCCCCACA CCCTCCAGGA AGTTCAGGTC 600 GCCATCATAA ACAACTCTAT GTGCAACCAC CTCTTCCTCA AGTACAGTTT CCGCAAGGAC 660 ATCTTTGGAG ACATGGTTTG TGCTGGCAAT GCCCAAGGCG GGAAGGATG C CTGCTTCGGT 720 GACTCAGGTG GACCCTTGGC CTGTAACAAG AATGGACTGT GGTATCAGAT TGGAGTCGTG 780 AGCTGGGGAG TGGGCTGTGG TCGGCCCAAT CGGCCCGGTG TCTACACCAA TATCAGCCAC 840 CACTTTGAGT GGATCCAGAA GCTGATGGCC CAGAGTGGCA TGTCCCAGCC AGACCCCTCC 900 TGGCCACTAC TCTTTTTCCC TCTTCTCTGG GCTCTCCCAC TCCTGGGGCC GGTCTGAGCC 960 TACCTGAGCC CATGCAGCCT GGGGCCACTG CCAAGTCAGG CCCTGGTTCT CTTCTGTCTT 1020 GTTTGGTAAT AAACACATTC CAGTTGATGC CTTGCAGGGC ATTCTTCAAA AAAAAAAAAA 1080 AAAAA 1085

SEQ ID NO: 4 Sequence length: 1085 Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Homo Sapiens Cell: Eosinophil Sequence characteristics Characteristic symbol: CDS Location: 10. .954 Method for determining characteristics: S Symbol representing characteristics: sig peptide Location: 10..87 Method for determining characteristics: Symbol representing S characteristics: proform peptide Location: 88..954 Method for determining characteristics: S Symbol indicating feature: mat. Peptide Location: 133..954 Method for determining characteristics: S Symbol indicating feature: active center (His) Location: 253..255 Method for determining features: S Indicates feature Symbol: Active center (Asp) Location: 418..420 Method for determining features: S Symbol representing feature: Active center (Ser) Location: 724..726 Method for determining features: S Symbol representing feature: ω, ω1, ω2 sites Location: 868.876 Features Determined method: S Symbol indicating feature: ω, ω1, ω2 site Location: 877..885 Method determining feature: S Symbol indicating feature: hydrophilic region Location: 883..900 Method determining feature : S Symbol representing characteristics: Hydrophobic region Location: 901..954 Method for determining characteristics: S sequence GAGGAGGCC ATG GGC GCG CGC GGG GCG CTG CTG CTG GCG CTG CTG CTG 48 Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu -26 -25 -20 -15 -15 GCT CGG GCT GGA CTC AGG AAG CCG GAG TCG CAG GAG GCG GCG CCG TTA 96 Ala Arg Ala Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala Pro Leu -10 -5 1 TCA GGA CCA TGC GGC CGA CGG GTC ATC ACG TCG CGC ATC GTG GGT GGA 144 Ser Gly Pro Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly 5 10 15 GAG GAC GCC GAA CTC GGG CGT TGG CCG TGG CAG GGG AGC CTG CGC CTG 192 Glu Asp Ala Glu Leu Gly Arg Trp Pro Trp Gln Gly Ser Leu Arg Leu 20 25 30 35 TGG GAT TCC CAC GTA TGC GGA GTG AGC CTG CTC AGC CAC CGC TGG GCA 240 Trp Asp Ser His Val Cys Gl y Val Ser Leu Leu Ser His Arg Trp Ala 40 45 50 CTC ACG GCG GCG CAC TGC TTT GAA ACC TAT AGT GAC CTT AGT GAT CCC 288 Leu Thr Ala Ala His Cys Phe Glu Thr Tyr Ser Asp Leu Ser Asp Pro 55 60 65 TCC GGG TGG ATG GTC CAG TTT GGC CAG CTG ACT TCC ATG CCA TCC TTC 336 Ser Gly Trp Met Val Gln Phe Gly Gln Leu Thr Ser Met Pro Ser Phe 70 75 80 TGG AGC CTG CAG GCC TAC TAC TAC ACC CGT TAC TTC GTA TCG AAT ATC TAT 384 Trp Ser Leu Gln Ala Tyr Tyr Thr Arg Tyr Phe Val Ser Asn Ile Tyr 85 90 95 CTG AGC CCT CGC TAC CTG GGG AAT TCA CCC TAT GAC ATT GCC TTG GTG 432 Leu Ser Pro Arg Tyr Leu Gly Asn Ser Pro Tyr Asp Ile Ala Leu Val 100 105 110 115 AAG CTG TCT GCA CCT GTC ACC TAC ACT ACT AAA CAC ATC CAG CCC ATC 480 Lys Leu Ser Ala Pro Val Thr Tyr Thr Thr Lys His Ile Gln Pro Ile 120 125 130 TGT CTC CAG GCC TCC ACA TTT GAG TTT GAG AAC CGG ACA GAC TGC TGG 528 Cys Leu Gln Ala Ser Thr Phe Glu Phe Glu Asn Arg Thr Asp Cys Trp 135 140 145 GTG ACT GGC TGG GGG TAC ATC AAA GAG GAT GAG GCA CTG CCA TCT CCC 576 Val Thr Gly Trp Gly Tyr Il e Lys Glu Asp Glu Ala Leu Pro Ser Pro 150 155 160 CAC ACC CTC CAG GAA GTT CAG GTC GCC ATC ATA AAC AAC TCT ATG TGC 624 His Thr Leu Gln Glu Val Gln Val Ala Ile Ile Asn Asn Ser Met Cys 165 170 175 AAC CAC CTC TTC CTC AAG TAC AGT TTC CGC AAG GAC ATC TTT GGA GAC 672 Asn His Leu Phe Leu Lys Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp 180 185 190 195 ATG GTT TGT GCT GGC AAT GCC CAA GGC GGG AAG GAT GCC TGC TTC GGT 720 Met Val Cys Ala Gly Asn Ala Gln Gly Gly Lys Asp Ala Cys Phe Gly 200 205 210 GAC TCA GGT GGA CCC TTG GCC TGT AAC AAG AAT GGA CTG TGG TAT CAG 768 Asp Ser Gly Gly Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp Tyr Gln 215 220 225 ATT GGA GTC GTG AGC TGG GGA GTG GGC TGT GGT CGG CCC AAT CGG CCC 816 Ile Gly Val Val Ser Trp Gly Val Gly Cys Gly Arg Pro Asn Arg Pro 230 235 240 GGT GTC TAC ACC AAT ATC AGC CAC CAC TTT GAG TGG ATC CAG AAG CTG 864 Gly Val Tyr Thr Asn Ile Ser His His Phe Glu Trp Ile Gln Lys Leu 245 250 255 ATG GCC CAG AGT GGC ATG TCC CAG CCA GAC CCC TCC TGG CCA CTA CTC 912 Met Ala Gln Se r Gly Met Ser Gln Pro Asp Pro Ser Trp Pro Leu Leu 260 265 270 275 275 TTT TTC CCT CTT CTC TGG GCT CTC CCA CTC CTG GGG CCG GTC 954 Phe Phe Pro Leu Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 280 285 TGAGCCTACC TGAGCCCATG CAGCCTGGGG CCACTGCCAA GTCAGGCCCT GGTTCTCTTC 1014 TGTCTTGTTT GGTAATAAAC ACATTCCAGT TGATGCCTTG CAGGGCATTC TTCAAAAAAA 1074 AAAAAAAAAA A 1085

SEQ ID NO: 5 Sequence length: 46 Sequence type: Number of nucleic acid chains: 1 Topology: Linear sequence AACTGGAAGA ATTCGCGGCC GCAGGAATTT TTTTTTTTTT TTTTTV

SEQ ID NO: 6 Sequence length: 30 Sequence type: Number of nucleic acid chains: 1 Topology: Linear sequence AAGCAGTGGT ATCAACGCAG AGTACGCGGG

SEQ ID NO: 7 Sequence length: 23 Sequence type: Number of nucleic acid chains: 1 Topology: Linear sequence AAGCAGTGGT ATCAACGCAG AGT

SEQ ID NO: 8 Sequence length: 19 Sequence type: Number of nucleic acid strands: 1 Topology: Linear sequence CCAAACTGGA CCATCCACC

SEQ ID NO: 9 Sequence length: 21 Sequence type: Number of nucleic acid strands: 1 Topology: Linear sequence CGGAGGGATC ACTAAGGTCA C

SEQ ID NO: 10 Sequence length: 23 Sequence type: Number of nucleic acid strands: 1 Topology: Linear sequence CTGGAAGAAT TCGCGGCCGC AGG

SEQ ID NO: 11 Sequence length: 21 Sequence type: Number of nucleic acid strands: 1 Topology: Linear sequence GGAGACATGG TTTGTGCTGG C

SEQ ID NO: 12 Sequence length: 20 Sequence type: Number of nucleic acid strands: 1 Topology: Linear sequence CGGGAAGGAT GCCTGCTTCG

SEQ ID NO: 13 Sequence length: 19 Sequence type: Number of nucleic acid strands: 1 Topology: Linear sequence GAGGAGGCCA TGGGCGCGC

SEQ ID NO: 14 Sequence length: 25 Sequence type: Number of nucleic acid strands: 1 Topology: Linear sequence CCTGCAAGGC ATCAACTGGA ATGTG

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] August 7, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0037

[Correction method] Change

[Correction contents]

[0037]

[Application to pharmaceuticals] The serine protease of the present invention
It is useful for preventing and / or treating allergic diseases, infectious diseases, neoplastic diseases, granulomatous diseases, collagen diseases, vasculitis and the like. It is also useful for diagnosis of the above diseases.
The serine protease of the present invention, or an antibody against the serine protease of the present invention, is usually administered systemically or locally, generally orally or parenterally. Preferably, oral administration and intravenous administration.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0052

[Correction method] Change

[Correction contents]

Example 8: Identification of 3 'end NotI (d) was obtained in the same manner as in Example 7 (modified 3' RACE) using the cDNA obtained in Example 7 as a template.
T) Primer: 5'-CTGGAAGAATTCGC
GGCCGCAGG-3 ′ (SEQ ID NO: 10) and a partial sequence ESP-1 (424) of the sequence obtained in Example 6:
5'-GGAGACATGGTTTTGTGCTGGC-
PCR (First PCR) was performed for 35 cycles using 3 '(SEQ ID NO: 11), and after purification, the above NotI
(DT) primer and partial sequence ESP-1 (456) of the sequence obtained in Example 6: 5'-CGGGAAGG
20 cycles of PCR (Second PCR) were performed using ATGCCTGCTTTCG-3 '(SEQ ID NO: 12). The product was subjected to agarose electrophoresis and treated with an ethidium bromide solution to obtain a band of about 400 bp. The obtained band was cut out, and the recovered cDNA was subcloned into a T vector by the same operation as in Example 5, and
L-2Blue was transformed. L containing ampicillin
The mixture was shaken in a B culture solution at 37 ° C. overnight. Wizard miniprepkit, Promega
Was used to extract the plasmid. Plasmid E
Cleavage using the coRI restriction enzyme, agarose electrophoresis, treatment with ethidium bromide solution,
The bp insert was confirmed. Sequencing was performed on the two clones obtained in the same manner as in Example 6. As a result, two clones were identical to a part of the sequence obtained in Example 6.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0053

[Correction method] Change

[Correction contents]

Example 9: Confirmation of full length Partial sequence ES of the sequence obtained from Examples 6, 7 and 8
P-1 (-9): 5'-GAGGAGGCCATGGGG
CGCGC-3 '(SEQ ID NO: 13) and partial sequence ES
P-1 (1082): 5'-CCTGCAAGGCAT
The same operation as in Example 7 was carried out using CAACTGGAATTGTG-3 '(SEQ ID NO: 14) as a primer and the cDNA obtained in Example 2 as a template. A total length of 1082 bp was obtained in 3 of 8 clones. Was. The sequence shown in SEQ ID NO: 3 was obtained by referring to the sequences of four clones including one clone having a deletion. The amino acid sequence deduced from this nucleic acid sequence is shown in SEQ ID NO: 1, the sequence of the translated part is shown in SEQ ID NO: 2, and the amino acid and nucleic acid together are shown in SEQ ID NO: 4.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0054

[Correction method] Change

[Correction contents]

The deduced amino acid sequence of the present invention consists of a total of 314 amino acids including a signal peptide (Met ^{-26 to} Ala ^-1 ), and has a serine protease profile (active center, His ⁵⁶ , Asp ¹¹¹ , Ser ²¹² ). And 18 hydrophobic regions (Trp ^{271 to} Va) at the C-terminus.
^l288 ). On the other hand, Ala ^{260 to} Ser ²⁶² or Gly ^{263 to} Ser ²⁶⁵ have ω, ω1, and ω2 sites for GPI uncarded membrane protein, and have both characteristics as a membrane protein and characteristics as a secretory protein. . By comparison with the known sequence, the serine protease is considered to be produced in the form of a proform (Ala
^{1 ~Val 288),} as a mature protein, believed to be from Ile ^16.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0056

[Correction method] Change

[Correction contents]

[0056]

[Sequence list] SEQ ID NO: 1 Sequence length: 314 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu Ala Arg Ala -26 -25 -20 -15 Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala Pro Leu Ser Gly Pro -10 -5 1 5 Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly Glu Asp Ala 10 15 20 Glu Leu Gly Arg Trp Pro Trp Gln Gly Ser Leu Arg Leu Trp Asp Ser 25 30 35 His Val Cys Gly Val Ser Leu Leu Ser His Arg Trp Ala Leu Thr Ala 40 45 50 Ala His Cys Phe Glu Thr Tyr Ser Asp Leu Ser Asp Pro Ser Gly Trp 55 60 65 70 Met Val Gln Phe Gly Gln Leu Thr Ser Met Pro Ser Phe Trp Ser Leu 75 80 85 Gln Ala Tyr Tyr Thr Arg Tyr Phe Val Ser Asn Ile Tyr Leu Ser Pro 90 95 100 Arg Tyr Leu Gly Asn Ser Pro Tyr Asp Ile Ala Leu Val Lys Leu Ser 105 110 115 Ala Pro Val Thr Tyr Thr Lys His Ile Gln Pro Ile Cys Leu Gln Ala 120 125 130 Ser Thr Phe Glu Phe Glu Asn Arg Thr Asp Cys Trp Val Thr Gly Trp 135 140 145 150 Gly Tyr Ile Lys G lu Asp Glu Ala Leu Pro Ser Pro His Thr Leu Gln 155 160 165 Glu Val Gln Val Ala Ile Ile Asn Asn Ser Met Cys Asn His Leu Phe 170 175 180 Leu Lys Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp Met Val Cys Ala 185 190 195 Gly Asn Ala Gln Gly Gly Lys Asp Ala Cys Phe Gly Asp Ser Gly Gly 200 205 210 Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp Tyr Gln Ile Gly Val Val 215 220 225 230 Ser Trp Gly Val Gly Cys Gly Arg Pro Asn Arg Pro Gly Val Tyr Thr 235 240 245 Asn Ile Ser His His Phe Glu Trp Ile Gln Lys Leu Met Ala Gln Ser 250 255 260 Gly Met Ser Gln Pro Asp Pro Ser Trp Pro Leu Leu Phe Phe Pro Leu 265 270 275 Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 280 285

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0057

[Correction method] Change

[Correction contents]

SEQ ID NO: 2 Sequence length: 942 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: cDNA to mRNA sequence ATGGGCGCGC GCGGGGCGCT GCTGCTGGCG CTGCTGCTGG CTCGGGCTGG ACTCAGGAAG 60 CCGGAGTCGC AGGAGGCGGC GCCGTTATCA GGACCATCG GC CATCACGTCG 120 CGCATCGTGG GTGGAGAGGA CGCCGAACTC GGGCGTTGGC CGTGGCAGGG GAGCCTGCGC 180 CTGTGGGATT CCCACGTATG CGGAGTGAGC CTGCTCAGCC ACCGCTGGGC ACTCACGGCG 240 GCGCACTGCT TTGAAACCTA TAGTGACCTT AGTGATCCCT CCGGGTGGAT GGTCCAGTTT 300 GGCCAGCTGA CTTCCATGCC ATCCTTCTGG AGCCTGCAGG CCTACTACAC CCGTTACTTC 360 GTATCGAATA TCTATCTGAG CCCTCGCTAC CTGGGGAATT CACCCTATGA CATTGCCTTG 420 GTGAAGCTGT CTGCACCTGT CACCTACACT AAACACATCC AGCCCATCTG CCTCCAGGCC 480 TCCACATTTG AGTTTGAGAA CCGGACAGAC TGCTGGGTGA CTGGCTGGGG GTACATCAAA 540 GAGGATGAGG CACTGCCATC TCCCCACACC CTCCAGGAAG TTCAGGTCGC CATCATAAAC 600 AACTCTATGT GCAACCACCT CTTCCTCAAG TACAGTTTCC GCAAGGACAT CTTTGGAGAC 660 ATGGTTTGTG CTGGCAATGC CCAAGGCGGG AAGGATGCCT GCTTCGGTGA C TCAGGTGGA 720 CCCTTGGCCT GTAACAAGAA TGGACTGTGG TATCAGATTG GAGTCGTGAG CTGGGGAGTG 780 GGCTGTGGTC GGCCCAATCG GCCCGGTGTC TACACCAATA TCAGCCACCA CTTTGAGTGG 840 ATCCAGAAGC TGATGGCCCA GAGTGGCATG TCCCAGCCAG ACCCCTCCTG GCCACTACTC 900 TTTTTCCCTC TTCTCTGGGC TCTCCCACTC CTGGGGCCGG TC 942

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0058

[Correction method] Change

[Correction contents]

SEQ ID NO: 3 Sequence length: 1082 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: cDNA to mRNA sequence GAGGAGGCCA TGGGCGCGCG CGGGGCGCTG CTGCTGGCGC TGCTGCTGGC TCGGGCTGGA 60 CTCAGGAAGC CGGAGTGGCAGCGAGATCGACGCGTC CCGACGGGTC 120 ATCACGTCGC GCATCGTGGG TGGAGAGGAC GCCGAACTCG GGCGTTGGCC GTGGCAGGGG 180 AGCCTGCGCC TGTGGGATTC CCACGTATGC GGAGTGAGCC TGCTCAGCCA CCGCTGGGCA 240 CTCACGGCGG CGCACTGCTT TGAAACCTAT AGTGACCTTA GTGATCCCTC CGGGTGGATG 300 GTCCAGTTTG GCCAGCTGAC TTCCATGCCA TCCTTCTGGA GCCTGCAGGC CTACTACACC 360 CGTTACTTCG TATCGAATAT CTATCTGAGC CCTCGCTACC TGGGGAATTC ACCCTATGAC 420 ATTGCCTTGG TGAAGCTGTC TGCACCTGTC ACCTACACTA AACACATCCA GCCCATCTGC 480 CTCCAGGCCT CCACATTTGA GTTTGAGAAC CGGACAGACT GCTGGGTGAC TGGCTGGGGG 540 TACATCAAAG AGGATGAGGC ACTGCCATCT CCCCACACCC TCCAGGAAGT TCAGGTCGCC 600 ATCATAAACA ACTCTATGTG CAACCACCTC TTCCTCAAGT ACAGTTTCCG CAAGGACATC 660 TTTGGAGACA TGGTTTGTGC TGGCAATGCC CAAGGCGGGA AGGATGCCT G CTTCGGTGAC 720 TCAGGTGGAC CCTTGGCCTG TAACAAGAAT GGACTGTGGT ATCAGATTGG AGTCGTGAGC 780 TGGGGAGTGG GCTGTGGTCG GCCCAATCGG CCCGGTGTCT ACACCAATAT CAGCCACCAC 840 TTTGAGTGGA TCCAGAAGCT GATGGCCCAG AGTGGCATGT CCCAGCCAGA CCCCTCCTGG 900 CCACTACTCT TTTTCCCTCT TCTCTGGGCT CTCCCACTCC TGGGGCCGGT CTGAGCCTAC 960 CTGAGCCCAT GCAGCCTGGG GCCACTGCCA AGTCAGGCCC TGGTTCTCTT CTGTCTTGTT 1020 TGGTAATAAA CACATTCCAG TTGATGCCTT GCAGGGCATT CTTCAAAAAA AAAAAAAAAA 1080 AA 1082

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0059

[Correction method] Change

[Correction contents]

SEQ ID NO: 4 Sequence length: 1082 Topology: linear Sequence type: cDNA to mRNA origin Organism name: Homo Sapiens cell: Eosinophil Sequence characteristics Characteristic symbol: CDS Location: 10. .951 Method for determining characteristics: S Characteristic symbol: sig peptide Location: 10..87 Characteristic determination method: S Characteristic symbol: proform peptide Location: 88..951 Characteristic determination method: S Symbol representing feature: mat. Peptide Location: 133..951 Method for determining feature: S Symbol representing feature: active center (His) Location: 253..255 Method for determining feature: S Representing feature Symbol: Active center (Asp) Location: 418..420 Method for determining characteristics: S Symbol representing characteristics: Active center (Ser) Location: 721..723 Method for determining characteristics: S Symbol representing characteristics: ω, ω1, ω2 sites Location: 865.873 Features Determined method: S Symbol indicating feature: ω, ω1, ω2 site Location: 874..882 Method determining feature: S Symbol indicating feature: hydrophilic region Location: 880.897 Method determining feature : S Symbol representing characteristics: Hydrophobic region Location: 898..951 Method for determining characteristics: S sequence GAGGAGGCC ATG GGC GCG CGC GGG GCG CTG CTG CTG GCG CTG CTG CTG 48 Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu -26 -25 -20 -15 -15 GCT CGG GCT GGA CTC AGG AAG CCG GAG TCG CAG GAG GCG GCG CCG TTA 96 Ala Arg Ala Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala Pro Leu -10 -5 1 TCA GGA CCA TGC GGC CGA CGG GTC ATC ACG TCG CGC ATC GTG GGT GGA 144 Ser Gly Pro Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly 5 10 15 GAG GAC GCC GAA CTC GGG CGT TGG CCG TGG CAG GGG AGC CTG CGC CTG 192 Glu Asp Ala Glu Leu Gly Arg Trp Pro Trp Gln Gly Ser Leu Arg Leu 20 25 30 35 TGG GAT TCC CAC GTA TGC GGA GTG AGC CTG CTC AGC CAC CGC TGG GCA 240 Trp Asp Ser His Val Cys Gl y Val Ser Leu Leu Ser His Arg Trp Ala 40 45 50 CTC ACG GCG GCG CAC TGC TTT GAA ACC TAT AGT GAC CTT AGT GAT CCC 288 Leu Thr Ala Ala His Cys Phe Glu Thr Tyr Ser Asp Leu Ser Asp Pro 55 60 65 TCC GGG TGG ATG GTC CAG TTT GGC CAG CTG ACT TCC ATG CCA TCC TTC 336 Ser Gly Trp Met Val Gln Phe Gly Gln Leu Thr Ser Met Pro Ser Phe 70 75 80 TGG AGC CTG CAG GCC TAC TAC TAC ACC CGT TAC TTC GTA TCG AAT ATC TAT 384 Trp Ser Leu Gln Ala Tyr Tyr Thr Arg Tyr Phe Val Ser Asn Ile Tyr 85 90 95 CTG AGC CCT CGC TAC CTG GGG AAT TCA CCC TAT GAC ATT GCC TTG GTG 432 Leu Ser Pro Arg Tyr Leu Gly Asn Ser Pro Tyr Asp Ile Ala Leu Val 100 105 110 115 AAG CTG TCT GCA CCT GTC ACC TAC ACT AAA CAC ATC CAG CCC ATC TGC 480 Lys Leu Ser Ala Pro Val Thr Tyr Thr Lys His Ile Gln Pro Ile Cys 120 125 130 CTC CAG GCC TCC ACA TTT GAG TTT GAG AAC CGG ACA GAC TGC TGG GTG 528 Leu Gln Ala Ser Thr Phe Glu Phe Glu Asn Arg Thr Asp Cys Trp Val 135 140 145 ACT GGC TGG GGG TAC ATC AAA GAG GAT GAG GCA CTG CCA TCT CCC CAC 576 Thr Gly Trp Gly Tyr Ile Ly s Glu Asp Glu Ala Leu Pro Ser Pro His 150 155 160 ACC CTC CAG GAA GTT CAG GTC GCC ATC ATA AAC AAC TCT ATG TGC AAC 624 Thr Leu Gln Glu Val Gln Val Ala Ile Ile Asn Asn Ser Met Cys Asn 165 170 175 CAC CTC TTC CTC AAG TAC AGT TTC CGC AAG GAC ATC TTT GGA GAC ATG 672 His Leu Phe Leu Lys Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp Met 180 185 190 195 GTT TGT GCT GGC AAT GCC CAA GGC GGG AAG GAT GCC TGC TTC GGT GAC 720 Val Cys Ala Gly Asn Ala Gln Gly Gly Lys Asp Ala Cys Phe Gly Asp 200 205 210 TCA GGT GGA CCC TTG GCC TGT AAC AAG AAT GGA CTG TGG TAT CAG ATT 768 Ser Gly Gly Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp Tyr Gln Ile 215 220 225 GGA GTC GTG AGC TGG GGA GTG GGC TGT GGT CGG CCC AAT CGG CCC GGT 816 Gly Val Val Ser Trp Gly Val Gly Cys Gly Arg Pro Asn Arg Pro Gly 230 235 240 GTC TAC ACC AAT ATC AGC CAC CAC TTT GAG TGG ATC CAG AAG CTG ATG 864 Val Tyr Thr Asn Ile Ser His His Phe Glu Trp Ile Gln Lys Leu Met 245 250 255 GCC CAG AGT GGC ATG TCC CAG CCA GAC CCC TCC TGG CCA CTA CTC TTT 912 Ala Gln Ser Gl y Met Ser Gln Pro Asp Pro Ser Trp Pro Leu Leu Phe 260 265 270 275 TTC CCT CTT CTC TGG GCT CTC CCA CTC CTG GGG CCG GTC 951 Phe Pro Leu Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 280 285 TGAGCCTACC TGAGCCCATG CAGCCAGGCCCC GTCAGGCCCT GGTTCTCTTC 1011 TGTCTTGTTT GGTAATAAAC ACATTCCAGT TGATGCCTTG CAGGGCATTC TTCAAAAAAA 1071 AAAAAAAAAA A 1082

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 7/00 C12N 9/64 16/40 A61K 37/64 ABN C12N 9/64 ADZ

Claims (13)

[Claims] 1. A polypeptide which is an eosinophil serine protease, a homolog thereof, a fragment thereof or a homolog of a fragment thereof, comprising an amino acid sequence represented by SEQ ID NO: 1 in a substantially pure form. 2. The polypeptide according to claim 1, comprising the amino acid sequence represented by SEQ ID NO: 1. 3. A cDNA encoding the polypeptide according to claim 2. 4. The cDNA according to claim 3, which has the nucleotide sequence represented by SEQ ID NO: 2, or a fragment that selectively hybridizes to the sequence. 5. The cDNA according to claim 3, which has the nucleotide sequence shown in SEQ ID NO: 3, or a fragment that selectively hybridizes to the sequence. 6. A replication or expression vector into which the cDNA according to claim 3 has been incorporated. 7. A host cell transformed with the replication or expression vector according to claim 6. 8. The method for producing a polypeptide according to claim 1 or 2, comprising culturing the host cell according to claim 7 under conditions for expressing the polypeptide according to claim 1 or 2. . 9. A monoclonal or polyclonal antibody of the polypeptide according to claim 1 or 2. 10. The polypeptide according to claim 1 or 2, or the antibody or antisense according to claim 9,
And a pharmaceutically acceptable excipient and / or carrier. 11. A method for screening a serine protease inhibitor, comprising using the serine protease according to claim 1 or 2. 12. A method for diagnosing an allergic disease, comprising measuring the serine protease activity according to claim 1 or 2. 13. A method for diagnosing an allergic disease, comprising using the antibody according to claim 9.